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Small Molecule Inhibitors, Design and Selection of
Jianwei Che and Yi Liu, Genomics Institute of the Novartis Research Foundation, San Diego, California
Nathanael S. Gray, Harvard Medical School, Dana Farber Cancer Institute, Biological Chemistry and Molecular Pharmacology, Boston, Massachusetts
doi: 10.1002/9780470048672.wecb123
The discovery and optimization of small-molecule inhibitors for the treatment of human disease has been the major focus of the pharmaceutical industry for more than a century. In the last decade, they have also attracted increased attention from academia because they provide a powerful means to interrogate biologic systems in an acute fashion. Although pharmaceutical companies have identified many small-molecule inhibitors for drug targets, to find a desirable small-molecule inhibitor for a specific target is still a major challenge for drug discovery and for chemical biology. Here, we present an overview of the computational approaches that can be exploited to discover small-molecule inhibitors for protein targets of interest.
Small molecule inhibitors are of special value to chemical biology and to drug discovery. Thousands of small molecules have been developed as drugs to treat various human diseases. Increasingly, small-molecule inhibitors are used to perturb the function of proteins to elucidate their biologic functions (1, 2). Complementary to the conventional genetics, small molecules allow rapid, conditional, and tunable inhibition. Furthermore, small molecules can often be identified that alter specifically one particular function of a multifunctional protein. The recent completion of the human genome sequencing project has resulted in the identification of a plethora of new protein targets for which small-molecule modulators could be developed as potential therapeutics and as biologic "tools." Identifying a selective small-molecule inhibitor for each protein in human genome is a formidable challenge for drug discovery and for chemical biology. Experimentally, high-throughput screening is the most widely used technology currently to identify hits for a target. On the other hand, computational methods, such as virtual screening and structure-based design, have become equally important to identify and to optimize small-molecule inhibitors. In Fig. 1, we illustrate how computational and experimental approaches can complement each other in the compound discovery and optimization process. The numbers of compounds associated typically with each method is indicated. At early stages of hit identification, high throughput methods such as virtual ligand screening (VLS) and ultra high-throughput screening (uHTS) are used frequently. At later stages, high accuracy algorithms combined with biologic assays can be used to guide the selection and the synthesis of a limited set of analogs. As shown in Fig. 1, computational and experimental approaches are not mutually exclusive processes. They are best exploited by using computation and experiment in an iterative fashion to optimize compounds progressively for a particular target of interest. Without these interactions, none of the methods can achieve its maximum potential. In this review, we focus on computational approaches depicted in Fig. 1. As will become clear later in the article, most computational methods are not associated exclusively with predefined processes or protocols. Significant redundancies exist in terms of when and what methods should be applied. The structure of the article follows the common flow of identifying small-molecule inhibitors. We will discuss the particular computational methodologies used at different stages of the process in subsequent sections.
Figure 1. Overview of computational methods used in the process of identifying small-molecule inhibitors.
Virtual Screening in Selecting Small Molecule Inhibitors
Virtual screening (VS) uses computational or theoretic methods to select molecules with desired biologic properties. It has broad applications in pharmaceutical industry. In this article, we limit ourselves to VS to identify biologically active small molecules. The estimated chemical space for small organic molecules is over 1060, and the registered organic and inorganic molecules in CAS are already over 30 million. Despite advances in experimental high throughput screening technologies, currently it is impossible to screen more than a few million compounds. With the rapid improvement of computational power and algorithms, VS methods have become an important complementary technique to experimental uHTS. It has many distinct advantages, such as investigating virtual molecules that can cover a broader area of chemical space. Compared with uHTS, VS is also a relatively inexpensive means to probe many molecular structures.
Generally, two types of virtual screening are used. One is receptor based, such as docking, and the other is ligand based, such as similarity search. In practice, both methods are often used synergistically. In Fig. 2, we illustrate how these two types of methods are used to select "hits" from a given small-molecule library.
Figure 2. Workflow of computational algorithms used for identifying ''hit'' molecules from a compound library. The top panel indicates the compound collection from which the ''hit'' molecules are to be identified. The middle panel illustrates two virtual screening strategies. One is based on a known receptor 3-D structure (i.e., docking and scoring) and the other is based on active ligand molecules alone (i.e., pharmacophore searching and ranking). The docking/scoring uses existing 3-D structures of receptors to construct binding models, and the pharmacophore searching/ranking uses known active small molecules to construct pharmacophore models.
Receptor-Based VLS
In receptor-based VLS, the atomic coordinates of receptor molecules are known experimentally or theoretically (e.g., homology models). The interactions between a small molecule and a protein are often characterized by a "lock-key" mechanism, in which high affinity small molecules often fit the protein-binding site well. The shape complementarities provide important enthalpy contributions from favorable van der Waals interactions. In addition, correctly positioned charges, hydrogen bonds, and hydrophobic contacts usually are required for higher affinity. Based on computational efficiency, receptor-based VLS can be divided into three tiers. The first tier is the popular high-throughput docking and scoring that can be computed for each ligand molecule within a few minutes on a typical desktop computer. In the rest of this article, we refer to high-throughput docking as docking and scoring for simplicity. The second tier includes the medium throughput methods that calculate the interactions more thoroughly and accurately. It often includes algorithms that account more accurately for desolvation energies, entropic penalties, and receptor flexibility. For example, detailed implicit solvent models usually are applied in these calculations. It normally takes tens of minutes for each calculation. The third tier represents the slowest method but ultimately offers more accurate results. The typical examples are atomistic molecular dynamics (MD) or Monte Carlo simulations with explicit water molecules. The details of these calculations are out of the scope of this review. Interested readers can consulate recent reviews on these specific topics (3).
Among the three classes of methods, docking is the most widely used method in pharmaceutical industry. Many high throughput docking software packages are available publicly, such as Dock, Autodock, Gold, Glide, FlexX, QXP, and ICM. In addition, many companies have built their own proprietary docking technologies. A docking based VLS calculation usually consists of three stages: preparing, computing binding modes, and scoring. Currently, most docking programs treat receptors as rigid molecules. Under this approximation, a receptor field can be calculated for the binding site. A receptor field is the interaction potential created by receptor atoms. It consists of van der Waals, electrostatic, hydrogen bonding, and solvation contributions. Without atom thermal motions, the receptor field is also static, so it can be precalculated in the absence of ligand molecules. This feature provides enormous savings of computational time over explicit atom models. Different programs employ very different algorithms to sample ligand conformations, to orient small molecules in the binding site, to retain preferred binding modes, and to increase the computational efficiency (4-7). The predicted binding configuration is selected based on molecular interaction energies or an empirical scoring function. Finally, docking ranks all ligand molecules based on binding poses that possess the highest score, and virtual "hit" molecules are selected from the top of the list. This method allowed Hopkins et al. (8) to identify submillimolar Kinesin inhibitors from an 110,000 compound library. Novel casein kinase II inhibitors (9) and BCR-ABL tyrosine kinase inhibitors (10) were also discovered by docking. Docking can also be applied to discover small-molecule inhibitors for protein-protein interactions. Bonacci et al. (11) used docking (FlexX) to screen the chemical diversity set of 1990 compounds. Among 85 virtual hits, 9 of them inhibited SIGK binding with Gβ1γ2 with IC50 that ranged from 100 nM to 60 μM. It was also used by Trosset et al. (12) to identify β-Catenin inhibitors for Wnt signal pathway from 177,000 compounds. Among 22 tested virtual hits, 3 hits were confirmed as binders to β-Catenin and Tcf4 competitors. Without experimental receptor structures, docking can be carried out using carefully constructed model structures. Becker et al. (13) and Salo et al. (14) reported the successful identification of small-molecule inhibitors of several G-protein coupled receptors (GPCR) systems based on docking.
One major challenge in docking-based VLS is the accuracy of scoring functions. Conceptually, binding free energy is a rigorous measure for correct binding conformation and compound affinity. However, the inherent errors in classic force fields and the extremely lengthy computation required preclude its immediate use for VLS. In practice, an empirical function (i.e., a scoring function) normally is used to estimate free energy of binding. A few examples of commonly used scoring functions are PMF (15), Chemscore (16), Drugscore (17), Goldscore (18), and Glidescore (19). The specific mathematic forms of Chem- score, Goldscore, and PMF are shown here to highlight their similarity and difference. Other scoring functions can be found in respective references.
where ∆G0 is a fitting constant, ∆Ghbond is hydrogen bond energy between ligand and receptor, ∆Gmetai is the energy between ligand atoms and metal ions in a receptor active site, ∆Glipo is the ligand receptor lipophilic interaction, and ∆Grot is for the rotatable bond penalty.
where ∆Ehbond is the hydrogen bond energy between ligand and receptor, and ∆Evdw is the van der Waals energy of ligand and receptor, and ∆Einternal is the van der Waals energy and torsional strain of the ligand molecule.
where kl is a ligand receptor atom pair of type ij, fj(r) is ligand volume correction factor, pijseg(r) is the number density of a ligand receptor atom pair of type ij at inter atomic distance r, pijbulk is the reference number density of ligand receptor pair of type ij, and ∆Evdw is the van der Waals interaction between ligand and receptor. Although common features exist within different scoring functions, they often emphasize different aspects of the protein-ligand interaction. These empirical scoring functions bear significant errors because of crude approximations. Currently, no scoring functions exist that are able to rank diverse compound libraries consistently and accurately. The performance of docking based VLS varies considerably from system to system and from program to program (20). Although some programs tend to perform better than others do, it is difficult to predict the performance a priori on a specific system. In addition to ranking molecules based upon a single scoring function, combination of several scores (i.e., consensus score) or statistical methods (e.g., Bayesian statistics) are also used to select virtual hits.
Often, rigid receptor models provide a reasonable approximation and offer tremendous practical value. The majority of docking calculations used in industry adopt this approximation. In reality, however, receptor atoms undergo constant thermal motions, and some proteins have significant plasticity. One example is inactive and active conformations of kinases in which the activation loop moves in and out of active sites (21) with some atoms that move well over 10 A. It is common to see a variety of conformational states for a given protein in PDB databank. It is also possible that such conformations observed crystallographically represent only a fraction of the transient dynamic states that may exist in solution. Although several methods (22-24) have been developed to account for protein flexibility, considerably more computational and experimental work will be required to create algorithms that can incorporate receptor flexibility accurately. In addition to receptor plasticity, another complicating factor is the difficulty to account accurately for the enthalpic and entropic contributions of discrete water molecules in the ligand bound and unbound states. Water molecules have been observed crystallographically to play important roles in mediating hydrogen bonds between protein atoms and between protein atoms and ligands. In most docking calculations, the water molecules are removed from binding sites, which assumes that to displace these water molecules by small molecules would give favorable entropic contribution. However, the entropic gain comes at the expense of enthalpy loss if the water molecules make strong hydrogen bonds to receptor atoms or mediate ligand-protein interactions. Several programs exist that attempt to include movable water molecules in docking calculations, however, often this results in a significant loss of computational efficiency.
Besides virtual screening of ligand libraries, docking is extremely useful for lead optimization. Successful docking can elucidate the important interactions between lead molecules and the target, explain important features of the structure activity relationships (SAR), and consequently help to design new molecules. Other than docking, useful receptor based VLS tools include MD simulation, MC simulation, and QM/MM calculation.
Ligand-Based Virtual Screening (2-D Similarity Search, 3-D Pharmacophore Search)
Another category of VLS methods is to identify ligands for targets that have unknown 3-dimensional (3-D) structures. Generally, these methods are called ligand-based VLS. The most simple and probably the most used searches involve a physical, chemical, or topological similarity search based on one or several active compounds. The similarity between two molecules can be defined in various ways. Two-dimensional (2-D) methods refer to those that only consider atom connection tables to describe molecular structures, because atom connection tables are best represented by 2-D pictures such as the ones in the top panel of Fig. 2. These methods consider topological relationships between atoms in a molecule regardless their actual 3-Dl coordinates. The simplicity of these methods allows computationally efficient comparisons. It can screen millions of molecules in seconds. Here, we outline a few representative examples.
1. Molecular 2-D fingerprints are a class of methods that use chemical substructures to characterize similarities between molecules. A fingerprint is a signature that encodes the chemical topological information of a small molecule. It is usually a long binary bit string in which each bit represents the presence 1 or absence 0 of a particular chemical substructure. For example, an ATP molecule has substructures such as a pyrimidine and imidazole heterocycle but not benzene; therefore, the bits that represent pyrimidine and imidazole would be set to "1" and the bit that represents benzene would be set "0" in the fingerprint bit string of ATP. Many variations in detailed definitions of 2-D fingerprints exist. In one straightforward way, molecular similarities are measured by the number of bits two compounds have in common.
