Functional Assays as Prismatic Views of Drug Activity: Relevance to New Drug Discovery

The two main processes involved in new drug discovery are screening and lead optimization; the latter process encompasses the dialogue between pharmacologists and medicinal chemists with the aim of maximizing primary activity and druglike properties of compounds. The pharmacological assay is the main tool in both of these processes; this will be discussed with special attention to new information regarding the signaling and networking of seven transmembrane receptors. In particular, the assays used for lead optimization will be discussed in terms of detecting and developing drugs that specifically emphasize some signaling pathways and not others, all through the stabilization of unique receptor protein conformations. There is considerable data to indicate that these are real phenomena that may be exploited for therapeutic advantage. A therapeutically relevant example will be discussed from a recent program designed to block entry of HIV-1 for the prevention and therapy of AIDS. The allosteric modulator aplaviroc (873140) and other allosteric modulators will be highlighted with reference to the development of future drugs that block the involvement of receptors in pathological process but otherwise allow them to function normally. In AIDS, this would allow the natural chemokine systems to assist in the protection against further HIV-1 infection.     

Human immunodeficiency virus type-1 and chemokines: beyond competition for common cellular receptors

The chemokines and their receptors have been receiving exceptional attention in recent years following the discoveries that some chemokines could specifically block human immunodeficiency virus type 1 (HIV-1) infection and that certain chemokine receptors were the long-sought coreceptors which, along with CD4, are required for the productive entry of HIV-1 and HIV-2 isolates. Several chemokine receptors or orphan chemokine receptor-like molecules can support the entry of various viral strains, but the clinical significance of the CXCR4 and CCR5 coreceptors appear to overshadow a critical role for any of the other coreceptors and all HIV-1 and HIV-2 strains best employ one or both of these coreceptors. Binding of the HIV-1 envelope glycoprotein gp120 subunit to CD4 and/or an appropriate chemokine receptor triggers conformational changes in the envelope glycoprotein oligomer that allow it to facilitate the fusion of the viral and host cell membranes. During these interactions, gp120 appears to be capable of inducing a variety of signaling events, all of which are still not defined in detail. In addition, the more recently observed dichotomous effects, of both inhibition and enhancement, that chemokines and their receptor signaling events elicit on the HIV-1 entry and replication processes has once again highlighted the intricate and complex balance of factors that govern the pathogenic process. Here, we will review and discuss these new observations summarizing the potential significance these processes may have in HIV-1 infection. Understanding the complexities and significance of the signaling processes that the chemokines and viral products induce may substantially enhance our understanding of HIV-1 pathogenesis, and perhaps facilitate the discovery of new ways for the prevention and treatment of HIV-1 disease.     

HIV chemokine receptor inhibitors as novel anti-HIV drugs

The chemokine receptors CXCR4 and CCR5 are the main coreceptors used by the T-cell-tropic (CXCR4-using, X4) and macrophage-tropic (CCR5-using, R5) HIV-1 strains, respectively, for entering their CD4⁺ target cells. In this review, we focus on the function of these chemokine receptors in HIV infection and their role as novel targets for viral inhibition. Besides some modified chemokines with antiviral activity, several low-molecular weight CCR5 and CXCR4 antagonistic compounds have been described with potent antiviral activity. The best CXCR4 antagonists described are the bicyclam derivatives, which consistently block X4 but also R5/X4 viral replication in PBMCs. We believe that chemokine receptor antagonists will become important new antiviral drugs to combat AIDS. Both CXCR4 and CCR5 chemokine receptor inhibitors will be needed in combination and even in combinations of antiviral drugs that also target other aspects of the HIV replication cycle to obtain optimum antiviral therapeutic effects.     

