Targeting enzyme inhibitors in drug discovery

Drugs that function as enzyme inhibitors constitute a significant portion of the orally bioavailable therapeutic agents that are in clinical use today. Likewise, much of drug discovery and development efforts at present are focused on identifying and optimizing drug candidates that act through inhibition of specific enzyme targets. The attractiveness of enzymes as targets for drug discovery stems from the high levels of disease association (target validation) and druggability (target tractability) that typically characterize this class of proteins. In this expert opinion the authors describe the existing practices and future directions in drug discovery enzymology, with emphasis on how a detailed understanding of the catalytic mechanism of specific targets can be used to identify and optimize small-molecule compounds that interact with conformationally distinct forms of the enzyme, thus resulting in high potency, high selectivity inhibitors.     

The hunt for antimitotic agents: an overview of structure-based design strategies

ABSTRACT

Introduction: Structure-based drug discovery offers a rational approach for the design and development of novel anti-mitotic agents which target specific proteins involved in mitosis. This strategy has paved the way for development of a new generation of chemotypes which selectively interfere with the target proteins. The interference of these anti-mitotic targets implicated in diverse stages of mitotic cell cycle progression culminates in cancer cell apoptosis.

Areas covered: This review covers the various mitotic inhibitors developed against validated mitotic checkpoint protein targets using structure-based design and optimization strategies. The protein-ligand interactions and the insights gained from these studies, culminating in the development of more potent and selective inhibitors, have been presented.

Expert opinion: The advent of structure-based drug design coupled with advances in X-ray crystallography has revolutionized the discovery of candidate lead molecules. The structural insights gleaned from the co-complex protein-drug interactions have provided a new dimension in the design of anti-mitotic molecules to develop drugs with a higher selectivity and specificity profile. Targeting non-catalytic domains has provided an alternate approach to address cross-reactivity and broad selectivity among kinase inhibitors. The elucidation of structures of emerging mitotic drug targets has opened avenues for the design of inhibitors that target cancer.     

Exploiting Enzyme Plasticity in Virtual Screening: High Efficiency Inhibitors of Glutamate Racemase

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Seeing small molecules in action with bioorthogonal chemistry

Chemical probes that target specific protein families offer powerful tools to accelerate drug discovery. Small molecules modified with uniquely reactive functional groups and detection tags provide novel tools to characterize complex proteomes functionally and also to help determine the specificity of small molecule inhibitors toward various enzyme/protein classes. This review highlights the application of bioorthogonal chemistries in combination with chemical probes, which together are offering unprecedented opportunities to dissect the functions of enzyme/protein families in vivo and enabling more precise target identification of small molecules. Advances in chemical probes and bioorthogonal reactions are poised to reveal new therapeutic targets and to facilitate the discovery and characterization of small molecules aimed at disease.     

A rational approach to maximize success rate in target discovery

To overcome the problem of high attrition rates in the drug discovery process, an efficient strategy how to identify, select, characterize and validate the most suitable drug targets before embarking on the resource-intense steps of lead discovery and lead optimization is mandatory. We have implemented such an efficient strategy consisting of (i) Target Selection based on gene expression analyses of drugable target genes in clinical samples and relevant in vitro model systems, to identify candidate targets with a specific tissue distribution and presence in human disease; (ii) Target Assessment exploiting the three-dimensional structure of proteins for detailed binding site analysis, to estimate the drugability of the protein for small-molecule inhibitor binding as well as selectivity profiles; and (iii) Target Validation providing evidence for a functional role in in vitro model systems, thus corroborating the biological hypothesis underlying the therapeutic concept. This rational approach has led to the discovery of drug targets for Lead Discovery, maximizing the likelihood for achieving target-selective inhibition by small-molecule inhibitors with minimal in vivo side effects and a therapeutic effect based on a sound biological hypothesis.     

Current possibilities and unresolved issues of drug target validation in Mycobacterium tuberculosis

Introduction: Target driven drug discovery is a long and arduous task requiring a huge investment of time, energy and resources. Therefore, it is very important to select targets which provide the maximum chance of obtaining inhibitors that will be efficacious in animal models and finally in tuberculosis (TB) patients. Areas covered: The article discusses the necessity for new targets in Mycobacterium tuberculosis (Mtb) drug discovery and how the functional redundancy of putative targets in Mtb adds a new dimension to the complexity of validation. The article also reviews survival kinetics using conditional knockout (KO) or knockdown (KD) strains and discusses how this has provided crucial information on target vulnerability. Furthermore, the article also comments on how the chemical validation of new targets using specific inhibitors has greatly supplemented the genetic validation efforts. Expert opinion: Because of complexity of pathogenesis of TB, the putative drug targets need to be validated under multiple physiological conditions. Target protein depletion can mimic chemical inhibition and, therefore, will be a valuable tool in predicting the vulnerability of a target. Conditional KO or KD makes it possible to study the phenotypes of Mtb strains under a variety of physiological states. The phenotype of these strains should also be tested in animal models which mimic human TB more closely. Finally, inhibitors with confirmed mode of action can be important tools for validating Mtb drug targets.     