2. Pharmacophores are defined as essential structural features in a molecule that are responsible for its binding activity, and they can also be used to generate 2-D fingerprints. Examples of commonly used pharmacophores are specific molecular fragments (i.e., an isopropyl group), hydrogen bond donors and acceptors, and negatively and positively charged atoms or groups. In two dimensions, the minimum number of bonds between two pharmacophores can be used as a distance metric. For example, the exocyclic amino group of ATP is located 3, 4, and 5 bonds away to the three acceptor nitrogens in the purine ring. This information is coded readily in a fingerprint bit string.
3. In addition to 2-D fingerprints, the topological relationships of atoms in a molecule can be explored in high feature dimensions and nonlinear fashion by other means, such as kernel methods (25), support vector machines (26), and self-organizing maps (27).
However, molecular recognition occurs in a 3-D world. It is well known that minor chemical modifications can alter the activity of a molecule drastically. On the other hand, very different chemical structures can have similar activity against the same target. The mere existence of particular pharmacophores and/or their topological relations are not sufficient for a molecule to be active against its target. To account for 3-D information, various VLS techniques based on activities of small molecules without receptor structures have been developed, such as 3-D quantitative structure activity relationship (QSAR) models. It is commonly accepted that a small molecule must exhibit a high degree of shape complementarity to bind to its protein target with high affinity. Finding the correct superposition of active ligand molecules is the key to a successful 3-D QSAR model. Of course, shape alone does not account for all contributions of small molecules. Other factors such as locations of hydrogen bond donors and acceptors, types of functional groups, electrostatic properties, and polarizabilities all play important roles for small-molecule activities. One attempt to capture this information is 3-D pharmacophore method. Like docking, many software programs exist to develop 3-D pharmacophore models, such as DISCO (28), Catalyst (28), GASP (28), PHASE (29), and many others (30, 31). Pharmacophore features are not mutually exclusive; for instance, a hydrogen bond acceptor (i.e., donating electron pair) can be negatively charged (e.g., side chain carboxylate from Asp or Glu). Most programs provide users with the flexibility to define their own pharmacophore features. The main merit of 3-D pharmacophore models is the ability to identify lead compounds that are diverse structurally.
Although software packages differ in how they construct pharmacophore models, they go through the following common steps: generate conformers, enumerate pharmacophore features, generate a hypothesis, and screen the ligand library. Usually, the active conformation of a small molecule is unknown without experimental evidence. A key component of a good pharmacophore method is to sample active conformations rapidly. Studies have shown that the active conformation of a small molecule may not necessarily be the same as the lowest energy one in the unbound state (32). Therefore, a set of possible conformers within a certain energy window (e.g., 15 kcal/mol above the lowest energy conformer) are collected and clustered. The feature-enumerating step is relatively straightforward. It identifies all the possible pharmacophore features in small molecules. Hypothesis generation is another challenging step in building pharmacophore models. In essence, this step involves finding a proper alignment of active molecules with a high degree of overlap in 3-D space in terms of their pharmacophore features. During this step, the spatial arrangements of pharmacophore features are identified so that they are common for all or a significant subset of active molecules. This step assumes that active molecules must satisfy a common pattern of pharmacophore features. However, it should not require a molecule to have all the features defined in a model to be active. The proper alignment of active molecules determines the quality of a 3-D pharmacophore model. Several algorithms have been developed to identify pharmacophore patterns (33-36) among the set of possible conformations for each active molecule. Pharmacophore hypothesis algorithms try to find and to rank the most probable pharmacophore models for known active molecules. It should also be noted that active molecules do not necessarily share a common pharmacophore pattern. Distinct binding modes have been observed in cocrystal structures (37). Moreover, different small molecules may bind to different binding sites of a receptor. Although a few algorithms exist to identify multiple pharmacophore patterns within a set of actives (29, 36), more robust methods are needed. The conformations of small molecules can be precomputed or sampled "on the fly." Once a valid pharmacophore model is constructed, it can be used to screen millions of small molecules. This strategy was used successfully by Singh et al. to identify some potent and novel VLA-4 antagonists (38). Three-dimensional pharmacophore models are used commonly for GPCR systems because generally, these membrane-localized target structures are unknown. A three-point pharmacophore model was constructed for Urotensin II receptor (39) and was used to search Aventis compound collection. Of 500 virtual "hits," 10 hits were active with the most potent compound exhibiting an IC50 of 400 nM. Recently, the first GPR30 specific agonist was discovered by 2-D and shape similarity in combination with a pharmacophore search (40).
Not only can active molecules be used in pharmacophore modeling, structures of inactive molecules can also provide valuable information. In common pharmacophore models, information provided by inactive compounds is captured in the concept of "excluded volume" (29, 41). An "excluded volume" is a region that is so close to the receptor that no ligand atoms are tolerated. "Excluded volume" can be obtained automatically or defined manually. The method relies on the assumption that the binding modes of the compound series are similar to each other. Although many examples from cocrystal structures and SAR have confirmed this assumption, many exceptions to this empirical observation exist. Chemical similarity does not always predict similarity in biologic activities. When active compounds consist of multiple scaffolds because of different modes of action, it is very challenging to develop pharmacophore models and to classify the actives into proper categories automatically. In addition, a compound that satisfies a good pharmacophore model is not guaranteed to have activity. For example, entropic contributions, solubility, cell permeability, and stability can also have important implications on a compound's biologic activity.
Even though most pharmacophore models are built without receptor structures, binding site structures do help to construct proper pharmacophore models. The critical residues provide a complementary image of ligand pharmacophore field. This "negative image" can be converted to a ligand pharmacophore model. One advantage of receptor structure based pharmacophore models is the natural definition of "excluded volume." The pharmacophore model derived from a receptor structure can be used not only to search small-molecule libraries directly, but also to guide docking calculations (42). Similarly, docking can help to derive receptor based pharmacophore models as well. For instance, docking active compounds can help to identify the important pharmacophore features. Submicromolar tRNA-guanine transglycosylase inhibitors were discovered by combinations of pharmacophore searching and docking (43). Pharmacophore models can also be used for de novo design (44, 45) and building 3-D QSAR models (29, 46).
Design of Small Molecule Inhibitors
VLS is a cost-effective and time-efficient approach to identify inhibitors for a given target from a small-molecule library. However, the hits from virtual screening usually are of low affinity and have properties that are not ideal for drug development or even for use as research tools (such as lacking cell permeability, potency, selectivity, and solubility). Furthermore, VLS is often performed using existing small-molecule libraries, which are biased heavily toward thoroughly explored regions of chemical space. An alternative and very important method to identify small-molecule inhibitors is through design. Two strategies are used in lead design. One strategy is based on known scaffolds or fragments from VLS, HTS, and fragment based screening to improve potency and desired pharmacological properties. The other strategy is de novo design, which generates small molecule leads from scratch. Figure 3 shows the relationships between these strategies and shows when they can be used appropriately.
Figure 3. Workflow of computational algorithms used to design lead molecules. The top panels indicate various sources of starting points on which ''lead'' design can be based. The middle panels illustrate the corresponding design strategies under the different starting conditions. The details of each method are described in the main text.
Scaffolds and Fragments-Based Lead Design
Computational methods offer ways to investigate chemical modifications automatically and rapidly that may be beneficial to the small-molecule binding. Based on a core fragment or scaffold of a hit, substituents are introduced to the core structure from a small fragment library to the core structure. The selection of substituents is biased toward favorable binding interactions. For each modification, a local energy optimization or full docking can be carried out. The new molecule is then evaluated based on a scoring function that measures interactions with the protein-binding site. The scoring function is similar to the one used in docking with necessary modifications to accommodate additional property requirements. The substituents can be added continuously until a given criteria, such as the number of iterations, desired score, or maximum size of a molecule, is satisfied. LUDI (47) by Accelrys, Rachel and LeapFrog by Tripos, and molecular evolution developed internally at GNF are examples of the method. Programs differ in the details of how molecular structures are modified, optimized, and ranked. This strategy has been used to improve activities of HIV protease inhibitors (47) and other molecules (48).
Besides obtaining scaffolds from VLS, experimental fragm ent-based screening by NMR and crystallography offers a good starting point to design novel small-molecule inhibitors. The key to this method is cocrystal structures of the target protein and low-molecular-weight compounds, typically limited to molecular weights of between 120-250 Da to allow for subsequent additions. Usually, these compounds have weak affinities (Kds = 10 μM-10 mmol/L). To explore modifications that have the potential to increase binding affinities, the aforementioned computational methods can be applied readily. For structures with a single small molecule in a complex, the small compound can be extended automatically to maximize its interaction with target protein. During the progressive modifications, the binding mode for the initial fragment can be altered. However, certain constraints are applied to avoid drastic changes to the binding modes unless substantial modifications are introduced to the original structure. When evidence exists that multiple ligands bind to different pockets of an extended binding site in one or multiple crystal structures, computational methods can be used to propose proper linkers or fuse-disjointed fragments to yield the synergistic effects. Gill et al. (49) have used this strategy to design P38a MAP kinase inhibitors based on hits with affinities in the millimolar range. For example, they identified a lead molecule with IC50 = 65 nM by extending the base fragment to acquire additional interactions, and also to achieve a 100-fold increasing in potency rapidly by cojoining two overlapping fragment hits for an indole-derived compound. The structures are depicted in Fig. 4. It should be emphasized that crystal structures are extremely important in fragment-based design iterations. Because fragment hits are usually small and possess low affinity for the targets, to predict the correct binding modes by docking is difficult. In addition, the modifications to the base fragment may alter its binding modes fundamentally.
Figure 4. Inhibitor design based on low-molecular-weight compounds. In the upper panel, the base fragment is shown in black and the added groups are shown in red. In the lower panel, a common core fragment for cojoining two fragments is shown in black and the different substituents are shown in red and light blue.
Hybrid Design Based on Known Inhibitors
Through many years of drug discovery efforts, pharmaceutical companies have synthesized millions of small-molecule inhibitors. A large amount of information has been accumulated on how these inhibitors bind to their protein targets and to the QSAR of these inhibitors. Hybrid design is an emerging technique that attempts to take full advantage of this information to design new inhibitors for existing or new protein targets (50). The essence of the hybrid design is to recombine known inhibitors for a particular target in a rational way to create new inhibitors for the same target or for a new target. In a manner analogous to the evolutionary recycling of protein domains, the hope for chemical evolution through hybrid design is to create new inhibitors by recombining pharmacophores derived from different classes of compounds. Hybrid design starts by using inhibitor-receptor binding information obtained experimentally from crystallography or computationally by docking to overlay multiple known ligands, and then to predict how to recombine inhibitor substructures to create new compounds. This strategy is illustrated in Fig. 5, in which a potent Aurora A kinase inhibitor can be thought of as a hybrid of two inhibitors developed originally for two other kinases. This strategy has also been applied successfully to design novel kinase inhibitors that bind to a specific inactive kinase conformation (51, 52). Gleevec is a Bcr-Abl inhibitor that has been demonstrated crystallographically to bind to the nucleotide binding cleft of Abl, which uses both the adenine binding region and an adjacent hydrophobic pocket created by the activation loop being in a unique inactive conformation (53). A hybrid strategy involved appending a 3-trifluoromethylbenzamide group to a known ATP binding site inhibitor (51). Of the hybrid compounds thus created, potent and selective inhibitors have been identified for Abl, c-Kit, p38, PDGFR, and Aurora kinases (52). Figure 5 shows how a benzamide group is attached to a quinazoline scaffold to create an inhibitor that binds to an inactive Aurora kinase conformation similar to that observed on Abl-Gleevec complex.
A computer program named BREED automates the process for hybrid design (54). The method imitates the common medicinal chemistry practice of joining fragments of two known ligands to generate a new inhibitor. The known active ligands are superimposed in their active conformation to identify all overlapping bonds, and the fragments on each side of each matching bond are swapped to generate a large set of novel inhibitors. This method has been demonstrated to have a high rate of success to identify novel inhibitors for HIV protease and protein kinases (54).
Figure 5. Illustration of hybrid design strategy for small-molecule inhibitors. The activation loops of the proteins are colored differently to indicate active (blue) and inactive (orange) states. The important fragments of inhibitors are colored blue and orange to highlight the hybrid design concept.
De Novo Design
Small molecule inhibitors can also be generated by de novo design without the prior knowledge of other active ligands. In essence, the procedures are similar to those mentioned previously. For example, LeapFrog can start without a given small molecule. It will select a core randomly and will grow a ligand from its fragment library. Although it is still rare for a computer program to generate practical and active molecules from scratch, reports of novel molecules from "raw" output of automated computer program exist. SkelGen (55) was able to generate small-molecules inhibitors for estrogen receptor binding sites without hints from known actives. Among the 17 highest scoring structures, 5 structures were active with 4 structures that posses novel structures. We have also implemented a de novo ligand design algorithm in our in-house software GModE using evolutionary algorithm. A molecule is represented by a unique genetic code that is subject to normal genetic operations such as mutation, addition, deletion, and translocation. The population of ligand molecules is evolved toward increasing fitness. Our retrospective studies have shown that the method can produce lead molecules that are extremely similar to experimental nanomolar inhibitors.