HIV-1进攻靶细胞的机制及相应环节抑制剂

HIV－1是导致获得性免疫缺陷综合症（AIDS）的流行最广、破坏力最强的病毒。HIV－1分两个步骤特异性地进攻CD4^ 细胞：一是利用表面糖蛋白gp120和靶细胞膜上的受体结合;二是通过跨膜糖蛋白gp41使病毒的包膜和靶细胞的质膜发生融合,经过上述步骤,病毒的核心蛋白和遗传物质得以进入人体,然其中进行复制,遇时,细胞膜的稳定性被破坏,细胞的内外环境失去平衡,最终导致细胞死亡。HIV－1进攻靶细胞的机制研究所取得的成就为研制安全有效的抗HIV／AIDS药物提供了新的思路和方向。     

Effects of HIV-1 protease on cellular functions and their potential applications in antiretroviral therapy

ABSTRACT: Human Immunodeficiency Virus Type 1 (HIV-1) protease inhibitors (PIs) are the most potent class of drugs in antiretroviral therapies. However, viral drug resistance to PIs could emerge rapidly thus reducing the effectiveness of those drugs. Of note, all current FDA-approved PIs are competitive inhibitors, i.e., inhibitors that compete with substrates for the active enzymatic site. This common inhibitory approach increases the likelihood of developing drug resistant HIV-1 strains that are resistant to many or all current PIs. Hence, new PIs that move away from the current target of the active enzymatic site are needed. Specifically, allosteric inhibitors, inhibitors that block HIV-1 protease active site, should be sought. Another common feature of current PIs is they were all developed based on the structure-based design. Drugs derived from a structure-based strategy may generate target specific and potent inhibitors. However, this type of drug design can only target one site at a time and drugs discovered by this method are often associated with strong side effects such as cellular toxicity, limiting its number of target choices, efficacy, and applicability. In contrast, a cell-based system may provide a useful alternative strategy that can overcome many of the inherited shortcomings associated with structure-based drug designs. For example, allosteric PIs can be sought using a cell-based system without considering the site or mechanism of inhibition. In addition, a cell-based system can eliminate those PIs that have strong cytotoxic effect. Most importantly, a simple, economical, and easy-to-maintained eukaryotic cellular system such as yeast will allow us to search for potential PIs in a large-scaled high throughput screening (HTS) system, thus increasing the chance of success. Based on our many years of experience in using fission yeast as a model system to study HIV-1 Vpr, we propose the use of fission yeast as a possible surrogate system to study the effects of HIV-1 protease on cellular functions and to explore its utility as a HTS system to search for new PIs to battle HIV-1 strains resistant to the current PI drugs.     

Plasma membrane signaling in HIV-1 infection

Plasma membrane is a multifunctional structure that acts as the initial barrier against infection by intracellular pathogens. The productive HIV-1 infection depends upon the initial interaction of virus and host plasma membrane. Immune cells such as CD4 + T cells and macrophages contain essential cell surface receptors and molecules such as CD4, CXCR4, CCR5 and lipid raft components that facilitate HIV-1 entry. From plasma membrane HIV-1 activates signaling pathways that prepare the grounds for viral replication. Through viral proteins HIV-1 hijacks host plasma membrane receptors such as Fas, TNFRs and DR4/DR5, which results in immune evasion and apoptosis both in infected and uninfected bystander cells. These events are hallmark in HIV-1 pathogenesis that leads towards AIDS. The interplay between HIV-1 and plasma membrane signaling has much to offer in terms of viral fitness and pathogenicity, and a better understanding of this interplay may lead to development of new therapeutic approaches. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.     

Coupling of agonist binding to effector domain activation in metabotropic glutamate-like receptors

Many membrane receptors are made of a ligand binding domain and an effector domain mediating intracellular signaling. This is the case for the metabotropic glutamate-like G-protein-coupled receptors. How ligand binding leads to the active conformation of the effector domain in such receptors is largely unknown. Here, we used an evolutionary trace analysis and mutagenesis to identify critical residues involved in the allosteric coupling between the Venus flytrap ligand binding domain (VFT) and the heptahelical G-protein activating domain of the metabotropic glutamate-like receptors. We have shown that a conserved interdomain disulfide bridge is required for this allosteric interaction. Taking into account that these receptors are homodimers, this finding provides important new information explaining how the different conformations of the dimer of VFT lead to different signaling of such dimeric receptors.     