Drug Adverse Reaction Target Database (DART) : proteins related to adverse drug reactions.

An adverse drug reaction (ADR) often results from interaction of a drug or its metabolites with specific protein targets important in normal cellular function. Knowledge about these targets is both important in facilitating the study of the mechanisms of ADRs and in new drug discovery. It is also useful in the development and testing of rational drug design and safety evaluation tools. The Drug Adverse Reaction Database (DART) is intended to provide comprehensive information about adverse effect targets of drugs described in the literature. Moreover, proteins involved in adverse effect targets of chemicals not yet confirmed as ADR targets are also included as potential targets. This database gives physiological function of each target, binding drugs/agonists/antagonists/activators/inhibitors, IC(50) values of the inhibitors, corresponding adverse effects, and type of ADR induced by drug binding to a target. Cross-links to other databases are also introduced to facilitate the access of information about the sequence, 3-dimensional structure, function, and nomenclature of each target along with drug/ligand binding properties, and related literature. The database currently contains entries for 147 ADR targets and 89 potential targets. A total of 187 adverse reaction conditions, 257 drugs, and 1080 ligands known to bind to each of these targets are also currently described. Each entry can be retrieved through multiple search methods including target name, target physiological function, adverse effect, ligand name, and biological pathways. A special page is provided for contribution of new or additional information. This database can be accessed at http://xin.cz3.nus.edu.sg/group/drt/dart.asp.     

Thymidylate synthase structure, function and implication in drug discovery

Recent methodologies applied to the drug discovery process, such as genomics and proteomics, have greatly implemented our basic understanding of drug action and are giving more input to medicinal chemists, in finding genuinely new targets and opportunities for the development of drugs with original mechanisms of action. In this paper, an example of the successful application of some new techniques to the target enzymes with the Thymidylate Synthase (TS) function is given. The improved knowledge of the complex mechanism of the biological pathways in which thymidylate synthase is involved represents a unique chance to find new mechanism-based inhibitors, aimed to treat not only cancerous diseases, but also infectious pathologies. Thymidylate synthase (TS or ThyA) has long been considered as one of the best-known drug targets in the anti-cancer area, after which old and new drugs, such as 5-fluoro uracil and the anti-folate ZD1694, have been introduced into chemotherapy to treat solid tumours. Only a few attempts have been made to find non-classical anti-folate inhibitors that are dissimilar to the folate co-factor, with the aim of finding unshared protein target domains on the enzyme structure, in order to specifically inhibit TS enzymes from pathogens. Only recently from omic studies, a new Thymidylate Synthase Complementing Protein (TSCP or ThyX) has been identified in a number of pathogens, showing a different structure with respect to human TS, thus opening new avenues to specific inhibitions. A depiction of the most recent progress in the study of Thymidylate Synthase enzymes is presented in the following sections.     

Selecting protein tyrosine phosphatases as drug targets

Protein tyrosine phosphatases (PTPs) have emerged as a new and promising class of signaling targets, since the discovery of PTP1B as a major drug target for diabetes and obesity. Blocking individual PTPs results in the activation of specific tyrosine phosphorylation events, but matching PTPs with such pathways and therapeutic indications is a complex undertaking. The history of PTP1B shows that its unusual knockout phenotype and observations with generic and antisense inhibitors in vivo, but not its classical molecular biology, triggered the rapid development of inhibitors that are today being developed for the clinic.     

Death-associated protein kinase as a potential therapeutic target

Death associated protein kinase (DAPK) is a calmodulin (CaM)-regulated serine/threonine protein kinase implicated in diverse apoptosis pathways, including those involved in neuronal cell death and tumour suppression. The requirement of DAPK catalytic activity for its proposed cell functions and the validation of protein kinases as therapeutic targets demand that DAPK be examined as a potential therapeutic target in human disease. The relevant placement of DAPK activity in apoptosis pathways is at an early stage of investigation, making its study as a therapeutic target tenuous. However, the current body of knowledge raises the possibility of DAPK as a therapeutic target for diseases characterised by rapid neurodegeneration, such as stroke or traumatic brain injury. The unmet need in these diseases is for an acute treatment schedule that might reduce neuronal loss. Bioavailable inhibitors of DAPK catalytic activity that target the central nervous system have a potential to fill this need. The development of such DAPK inhibitors is now feasible based on the recent emergence of enabling technology and knowledge. These include a quantitative and selective enzyme assay, a high resolution structure of the active catalytic domain and discovery of cell-permeable, low molecular weight inhibitors of CaM kinases that cross the blood-brain barrier. DAPK as a potential therapeutic target for cancer is less attractive due to the incomplete state of knowledge about DAPK and inherent limitations in drug development for the discovery of specific activators of genes downregulated by promoter hypermethylation. This article provides a brief summary of relevant research and the rationale that is at the foundation of this opinion.     