If the target binding site structure is unavailable, de novo design can be carried out based on known active molecules. This circumstance may occur because none of the known actives that pose desired properties or that pose novel inhibitors are sought for patentability. However, the known inhibitors provide a basis to construct 3-D pharmacophore and other models such as Comparative Molecular Field Analysis (CoMFA) that can provide a template to guide the computational algorithm to maximize the fitness of the molecule "population" toward a given model. For example, LeapFrog can use CoMFA models in place of target binding site structures. It is also straightforward to apply pharmacophore models to novel molecular design with and without starting fragments. Novel structures are chosen based on its fitness to the given model. Unlike de novo design with known binding site structures, it is important to minimize the possibilities for resulting molecules to have steric clashes with receptor atoms. Even though these programs have produced promising results, the major drawback for such automated computer algorithms is that the "raw" computer derived molecules often are undesirable synthetically or implausible chemically. Some programs (55) have implemented chemistry rules to guide the generation of new molecules, but it is still far from satisfactory. Moreover, because a drug molecule interacts with its intended target in a complex biologic environment, a good lead molecule is selected by not only its binding affinity to its intended target, but also other properties such as crucial physical chemical properties, ability to expand SAR, selectivity relative to undesired off targets such as cytochrome P450 enzymes, and ion channels such as HERG. All are important considerations to advance a molecule toward clinical development. Various computational methods have been developed to predict these properties; however, normally they are not integrated into automated de novo design program.
Hit and Lead Optimization
Computational algorithms and modeling have advanced significantly over the last two decades in terms of accuracy, efficiency, and ease of use. Many modeling procedures are automated; however, they are far from replacing the intuition and knowledge of an experienced computational chemist. Although automated programs are used widely in early stages of lead discovery with limited human intervention, lead optimization is a process in which a computational chemist makes most contributions out of his knowledge, insight, experience, and intuition. Before lead optimization, computational algorithms are designed to explore wide areas of chemical space and a wide range of activity data. During lead optimization, focus is placed on one or a few particular compound series. Small modifications are introduced to investigate local SAR and to improve other pharmacologic properties. Incremental improvements are often the objectives at this stage. Whereas the automated computational methods described previously are still important, they are not sufficiently precise to distinguish between modifications that result in 2kcal/mol changes in binding affinities. Manual intervention, such as visual inspection and manual docking from an experienced chemist, are often of great importance for this phase.
Structure Based
As we can observe from previous discussion, protein structures have played invaluable roles in small-molecule inhibitor selection and design. Currently, over 40,000 biomacromolecular structures are deposited in RCSB protein databank. Over 5000 new structures were deposited in 2006. The atomic level structural information provides an enormous wealth of mechanistic insights with regard to protein function. Approximately 6500 protein structures in the database are in complex with an inhibitor or substrate to provide chemists with important insights into the mechanism of ligand recognition. Because biologic systems are very diverse, each system requires specialized knowledge and treatment. Successful ligand design requires an intimate knowledge of all known inhibitors, which include how their activity and selectivity is changed as a function of chemical modification (SAR), rules for which interactions are required for high affinity binding, knowledge about the potential for conformational rearrangements, and insight into what is tractable synthetically. Success therefore requires the close collaboration of computational chemists, medicinal chemists, and structural biologists. Here, we will attempt only to describe a few common techniques that computational chemists use to optimize the activity of a small-molecule inhibitor.
Visual inspection is probably one of the oldest and most powerful methods in computer modeling. Large amounts of information can be acquired by looking at the 3-D structures of an inhibitor and a protein complex, such as important hydrogen bonds, hydrophobic contacts, and strong electrostatic interactions. It allows one to design a derivative to make stronger or additional hydrogen bonds, to explore unoccupied pockets, and even to alter the scaffold while maintaining important interactions. Of course, regular molecular modeling methods are used here to ensure the proposed molecules possess sensible conformations. In contrast to the automatic molecular generating programs mentioned previously, the modifications here are proposed directly by chemists; therefore, the molecules are tractable synthetically and often follow a particular experimentally derived SAR series. The iterative use of model building, synthesis, and biologic evaluation is the most powerful and efficient means to obtain small molecules with a desired biologic activity. When multiple cocrystal structures with different ligands exist, one can gain even more insights into conserved interactions. This insight can be very useful to identify key interactions and to help design new compounds to maximize possible key interactions. An algorithm developed by Deng et al. (56) uses a binary string to represent ligand protein interactions. By clustering the binary strings of different small-molecule binders, the authors were able to illustrate the similarity and diversity of small-molecule binding modes. This information can be used to select correct binding modes as well as to propose new molecules.
"Anchored docking" is another common technique used in lead optimization. Unlike automated docking, "anchor" points such as particular hydrogen bonding are specified by the user to limit the configuration space sampled by docking program. This limit is to ensure that the knowledge of a chemist about the system is enforced, because usually automated docking is sufficiently accurate to find correct binding modes consistently. The "anchor" points can be defined for a receptor, a small molecule, or both. The correct definition of "anchor" points largely relies on the users' knowledge of the system.
Qsar Based
Even with amount of protein structures available, many interesting protein targets without 3-D structural information exist. GPCRs are typical examples. In this case, only SAR derived from the biologic assay can be used to guide optimization. Under these circumstances, QSAR has been an important tool. It is probably one of the earliest tools for computer aided drug design. Properly choosing descriptors for QSAR models is much like an art and requires a deep understanding of the system of interest. Most common descriptors are based on physical, chemical, geometric, and topological properties. Three-dimensional QSAR models may require active ligands to be aligned properly just like the pharmacophore model building. In fact, 3-D QSAR models can be generated directly by pharmacophore models, and many pharmacophore-generating programs provide this capability. One method developed early and widely used for 3-D QSAR is CoMFA.
Design of Selective Small Molecule Inhibitors
For a small-molecule inhibitor to be useful as a therapeutic drug or as a tool for chemical biology study, it must have specificity for its intended target. A small-molecule drug often causes side effects or toxicity if it also modulates other proteins in the body (i.e., off-targets). Nonselective inhibitors will confound the analysis of resulting phenotypes. Therefore, the selectivity is a very important feature when considering design and selection of small-molecule inhibitors. Because ~30,000 proteins are encoded in the human genome, which can possess multiple ligand binding sites and can be present in vastly different concentrations, finding specific inhibitors can be an extremely difficult task. Analyzing the sequence and structural differences between a targeted protein and its closely related homologs is an effective way to design selectivity rationally.
Targeting the Differences in the Binding Pocket
The binding affinity of a ligand is determined by its complementarity to the size, shape, and physicochemical properties of the protein-binding site. To design selective small-molecule inhibitors rationally, the 3-D structure of the target-binding site is required. In principle, the structure of a protein-binding pocket is determined by its amino acid sequence, and it is straightforward to identify unique amino acids by sequence alignment. Once the unique residues are identified, they can be analyzed in the context of small-molecule binding to determine the residues that are important for protein ligand interaction. The inhibitors can then be designed to target them specifically for selectivity. Taunton et al. have used this approach to design selective inhibitors for p90 ribosomal protein S6 kinases (RSKs) (57). They identified two key selective determinants for RSKs relative to other kinases: One determinant is a small "gatekeeper" The amino acid, and the other is a Cys in the glycine-rich loop. Targeting these two residues, they were able to make very potent and selective inhibitors for RSKs.
Although successful for RSKs, it is often very difficult to identify unique residues suitable for selectivity for all proteins in a family. In many cases, a mono-selective inhibitor among the protein family members is not feasible, so selectivity against a subset of the family members is desired. In this case, selectivity can be achieved by targeting the sequence differences in the binding sites between the target protein and the unwanted proteins. This strategy has been applied successfully to design selective inhibitors for targets within a closely related subfamily, such as p38 kinase α, β, γ, and δ isoforms (58); protein kinase CDK2, and CDK4 (59); and COX-1 and COX-2 (60).
For protein targets that are closely related, the binding sites may look the same and no sequence difference is around the binding pocket. Therefore, a small molecule that binds to this primary site has very little selectivity. In these cases, a commonly used strategy is to search for a secondary site near the primary binding pocket. If it is less conserved, selective inhibitors can be designed by targeting the secondary site. For example, it is very difficult to develop a selective protein tyrosine phosphatase-1 B (PTP-1B) inhibitor, but a crystal structure of PTP-1B in complex with bis-(para-phosphophenyl) methane reveals a secondary aryl phosphate-binding pocket adjacent to the active site that can be targeted to design selective PTP-1B inhibitors (61).
Many protein structures are highly dynamic, and induced-fit effects are well known in small-molecule and protein binding (21). Although two proteins can have very similar binding pockets in static form, they can have different plasticity. Although protein flexibility is one major obstacle to predict the binding mode and the affinity of a ligand accurately, it provides a structural basis to design selective inhibitors. Usually, the conformations of the active enzymes within a family are more alike, but the inactive conformations are more different. For example, the ATP binding pockets of protein kinases in active states are highly similar, but considerable conformational variability exists among kinases in the inactive state that can be exploited for selective inhibitor design. The unique "DFG-out" inactive conformation has been applied already to the design of selective kinase inhibitors, which includes the already approved drug Gleevec and Nexavar. Lapatinib, one of the most selective kinase inhibitors, achieves its high selectivity by binding to an inactive conformation of EGFR with a methionine in the a-C helix moved away from its normal position to accommodate the benzyloxy group of lapatinib (62). Exploiting differentially accessible conformational states among closely related targets has also been the structural basis for observed high selectivity of PI3δ kinase inhibits (63).
Figure 6. Flowchart of methods employed for designing selectivity of small-molecule inhibitors.
Engineering Selectivity Using
Identifying a selective small-molecule inhibitor can be time consuming. For a target in a large protein family, it is almost impossible to achieve monoselectivity. Inhibiting multiple targets by a small molecule leads to a significant complication in the analysis of target validation and studies of protein functions. To overcome the difficulty of identifying monoselective small-molecule inhibitors, a chemical genetic approach is applied to design as orthogonal receptor-ligand pairs (64, 65). In this approach, the targeted protein is mutated in the active site to create a structural distinction between the target and all other members of proteins in the same family. Then, a small molecule is designed to target the distinct structure so that it only binds the mutated protein. The first step is to study the binding mode of a small molecule to its target and identify an important interaction that is highly conserved for the ligand binding to all members in the protein family. Then, the ligand is modified in the conserved interaction region to disrupt the interaction so that it cannot bind effectively to any wild type protein, which includes the target. Two modifications are used commonly. One is to add a bulky substitute (a "bump") at the interacting position to create steric clashes, and the other is to disrupt the critical hydrogen bonding between the ligand and the protein. The next step is to mutate the residues around the bump to smaller ones (which creates a "hole") to accommodate the "bump" or to mutate residues to restore the critical hydrogen bonds. Although both approaches have introduced orthogonal protein-ligand pairs successfully, the "bump-hole" strategy is more suitable for design and is applicable to many different protein families. Shokat's lab has successfully applied the "bump-hole" strategy to generate monospecific inhibitors for engineered kinases (64). The monospecific inhibitors have been used widely in kinase signaling pathway elucidation and target validation (66). Figure 6 outlines the strategies and workflows that we discussed for selective inhibitor design, in which the method details of each step in the flow chart have to be tailored towards specific systems.
In this review, we described briefly some common computational techniques to select and to design small-molecule inhibitors. It is not a comprehensive list of all methods available. Instead, we focused on basic methods that are used commonly in drug discovery research. Because of the size limitation, it is also impossible to discuss all the details in each method. We hope that this short review can provide some fundamental concepts used in virtual screening and rational design, and can provide the interested reader with an entry point to explore the field of computational inhibitor design and selection. Although we described the computational methods in a linear flow, the methods used in one stage of small-molecule inhibitor discovery process can be applied at different stages whenever appropriate. To choose the proper methods for different purposes at different stages is crucial for computational modeling to be effective. In addition, computational modeling should always interact tightly with experimental investigation in order to make significant contributions. VS and rational design is also an ever-changing field. New algorithms and strategies are emerging continuously. We believe that computational methods will play increasingly important roles in discovering new small-molecule inhibitors.
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Computation and Modeling of Molecular Recognition
Intermolecular Interactions in Molecular Recognition
Mechanisms of Enzyme Inhibition
Protein-Ligand Interactions
Structural and Functional Aspects of Molecular Recognition