9H-Xanthene-9-carboxylic acid [1,2,4]oxadiazol-3-yl- and (2H-tetrazol-5-yl)-amides as potent, orally available mGlu1 receptor enhancers

Small molecule mGluR1 enhancers based on the lead compound (9H-xanthene-9-carbonyl)-carbamic acid butyl ester derived from random-screening hit diphenylacetyl-carbamic acid ethyl ester were designed and synthesized as useful pharmacological tools for the study of the physiological roles mediated by mGlu1 receptors. The synthesis and the structure-activity relationship of this new class of positive allosteric modulators of mGlu1 receptors will be discussed in detail.     

Receptor dimerization: a key step in chemokine signaling.

Chemokines exert their effects through their interaction with seven transmembrane domain receptors coupled to G-proteins, GPCRs. Such receptor ligation leads to the regulation of numerous activities where chemokines play a key role, including hematopoiesis, T-cell activation, angiogenesis, inflammatory diseases or HIV-1 infection. Here we discuss the molecular mechanisms that underlie chemokine receptor activation. As occurs with other GPCRs, chemokines initiate the signaling cascades by inducing receptor dimerization. This dimerization enables the activation of the JAK/STAT pathway which allows the subsequent triggering of G-protein dependent signaling events. This mechanism provides a new context to explain some of the activities exerted by chemokines and introduces new targets for the development of drugs to fight those diseases were chemokines are implicated, such as inflammation and AIDS.     

G-protein-coupled receptors in drug discovery: nanosizing using cell-free technologies and molecular biology approaches

Signal transduction by G-protein-coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. Signal transduction has been studied extensively with both cell-based systems and assays comprising isolated signaling components. Interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high throughput screening, biosensors, and so on will focus greater attention on assay development to allow for miniaturization, ultra-high throughput and, eventually, microarray/biochip assay formats. Although cell-based assays are adequate for many GPCRs, it is likely that these formats will limit the development of higher density GPCR assay platforms mandatory for other applications. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR assay platforms adaptable for such applications as microarrays. The authors review current cell-free GPCR assay technologies and molecular biological approaches for construction of novel, functional GPCR assays.     

The role of immunosuppression of mesenchymal stem cells in tissue repair and tumor growth

Human Immunodeficiency Virus Type 1 (HIV-1) protease inhibitors (PIs) are the most potent class of drugs in antiretroviral therapies. However, viral drug resistance to PIs could emerge rapidly thus reducing the effectiveness of those drugs. Of note, all current FDA-approved PIs are competitive inhibitors, i.e., inhibitors that compete with substrates for the active enzymatic site. This common inhibitory approach increases the likelihood of developing drug resistant HIV-1 strains that are resistant to many or all current PIs. Hence, new PIs that move away from the current target of the active enzymatic site are needed. Specifically, allosteric inhibitors, inhibitors that prohibit PR enzymatic activities through non-competitive binding to PR, should be sought. Another common feature of current PIs is they were all developed based on the structure-based design. Drugs derived from a structure-based strategy may generate target specific and potent inhibitors. However, this type of drug design can only target one site at a time and drugs discovered by this method are often associated with strong side effects such as cellular toxicity, limiting its number of target choices, efficacy, and applicability. In contrast, a cell-based system may provide a useful alternative strategy that can overcome many of the inherited shortcomings associated with structure-based drug designs. For example, allosteric PIs can be sought using a cell-based system without considering the site or mechanism of inhibition. In addition, a cell-based system can eliminate those PIs that have strong cytotoxic effect. Most importantly, a simple, economical, and easy-to-maintained eukaryotic cellular system such as yeast will allow us to search for potential PIs in a large-scaled high throughput screening (HTS) system, thus increasing the chances of success. Based on our many years of experience in using fission yeast as a model system to study HIV-1 Vpr, we propose the use of fission yeast as a possible surrogate system to study the effects of HIV-1 protease on cellular functions and to explore its utility as a HTS system to search for new PIs to battle HIV-1 resistant strains.     