text:
Staff Intranet | Staff Mail | Single Registry
Call Us on our Toll Free Number 1533
enhancing synergies, improving lives
Introduction to Social Protection
Social Protection Secretariat
Kenya National Social Protection Policy
SP Secretariat Staff
Social Protection Components
National Safety Net Program
Older Persons Cash Transfer (OPCT)
Hunger Safety Net Programme (HSNP)
Cash Transfer for Orphans and Vulnerable Children (CT-OVC)
Cash Transfer for Persons With Severe Disabilities (PWSD-CT)
Single Registry
SP Conference
SP Conference 2015
HomeSingle Registry
Single Registry for Social Protection
Social Protection in Kenya has experienced significant growth in the past few years; this includes the exponential growth in the number of beneficiaries households covered to the current over 1.5 million in 2018 from all the counties. This growth, however, has been plagued by a number of challenges. The main challenge has been fragmentation and lack of coherent coordination mechanisms, which results in missed opportunities for ensuring efficiency of the investments made. Consequently, duplication of effort and parallel implementation structures do not optimize the limited capacity or support a coherent approach to capacity building across the sector.
As part of efforts to address this challenge, the Social Protection Secretariat (SPS) has developed a Single Registry System for the social protection programmes as part of the implementation of National Social Protection Policy and the Vision 2030. The objective of this system is to consolidate information from the different management information systems (MIS) for the SP programmes that are currently operated independently by different departments and ministries.
The goal of establishing the Single Registry MIS is to provide accurate and analytical reports on the performance of the social protection sector. The objectives of the program are as follows:
Provide increased harmonization and consolidation of fragmented social protection schemes through comprehensive oversight of all social protection initiatives
For better communication to the general population and target audiences
To provide a single platform where common and essential information across social protection programmes are stored, analyzed and reported for the benefit of the stakeholders.
To provide checks against one beneficiary receiving multiple benefits within and across programme
Helps in the elimination of fraud by ensuring that beneficiary details are verified electronically against the 30 million National Population Registry (IPRS) database. This ensures that the programmes pay only genuine beneficiaries.
Strengthen Monitoring &Evaluation of National safety Net Programme by providing reports against indicators on a bi-monthly, Bi-annually and annual basis based on established monitoring and evaluation framework.
Expose potential area(s) for social protection effort(s)
The system currently has information from the following programmes: Cash Transfer for Orphans and Vulnerable Children (CT-OVC), Older Persons Cash Transfer Programme (OPCT), Persons with Severe Disability Cash Transfer (PwSD-CT) – which are under the Ministry of Labour and East African Affairs; Hunger Safety Net Programme (HSNP) – under the National Drought Management Authority and the World Food Program (WFP) Cash for Assets Program. The Single Registry joins key information from the five cash transfers on registration and enrollment, payments, complaints and grievances and change management for the beneficiaries as well as a link to the Integrated Registration Service (IPRS) to validate beneficiary national ID details. Figure 1 illustrates the current data sources of the Single Registry.
At present, the single registry links programs in the social assistance component of social protection. Going forward, linkage to other components of social protection will be sought such as social security and health insurances.
Single Registry Data Sources
The Single Registry is a web portal that allows anyone with internet access to gather key statistics regarding the programs that are linked. Authorized users, such as the implementing officers can gain access to the restricted area which provides more information on beneficiaries of the cash transfer programs that are linked. Click here to access the Single Registry.
The following are some of the benefits of the Single Registry:
Efficient program monitoring
Reduced double registration
Increased transparency and accountability
Seamless and efficient transfer of data
Enhanced quality of operations and services for field officers
Baseline Data for future programs
Copyright © National Social Protection Secretariat All rights reserved. Concept by 24i

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: Wordpress facebook share - og:image blank I tried everything but I can't find the problem.
I got this site http://www.ilsanoquotidiano.com and if I try to share to facebook a news, the og:image is always blank, but up to a month ago this works fine.
I tried with plugins or manually but the og:image is always blank even in facebook debugger but the raw tag is correct.
Could anyone help me?
Thanks

A: You can scrap the facebook sharing information via debugger. Link to check the result is given below:
https://developers.facebook.com/tools/debug/sharing/?q=http%3A%2F%2Fwww.ilsanoquotidiano.com%2F

A: Thanks for the reply, reading online I found that a problem could be the Jetpack version update, I tried to deactivate the plugin and after few hours everithing worked fine. I can't figure why happened this.

redpajama_set_name: RedPajamaStackExchange


text:
Suzuki museum in Hamamatsu
More Suzuki machines at the Heartech Plaza
Jeff Alexander, Ph.D. Student in Vancouver, Canada, was kind enough to send us pictures he took when visiting the Suzuki museum in Hamamatsu, Japan. There's hundreds of Suzuki motorcycles and cars dating back to the 50's. Here's a couple of the pictures. Enjoy!
Click on the images for larger format.
The picture above shows a Suzuki loom. As we know, Michio Suzuki started his career as manufacturer of looming machines; machines that were used for making fabrics for clothes. Michio Suzuki was 22 years old when he constructed his first, pedal-driven wooden loom, and started to sell his product. The year was 1909.
In 1952, Suzuki Jidosha Kogyo (Suzuki Loom Manufacturing) started manufacturing motorbikes, under the brand name Colleda. The first models were standard bicycles (push bikes) with small engines attached to it. The green-red bike on the left shows one of these early bikes, a Diamond Free from the early fifties.
Apparently it's a special bike, used for showing how durable the Colleda bikes were. "Suzuki's Around the World trip bike" says the sign at the museum. Unfortunately I can only guess the rest of the story. Perhaps one of you readers can tell more about the "trip around the world"?
Here's more Suzuki cycle motor info.
1956 Colleda TT
The following picture shows the classic 1956 Colleda TT model, the ancestor of the T20, T250 and the GT250. You don't need to have all too wild imagination to guess that the designer of the bike had been inspired by American car design.
Here's more Suzuki Colleda info.
Winds of change...
Hundreds of classic Suzuki bikes bikes in mint condition.
Barry Sheene's GP racers
There's some real classic bikes at the museum. What about these two? Barry Sheene's TR750R Grand Prix racer from the early seventies — or Sheene's 1978 Grand Prix bike RGB700?
The Sheene "in action" picture is borrowed from the Mehli-Race website.
More Suzuki racers
And there's much more. Tons of winner bikes in a row, from early sixties to the present day.
Big board on the wall tell the story of Suzuki Motor Company, from the early cycle motors to today's street-legal racers. I wish I had the opportunity to visit the museum some day.
I would mind Suzuki Motor Company offering me a free trip to the museum... Do you read this, the big bosses at Suzuki?
All the photos on this page (unless mentioned otherwise) was sent to me by Allan Tucker, Barbados. The source unknown. If you recognize the pictures and know who has taken them, please let me know about it!
The museum, the Heartech Plaza, is not open to general visitors. It's for Suzuki internal use only. You have to contact Suzuki and be persuade them to open the doors for you if you want to see all the interesting bikes in the museum.
Some owners clubs in Japan, RG Gamma, DR, GSX-R and so on, made contacts to SUZUKI, and were permitted to visit the museum. The reports can be found on the internet (in Japanese language) here.
The museum has no website and apparently can't be contacted by e-mail either.
The postal (snail mail) address of the Heartech Plaza is the following:
The Heartech Plaza of Suzuki Motor co, Ltd.
300 Takatsuka, Hamamatsu, Shizuoka-Pref,
JAPAN.
More: Photo Gallery
More: Suzuki racing history
More: All Suzuki models


This post is a follow up to our previous post, this time however we pose our questions directly at Zimbabwe's opposition parties. The purpose is for the Opposition parties to communicate to us the citizenry exactly what they are about. We the 2018 voters we believe we need to know.
Our position is very simple, there is no nutritional value in slogans and promises. Only strategies and solutions with the possibility of tangible result that can be executed can give us full bellies.