G protein coupled receptors as allosteric proteins and the role of allosteric modulators

Seven transmembrane receptors (7TMRs) are proteins that convey signals through changes in conformation. These conformations are stabilized by external molecules (i.e. agonists, antagonists, modulators) and act upon other bodies (termed ‘guests’) which can be other molecules in the extracellular space, or proteins along the plane of the membrane (receptor oligomerization) or signaling proteins in the cytosol (i.e. G protein, β-arrestin). These elements comprise allosteric systems and a great deal of 7TMR pharmacology can be considered in terms of allosteric behavior. Allosteric ligands acting on 7TMRs possess four unique behaviors that can be valuable therapeutically; () the ability to alter the interaction of very large proteins, () probe dependence, () saturable effect, and () induction of separate changes in affinity and efficacy of other ligands. Two of these behaviors (namely probe dependence for CCR5-based HIV-1 entry inhibitors and functional selectivity for biased agonism) will be highlighted with examples.     

Structural proteomics in drug discovery

High-throughput, automated or semiautomated methodologies implemented by companies and structural genomics initiatives have accelerated the process of acquiring structural information for proteins via x-ray crystallography. This has enabled the application of structure-based drug design technologies to a variety of new structures that have potential pharmacologic relevance. Although there remain major challenges to applying these approaches more broadly to all classes of drug discovery targets, clearly the continued development and implementation of these structure-based drug design methodologies by the scientific community at large will help to address and provide solutions to these hurdles. The result will be a growing number of protein structures of important pharmacologic targets that will help to streamline the process of identification and optimization of lead compounds for drug development. These lead agonist and antagonist pharmacophores should, in turn, help to alleviate one of the current critical bottlenecks in the drug discovery process; that is, defining the functional relevance of potential novel targets to disease modification. The prospect of generating an increasing number of potential drug candidates will serve to highlight perhaps the most significant future bottleneck for drug development, the cost and complexity of the drug approval process.     

Bioinformatics studies on drug resistance against anti-HIV-1 drugs

More than 20 drugs have been available for anti-HIV-1 treatment in Japan. Combination therapy with these drugs dramatically decreases in morbidity and mortality of AIDS. However, due to high mutation rate of HIV-1, treatment with ineffective drugs toward patients infected with HIV-1 causes accumulation of mutations in the virus, and emergence of drug resistant viruses. Thus, to achieve appropriate application of the drugs toward the respective patients living with HIV-1, methods for predicting the level of drug-resistance using viral sequence information has been developed on the basis of bioinformatics. Furthermore, ultra-deep sequencing by next-generation sequencer whose data analysis is also based on bioinformatics, or in silico structural modeling have been achieved to understand drug resistant mechanisms. In this review, I overview the bioinformatics studies about drug resistance against anti-HIV-1 drugs.     

Allosteric Drugs Acting at Muscarinic Acetylcholine Receptors

The binding properties of muscarinic acetylcholine receptors are affected by various drugs acting at a second (allosteric) binding site, usually (but not always) at supratherapeutic concentrations. Allosteric drugs acting at GABA receptors present advantages over competitive drugs; this explains the interest raised by allosteric effects on muscarinic receptors. A theoretical and practicable definition of allosteric drugs acting at muscarinic receptors will be given in this work, together with a summary of recent data concerning the number, position, and structural requirements of their binding sites.     