You can buy a lot of Korean ingredients online these days, but a physical Korean grocery store is still the best place to buy ingredients and equipment for Korean cooking. They have the best selection and prices, and you can see what you are buying so you're sure to get the best quality and exactly what you need.
But Korean grocery stores can be kind of confusing, especially if you can't read Korean. Even if you speak Korean, there are plenty of tips and tricks for picking the best ingredients off the shelf. Choosing fruits and vegetables and reading packages is not always easy! So I made this series with the help of the Korean government's Agro-Trade Center to show you how to shop in a Korean grocery store, step by step.
I had always wanted to make a series like this, but it was kind of hard for me to organize by myself. So I was very happy when the Agro-Trade Center New York offered to help me. The plan was to make a 20-minute video, but after looking at the footage I realized there was a ton of great information there that I couldn't cut out. So it ended up as a series of four videos.
Special thanks to my readers Cora, Bridget, Lucy, Andrew C, Shawn, and Andrew Lee for coming along and helping me. I found them through my monthly letter, so be sure to sign up if you want to be the first to know about my next event.
And if you're looking for a Korean grocery store in your area, be sure to check out the Korean grocery store directory on my website, where all the stores are submitted by my readers around the world. If your local store is not there, please add it!
I show you how to choose the right rice for Korean food, and walk you through the produce section so you can learn how to choose the best Korean vegetables for your cooking and kimchi-making.
Making rice, Seaweed rice rolls (Gimbap), Sweetened rice with fruits and nuts (Yaksik), Mushroom bulgogi, Mung bean pancake (Bindaetteok), Sweet, sour, and spicy mushrooms with water dropwort (Beoseot minari chomuchim), Taro soup (Toran-guk), Eggplant sidedish (Gaji-namul), Cubed radish kimchi (Kkakdugi), and Napa cabbage kimchi.
These ingredients are essential to Korean cooking, but can be confusing to shop for. I show you how to buy them and we taste many brands to find the best!
Soup soy sauce (Guk-ganjang), Regular soy sauce (Ganjang), Fish sauce (Aekjeot), Rice syrup (Salyeot), Corn syrup (Mulyeot), Black bean paste (JJajang, or Chunjang), Fermented Bean paste (Doenjang), Hot pepper paste (Gochujang), Hot pepper flakes (Gochugaru), Korean spicy dipping sauce (Ssamjang), Sesame oil (Chamgireum), Toasted sesame seeds (Bokkeun-kkae), and Raw sesame seeds (Cham-kkae).
Making soup soy sauce (Guk-ganjang), Yuja tea (Yujacha), Braised lotus roots (Yeon-geun-jorim), Noodles with blackbean sauce (Jjajangmyeon), Fermented soybean paste stew (Doenjang-jjigae), Hot pepper paste (Gochujang), Korean spicy dipping sauce (Ssamjang), and Toasted sesame seeds (Bokkeun-kkae).
You can make many many delicious Korean dishes with these things, so we go over them one by one!
Noodle soup (Guksu), Cold noodles in chilled broth (Mul-naengmyeon), Cold, spicy, chewy noodles (Bibim-naengmyeon), Sweet potato starch noodles stir fried with vegetables (Japchae), Hotteok filled with vegetables and noodles (Yachae hotteok), Crispy and crunchy fried chicken (Dakgangjeong), Mung bean jelly side dishes (Cheongpomuk-muchim), Hot pepper paste (Gochujang), Rice dessert drink (Sikhye), Rice cake (Injeolmi), Kimchi stew (Kimchi-jjigae), Spicy whelks with noodles (Golbaengi muchim), Korean-style curry rice (Kare-raiseu), Multigrain rice (Japgokbap), Red bean porridge (Dongji-patjuk), Fermented soybean paste (Doenjang), Mung bean sprouts (Sukju-namul), Mung bean pancakes (Bindaetteok), Seaweed rice rolls (Gimbap), Triangle gimbap (Samgak gimbap), Rice cake soup (Tteokguk), Seaweed soup (Miyeokguk), Bibimbap, and Spicy bellflower root side dish (Doragi-muchim).
This video gets a little chilly in the frozen and refrigerated sections! I also look at a few things in the kitchenware section like earthenware pots and crocks, and tabletop butane gas burners.
Earthenware bowl (Ttukbaegi), Earthenware crock (Onggi), and a Gas Burner.
Posted on Sunday, April 30th, 2017 at 10:02 am. Last updated on June 13, 2017.
Posted April 8th, 2018 at 5:37 pm | Log in to reply.
Maangchi, excellent series. Would you be able to tell me which restaurant did you go to after shopping. Please let me know. Best.
Posted April 14th, 2018 at 9:37 am | Log in to reply.
That restaurant was right in the grocery store. It's a small cafeteria, but it has a large selection and all the food is delicious. When we ate there, the cook came out and told me she was one of my followers!
Posted March 6th, 2018 at 10:11 pm | Log in to reply.
Posted July 20th, 2017 at 7:11 pm | Log in to reply.
Posted June 11th, 2017 at 7:31 am | Log in to reply.
I really love your recipes and I have been a fan of Korean cuisine for a long time, they are just so delicious!!
I have been having a problem finding Gyeoja-garu, and I was wondering if you can make it your self?
Posted May 23rd, 2017 at 5:15 pm | Log in to reply.
Hi Maangchi, thank you for the useful info in those videos. I have a question regarding the black bean paste: is "fermented black bean paste" the same as "black bean paste"? I saw both types at P.A.T yesterday, which one should I buy to make Jjajangmyeon?
Posted May 29th, 2017 at 8:03 pm | Log in to reply.
This is a picture of the black bean paste that doesn't say "fermented". The korean characters look the same as the ones you show in the video.
Posted May 29th, 2017 at 8:45 pm | Log in to reply.
yes, it is roasted fermented black bean paste.
Posted May 30th, 2017 at 6:55 am | Log in to reply.
Posted May 18th, 2017 at 3:02 pm | Log in to reply.
Hi Maangchi! I'm Korean (moved to the states when I was 4) and even I have difficulty deciding what to choose. Usually trial and error. Every time I do find the one I like, it seems like they're out or discontinued :-( Thank you for making these videos; it's really informative!
Posted May 9th, 2017 at 7:14 am | Log in to reply.
Posted May 9th, 2017 at 12:04 pm | Log in to reply.
Thank you so much for your nice comment here!


Welcome to the CIHT Society of Road Safety Auditors (SoRSA) website. SoRSA represents an exciting initiative for the Chartered Institution of Highways and Transportation (CIHT), in providing a home for the development of professional highways safety auditing best practice.
SoRSA has been established by the Institution as a response to a growing call from professionals operating in the field of safety auditing and safety engineering practice in the UK, for a forum to exchange best practice and, importantly, to provide advice and ultimately routes to professional recognition for safety auditing practitioners.


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Turning An Eroding Factor into a Distinctive Competency
I met Class2Class Co-Founder Matthew Hightower when he came into to my office at Texas A&M a few years back seeking input on his first startup idea. He was a student at the time and I was gearing up to take a group of Aggies to Africa. Matthew joined the group and we've been conversing about business strategy ever since. That first startup didn't go so well but Matthew was determined to learn from the experience and keep pursuing his entrepreneurial dreams. Familiar with the V-REEL™ Framework for strategy development, Matthew approached his second venture thinking a bit differently.
"I started thinking about some of the things that I have under my belt resource wise that could help make this new venture a success," Matthew said.
Matthew explained that he'd taken a lot of entrepreneurship classes and listened to a number of podcasts with Steve Blank, the LeanStartup model, YCombinator and others. He employed those tools and then used V-REEL™ as a sort of filter to organize his thoughts and prioritize.
"The V-REEL™ process was a way for me to look at a venture and identify some core things that I can focus on," he said. "There is so much information out there and you're trying to learn as much as you can as fast as possible but, as an entrepreneur, your time is limited and you need to focus on the things that matter most. V-REEL™ helped me identify what I needed to focus on."
V-REEL™ helped Matthew prioritize. Together with his business partner, Matthew identified an opportunity to facilitate a growing movement toward collaborative international learning. While most of the emphasis in this space is in language learning, opportunities are broad across a wide range of disciplines and in both K-12 classrooms and universities.
"We realized how difficult it is for teachers to make these connections and believed we could build a platform to facilitate connections," Matthew explained.
Using Steve Blank's customer development process helped Matthew think about pain points and ways they might bring value to the customer, what that might look like, and how they might make things that teachers or someone else would pay for. Matthew then started thinking about all of this using V-REEL™. He quickly identified step one: how he could bring customers value. But then there is rareness to consider.
"Rareness is a tough one," he said. "As an entrepreneur starting off, that's something we need to focus on more. It allowed me to figure out how vulnerable we really are."
Matthew's realization gave birth to a solution that he has been developing into something rare and valuable in his market. Matthew realized that he had a real eroding factor because neither he nor his business partner has any background in education. But Matthew is great at relationship building and learning. So he has worked diligently to put those capabilities into play.
"We identified that we could increase our rareness with one strategic decision," he explained. "Surround ourselves with teachers and people with the educational background that we lack."
Matthew explained how Class2Class intentionally set out to build rareness through relationships and by educating themselves about their customers' needs. I've been impressed at the relationships they've been abled to create, not just in the U.S. but also abroad. By listening and really focusing on building strong relationships, Class2Class is setting itself up to have a strong distinctive competency in the marketplace.
It's probably not uncommon for a company to identify an eroding factor and turn that around with enablers such that it creates distinction in the marketplace. There is often opportunity to be found in weaknesses. Matthew recognized that early and set out to create rareness. Interestingly, during our conversation, he seemed to identify more opportunity to refine both his business and personal strategy by employing the V-REEL™ Framework.
We talked about using the resources from Think Beyond Value to capture eroding and enabling factors on paper, and then prioritize. We talked about using V-REEL™ to assess Matthew's own ability to create value and be rare, both personally and for his business. It was a great conversation rich with insights into value creation, rareness and how to build your way to distinction in the marketplace. Have a listen.


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"Dr. Angulo! Can't say enough how much *everyone* loved Rumbache and Leo Servin - they were the best! We're extremely grateful for everything. Manuela and I will definitely be coming around to see them in the future."
Ethan & Manuela Fassett
"I just wanted to say thank you for arranging the music for our wedding last February. I was so stressed about finding the perfect band for my new mixed Anglo & Mexican family. You made the perfect recommendation and even today, one year later, my entire family (both sides) rave about the wonderful band. It was absolutely perfect and we will always have beautiful memories of our reception. Thank you!"
Beatriz & Chris Pratt
"I wanted to send you a quick note on behalf of the services that were provided by Patricio at Oyé Productions. They provided a cigar roller for a high end reception I had in November. The cigar rollers came on time, set up before doors opened, engaged the guests and were overall very professional. The guests loved it so much that I can't wait to find another event to use them for! Invoice was delivered on time after the event, and all was wonderful! I would definitely use Patricio and his company again!"
Emilie, Four Seasons Hotel
"We are back from Hawaii and wanted to let you know that the Steel Band you recommended was amazing!!! Once the dinner was over, you couldn't get me off the dance floor. In fact, my 87 year old aunt had been on her way out, with her walker in her hands, and the next thing you know she was pushing it aside and getting down!!! It was fantastic!!"
"Thank you so much for providing the very entertaining Cigar Rolling at the Junior League Patron Party last night. What a fabulous touch! Everyone loved it."
Suzanne Rischman
"Thank you for making our Anniversary Dinner an evening to remember. The musical performance was outstanding! Many members said they truly enjoyed the music and it was a great touch to the dinner. Our warmest regards and heartfelt gratitude to Oyé Productions."
Barbary Coast Boat Club
"Oyé Productions – a unique and flavorful solution to your music needs, connected to some of the greater artists in our area. In addition to being ultra and splendidly talented, everyone is just a pleasure to work with, I felt so well taken care of and was completely confident that Oyé Productions was going to match the energy and class of my event. To this day, everyone talks about the music – it was outstanding!"
Michelle Mandel
"Oyé Productions was fantastic. They took care of all the details and our event was a hit! They selected the perfect band for an unforgettable evening of fun, dancing and laughter."
Taste of the Nation
"The wedding and reception were magical. Many, many people have told us that it was the most fun they even had at a wedding. I am so grateful to you for your suggestions, and for arranging it all. The music was perfect!"
Abby Levinson
Natalia and I would like to express our gratitude for the fantastic job done by Oyé Productions in lining up Azabache for our recent wedding. They were the perfect band choice for our Latin themed wedding and really delivered the hot salsa sound you said they would. All 170 of our wedding guests were in a frenzy at the end of the night! Choosing a band was tough with all the talent you represent. The depth and breadth of your band selection for Latin and World music is really unparalleled. Your guidance was crucial in making the final choice. Thanks again for the terrific job."
Jason & Natalia
"You are a thorough professional and deserve much success."


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Discover and read the best of Twitter Threads about #boycotttheguardian
Tim Skellett
@Gurdur
But now, onto my own personal summation of this all. The @Guardian, it is THE paper and site that gives most national (UK) and international (USA, Australia, the damned world) to coverage of issues like poverty, class. It has worldclass investigative #journalism. But.....
... but I admit to being more than fully cheesed off with the Guardian myself. I was a loyal funder, around 60 quid a month, I stopped completely, because the Guardian's new policies were so idiotic and off-putting, not to mention counter-productive.
But! ....
But, bluntly, the @Guardian still is THE major newspaper in the UK, the USA or Australia, to give a very wide range of opinion from the public, & to also give reasonably trustworthy investigative #journalism.
I refuse to contribute money, but it's still very much my first go-to.
The #BoycottTheGuardian is because of ... the Jews. Who woulda thunk it? What a surprise, so shocking.
https://twitter.com/bickypeg/status/1045389778047447040
Such a surprise, so shocking, it's coz of the Jews, innit
https://twitter.com/mochavoca/status/499032192904077312
Some seem very eager to attach yellow stars to the Guardian (by the way, for anyone interested in the truth, it's not Jews who own the Guardian at all, and the Guardian tends to be pro-Palestinian. But hey, #BoycottTheGuardian because Jews).
https://twitter.com/indyrikki/status/498912388578828289
Oh, and let's discuss that bit of trivia, that went trending on Twitter for a short while, only in the UK, naturally. The #BoycottTheGuardian garbage. Because maybe the UK could benefit from dosing up the tapwater with lithium and haloperidol too.
For those who don't know it: the @guardian is a broadsheet, i.e. a respectable newspaper, not a scandal-mongering tabloid, based in the UK, though its web-presence is heavy in the USA and Australia. It is the smallest of national broadsheets in the UK, and the best one.
The @Guardian, national broadsheet w/ the smallest subscription in the UK, is also the most "left"-wing & liberal of the UK national broadsheets. Known 1821 till 1959 as the Manchester Guardian, it has done a huge deal of v important investigative #journalism over the centuries.
#BoycottTheGuardian #poverty #fakenews #journalism