Structural proteomics in drug discovery

High-throughput, automated or semiautomated methodologies implemented by companies and structural genomics initiatives have accelerated the process of acquiring structural information for proteins via x-ray crystallography. This has enabled the application of structure-based drug design technologies to a variety of new structures that have potential pharmacologic relevance. Although there remain major challenges to applying these approaches more broadly to all classes of drug discovery targets, clearly the continued development and implementation of these structure-based drug design methodologies by the scientific community at large will help to address and provide solutions to these hurdles. The result will be a growing number of protein structures of important pharmacologic targets that will help to streamline the process of identification and optimization of lead compounds for drug development. These lead agonist and antagonist pharmacophores should, in turn, help to alleviate one of the current critical bottlenecks in the drug discovery process; that is, defining the functional relevance of potential novel targets to disease modification. The prospect of generating an increasing number of potential drug candidates will serve to highlight perhaps the most significant future bottleneck for drug development, the cost and complexity of the drug approval process.     

The impact of cryo-EM on determining allosteric modulator-bound structures of G protein-coupled receptors

G-protein coupled receptors (GPCRs) are important therapeutic targets for the treatment of human disease. Although GPCRs are highly successful drug targets, there are many challenges associated with the discovery and translation of small molecule ligands that target the endogenous ligand-binding site for GPCRs. Allosteric modulators are a class of ligands that target alternative binding sites known as allosteric sites and offer fresh opportunities for the development of new therapeutics. However, only a few allosteric modulators have been approved as drugs. Advances in GPCR structural biology enabled by the cryogenic electron microscopy (cryo-EM) revolution have provided new insights into the molecular mechanism and binding location of small molecule allosteric modulators. This review highlights the latest findings from allosteric modulator-bound structures of Class A, B, and C GPCRs with a focus on small molecule ligands. Emerging methods that will facilitate cryo-EM structures of more difficult ligand-bound GPCR complexes are also discussed. The results of these studies are anticipated to aid future structure-based drug discovery efforts across many different GPCRs.     

Real-time molecular and cellular analysis: the new frontier of drug discovery

The pharmaceutical industry is currently facing the challenge of maintaining increased efficiency and productivity while contending with a deluge of genomic and high-throughput screening data. To ease the bottlenecks at target validation and lead optimization, the industry must look to the living cell, the ultimate target of all drugs, as a source of new biological knowledge. This new 'cell-centric' perspective must integrate reagents that report on the state of molecular processes within the cell, automated detection and analysis of these processes, and cellular knowledge, building into a single platform.     

GPCR structures in drug design,emerging opportunities with new structures

In recent years, GPCR targets from diverse regions of phylogenetic space have been determined. This effort has culminated this year in the determination of representatives of all major classes of GPCRs (A, B, C, and F). Although much of the now well established knowledge on GPCR structures has been known for some years, the new high-resolution structures allow structural insight into the causes of ligand efficacy, biased signaling, and allosteric modulation. In this digest the structural basis for GPCR signaling in the light of the new structures is reviewed and the use of the new non-class A GPCRs for drug design is discussed.     

Molecular dynamics studies of the wild-type and double mutant HIV-1 integrase complexed with the 5CITEP inhibitor: mechanism for inhibition and drug resistance

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the virus and is an attractive target for the development of new drugs useful in acquired immunodeficiency syndrome multidrug therapy. Starting from the crystal structure of the 5CITEP inhibitor bound to the active site in the catalytic domain of the HIV-1 IN, two different molecular dynamics simulations in water have been carried out. In the first simulation the wild-type IN was used, whereas in the second one the double mutation T66I/M154I, described to lead to drug resistance, was introduced in the protein. Compelling differences have been observed in these two structures during analyses of the molecular dynamics trajectories, particularly in the inhibitor binding modes and in the conformational flexibility of the loop (residues 138-149) located near the three catalytic residues in the active site (Asp(64), Asp(116), Glu(152)). Because the conformational flexibility of this region is important for efficient biological activity and its behavior is quite different in the two models, we suggest a hypothetical mechanism for the inhibition and drug resistance of HIV-1 IN. These results can be useful for the rational design of more potent and selective integrase inhibitors and may allow for the design of inhibitors that will be more robust against known resistance mutations.