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Armaan's approach is simplicity. He takes us through the Amazon Affiliate Program basics, then right into his strategies for content and backlinks. He could have stopped there - his methods are simple, down-to-earth, very common sense. But he added on seven tips on conversion that any reader, IM newbie or old hand, can use as valuable tools in their online business. Did I say this book is gold? Nix that..it is GREEN! Evergreen! Everybit as useful and up-to-date as the day he wrote it. Thank you Armaan. OH! I almost didn't mention the giveaway at the end!!
This book is written by a hugely successful Amazon affiliate member, Armaan Kohli, and includes real case studies of how he's built a successful niche site empire that's monetized through the Amazon Associates Program. He shows you screenshots of how his income keeps growing along with an example of how he ultimately sold his 6 month old Amazon affiliate website for over $15000!
One of the challenges I came up against when writing about cameras regularly was that while a certain percentage of my readers were actively shopping for a new camera, many readers already owned one. In fact, writing a 'photography tips' blog means you attract more people wanting to learn how to use a camera that they already own, rather than buying a new one.
Amazon customers are loyal. Beyond that, Amazon customers--especially Amazon Prime members--are more than happy to convert. According to a 2015 report, Prime members are said to convert at a rate 22 times greater than customers that shop with other top online retailers. So, if you can tap into that audience and appeal to their willingness to spend money with Amazon, you should see a good return on your decision to join the program.
I stumble upon a roadblock signing up as an affiliate for Amazon. On step 3 they ask for a number for verification. They are restricted from making calls to the Phils for verification. I thought of using a Google voice number (US based). Will that help? I don't want to try until I am sure for fear of getting rejected even before I start. Or what do you suggest? I tried a live chat with Amazon and all the CS said was yeah no call verification for a Philippine number. appreciate your help. Thank you.
Shovel driveways and sidewalks in the winter if it snows in your area. Pick a shovel that's lightweight yet sturdy and has a non-stick blade. Come up with a shoveling strategy, whether it's working from the top of the driveway to the bottom or going in diagonal lines across the driveway. Push the snow from side to side and only pick it up when you need to avoid wearing yourself out fast.[9]
Their policies are strict. Be sure to read through Amazon's affiliate program policies very carefully. Any violation of their policies is grounds for account suspension--something which could be devastating if you build an entire website or store around affiliate links. Amazon has also been known to suspend accounts without any prior notice, so tread carefully.
Español: ganar dinero, Deutsch: Geld verdienen, Português: Ganhar Dinheiro, Italiano: Fare Soldi, Nederlands: Geld verdienen, Français: gagner de l'argent facilement et rapidement, Русский: много зарабатывать, 中文: 赚钱, Čeština: Jak vydělat peníze, Bahasa Indonesia: Menghasilkan Uang, العربية: ربح المال, Tiếng Việt: Kiếm tiền, 日本語: お金を稼ぐ, 한국어: 돈 버는 방법, Türkçe: Nasıl Para Kazanılır, हिन्दी: पैसे बनाएँ (Kaise Paise Kamaye, Make Money)
The other type of Amazon affiliate link I use is when I'm mentioning a product in passing and/or a new product is announced. For example, when Nikon announced the Nikon D300s we immediately posted about the news because it was a notable and anticipated camera announcement. The camera was not yet available in stores and we were not able to get a review sample yet – but it was available for Pre-Order on Amazon so we linked to it.
Adding to that, most of the country's highest-ranked teaching and research hospitals are affiliated with M.D. schools—including the 20 best in the nation as rated by U.S. News & World Report. — Carolyn L. Todd, SELF, "What Is a Doctor of Osteopathic Medicine, and Should You See One?," 6 Feb. 2019 He has been affiliated with churches in Winston-Salem, North Carolina, Lincoln Heights, Ohio, and Baltimore, Maryland, before being elected the bishop of the Episcopal Diocese of North Carolina. — Elizabeth Angell, Town & Country, "Who Is Bishop Michael Curry?," 5 Dec. 2018 Many of the trainers along your journey are affiliated with famed series villains Team Rocket, who enjoy a decidedly Saturday Morning Cartoon flavor in their cheesy shouts against you. — Sam Machkovech, Ars Technica, "Pokemon Let's Go: Well, I guess my adult ass is going to collect them all," 16 Nov. 2018 SpaceX is not affiliated with any of the startups currently working to build a real hyperloop, but the super-fast transportation system is still close to Musk's heart. — Andrew J. Hawkins, The Verge, "WARR Hyperloop pod hits 284 mph to win SpaceX competition," 23 July 2018 This giveaway is not affiliated with/supported by Kroger. — Susan Tompor, Detroit Free Press, "Amazon Prime Day: Gift cards, coupons and other scams to watch for," 14 July 2018 Most of the 20 teams do not currently use their full quota of 17 non-homegrown players, a status which means they have not been affiliated to an English or Welsh club for three complete seasons or 36 months before their 21st birthday. — Rob Harris, The Seattle Times, "Post-Brexit bid to curb foreign player imports splits soccer," 21 Nov. 2018 Evangelical pastor Silas Malafaia said on Twitter that de Oliveira was once affiliated with the left-wing Socialism and Liberty Party, which like most of Brazil's political parties criticized the attack and called for a thorough investigation. — Fox News, "Suspect in Brazil candidate stabbing moved to new prison," 8 Sep. 2018 The Daily Emerald reported he was affiliated with the Phi Gamma Delta fraternity. — The Oregonian/oregonlive, OregonLive.com, "University of Oregon student, 21, found dead at Shasta Lake," 19 May 2018
Tutor kids in a subject that you're an expert at if you like teaching. Think about the classes you do best in at school or the topics you know a lot about. Help out your peers or teach younger kids who want to get a head start before the next school year. Put up fliers around your neighborhood or school with the topics you're tutoring, the cost, and what services you offer.
There are many ways in which you can make money with WordPress. Creating a website for the purposes of promoting a business or selling goods through an online store is one such way. Monetizing a blog with display ads is another. While there are other ways in which you can use your WordPress site to make money, perhaps one of the easiest ones to get started with is affiliate marketing.
However, one day I had a reader offer to write a book review for me. I knew the reader so I was confident the review would be OK to publish. As with all my reviews, it had an affiliate link to Amazon in it. I was a little skeptical about whether the review would convert. I thought my readers might not respond as well to a stranger's review of the book. I was wrong.
You'll also need ecommerce software, fulfillment software, worry about warehousing, customer service and refunds. But that's not all. You'll also need traffic. Think search engine optimization, Facebook ads, and other social media campaigns. It is hard work, especially on your own. You could opt for Amazon's platform, which might be the easier route. But, then again, at the end of the day, this is a serious business, which could produce significant profits. So you're either all in or you're not.


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Covid may cause brain damage linked to strokes due to body battling to fight off virus, study claims
COVID may cause brain damage linked to strokes due to your body battling off the virus, a study claims.
Research has found that coronavirus infections might cause an inflammatory response in the brain, leading to 'stroke-like' damage to certain areas.
Covid may cause brain damage associated with strokes, the study foundCredit: Getty Images – Getty
Researchers from the National Institutes of Health used an MRI scanner and found damage to the olfactory bulb in the brain and also damage in the brain-stem.
And because no coronavirus was found in the tissue, the conclusion was made that the damage resulted from "the body's inflammatory response to the virus."
The researchers had found damage caused by thinning and leaky brain blood vessels in samples from patients who had battled coronavirus.
But the study revealed that the damage didn't look like it was associated with symptoms of "a lack of oxygen."
Avindra Nath, the clinical director at the National Institute of Neurological Disorders and Stroke and the senior author of the study, said: "We found that the brains of patients who contract infection from SARS-CoV-2 may be susceptible to microvascular blood vessel damage."
Writing in the New England Journal of Medicine, he added: "Our results suggest that this may be caused by the body's inflammatory response to the virus.
"We hope these results will help doctors understand the full spectrum of problems patients may suffer.
"This so that we can come up with better treatments.
"So far, our results suggest that the damage we saw may not have been not caused by the SARS-CoV-2 virus directly infecting the brain.
"In the future, we plan to study how COVID-19 harms the brain's blood vessels and whether that produces some of the short- and long-term symptoms we see in patients."
The study was conduced on brain tissue from 19 patients who had died from coronavirus, aged five to 73, who had suffered with the virus for varying amounts of time.
LASTING DAMAGE
Dr Nath described himself as being "completely surprised" at finding brain damage among the samples, adding that the harm was multifocal and "usually associated with strokes and neuroinflammatory diseases."
Stroke-like complications were revealed in ten patients, and the scans exposed damage that "corresponded to congested blood vessels".
The findings in the recent study have revealed more about the neurological symptoms that many coronavirus sufferers have experienced.
Meanwhile, every adult is on course to get the Covid vaccine by autumn with 200,000 Brits already getting jabbed each day, Matt Hancock said today.
The Health Secretary unveiled the UK's "exit strategy" out of the pandemic and revealed 350million doses of the vaccine are on order.
And he said he hopes every adult will have received the jab by autumn this year as part of the biggest vaccination programme ever.
But he refused to speculate on whether the lockdown rules could be strengthened amid claims from scientists they are "too lax" in stopping the spread.
Scientists believe the measures do not go far enough to stop cases exploding and are instead calling for a "total clampdown".
They say nurseries should be closed as well as places of worship with it made compulsory to wear face masks in public.
London mayor Sadiq Khan has also led calls for face coverings to be worn outside the home as he declared a major incident in the capital.
Covid cases today jumped by more than 50,000 for the 13th day in a row as deaths rose by 563 – pushing the total number of victims to more than 81,000.
There are now 3,072,349 people infected with the disease in the UK after 54,950 tested positive.
Deaths have continued to rise with the latest toll pushing the total number to 81,431.
Today's figure is the third-highest Sunday rise in deaths and there is a usually a lag in reporting at the weekend.
The damage to the brain tissue found in the study was similar to a stoke Credit: Getty Images – Getty
Crystal Palace star Eze 'facing FA investigation' over potentially breaking coronavirus rules by attending QPR match
People desert WhatsApp over fears personal data will now be shared with Facebook after users alerted to new changes
Mosques and temples to become jab hubs to tackle vaccine fake news
How to boost your pension by £1000s and stop the coronavirus crisis from delaying retirement
Wetherspoons to stay shut until APRIL as coronavirus lockdown restrictions continue
Tories will be seen as 'nasty party again' if they don't extend £20 a week benefits
Here's how to properly dispose of tampons
Priti Patel says 'we have a long way to go' before lifting Covid lockdown as vaccine rollout 'at pivotal stage'
Don't believe TikTok – wiggling your nose can't get you a free nose job
Fit List: Must-have fitness picks from sports bras to waterproof jackets


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22 1/2 by 30 1/2 in. 57.2 by 77.5 cm.
Executed in 1975, this work is registered in the archives of the Calder Foundation, New York, under application number A26002.
This work is in good condition overall. The sheet is hinged intermittently verso to the mat. The edges of the sheet are deckled and slightly dulled. There is evidence of time staining to the sheet throughout and particularly along the perimeter from what appears to be previous hinges. There is evidence of scattered liquid surface accretions throughout and there is a small tear at the lower right corner. Framed under glass.