Interleukin-5: a drug target for allergic diseases

There is a large body of evidence that eosinophils are a key component of the allergic response in asthma. Interleukin (IL) 5 is uniquely involved in the production of eosinophils, and with a variety of other cytokines and factors controls their activation, localization and survival. Thus, IL-5 is an important drug target for new anti-asthmatics. The routes to drug discovery are based on screens for inhibitors of IL-5 production, ligand antagonists, control of receptor expression and receptor activation. In this review, we will discuss specific targets and screening assays with examples of some of the compounds in development.     

计算机辅助药物设计在病毒酶抑制剂开发中的应用

计算机辅助药物设计是创新药物研究的新方法和新技术,近年来引起了研究机构和制药公司的高度重视。本文概述了CADD的设计方法,并例举yCADD在HIV-1整合酶抑制剂和感冒病毒神经氨酸酶抑制剂研发中的应用,阐述了CADD在酶抑制剂研究中应用前景。     

DNA repair inhibitors: the next major step to improve cancer therapy

Modern cancer therapies, mainly ionizing radiation and certain classes of chemotherapies target DNA. Although these treatments disrupt the genome, their rationale is clear. They prevent cancer cells from dividing and proliferating. Nevertheless, cancer cells can survive by over-activating a wide range of DNA repair pathways to eliminate the induced damage. In this context, DNA repair mechanisms are considered to be a vital target to improve cancer therapy and reduce the resistance to many DNA damaging agents currently in use as standard-of-care treatments. Here, we focus on two important DNA repair pathways, namely base excision repair (BER) and nucleotide excision repair (NER). Specifically, our focus is on two protein targets that are linked to the hallmark "relapse" and "drug resistance" phenomena. These are Excision Repair Cross-Complementation Group 1 (ERCC1), and DNA polymerase beta (pol β). The former is a key player in NER, while the latter is the error-prone polymerase of BER. Our objective is to list all known inhibitors for the two targets and provide an overview of the great efforts that were made in their discovery. While in the DNA pol β case more than sixty inhibitors were identified, very few inhibitors have been discovered on the ERCC1 side. It is hoped that this review will assist in the discovery of novel, potent and specific drug candidates aimed at improving existing cancer therapies including ionizing radiation, bleomycin, monofunctional alkylating agents and cisplatin.     

In silico three-dimensional pharmacophores for aiding the discovery of the Pfmrk (Plasmodium cyclin-dependent protein kinases) specific inhibitors for the therapeutic treatment of malaria

The resurgence of malaria and lack of effective antimalarial drugs affect millions of people worldwide every year, causing several million deaths. With the emergence of structure-based drug design methodologies, a major thrust in drug discovery efforts has shifted towards targeting specific proteins in parasites that are involved in their metabolic pathways. Although cyclin-dependent kinases (CDKs), due to their direct role in cell cycle regulations, have been targeted for the development of cancer therapeutics, CDKs for Plasmodium falciparum have only been recently identified to be attractive for the discovery of antimalarials. One of the plasmodium CDK targets is Pfmrk. Being a putative homolog of Cdk7 and, thus, having the possibility of dual functions, both in cell cycle control and gene expression within the parasite, pfrmk has become an interesting antimalarial chemotherapeutic target. This review discusses how in silico methodologies, without the knowledge of the X-ray crystallographic structure of Pfmrk, particularly based on the development of pharmacophores on known inhibitors can aid the discovery and design of Pfmrk-specific inhibitors through virtual screening of compound databases and provides insights into the understanding of the mechanism of binding in the active site of this enzyme.     

PDE inhibitors--Second William Harvey Research Conference. Drugs with an expanding range of therapeutic uses. 1-3 December 1999, Nice, France

This meeting underscored advances in the exploitation of cyclic nucleotide phosphodiesterases (PDEs) as drug targets. One highlight of the meeting was the disclosure of a new PDE isozyme, bringing to 11 the total number of genetically distinct isozyme families thus far identified. Also reported was the phenotypic characterization of a PDE4D murine genetic knockout. With respect to drug discovery and development, the most encouraging information presented centered on advances in targeting PDE4 with therapeutically useful inhibitors. Historically, the therapeutic utility of isozyme-selective PDE4 inhibitors has been limited by class-associated side effects, namely nausea and dyspepsia. New PDE4 inhibitors are being designed with the specific intent of improving upon the therapeutic ratio of first-generation agents. The profiles of two second-generation PDE4 inhibitors, SB-207499 (Ariflo; Smithkline Beecham plc) and PD-189659, were presented. SB-207499 demonstrated marked efficacy in phase II clinical trials in patients with moderate-to-severe chronic obstructive pulmonary disease (COPD), a disease of very high unmet medical need. PD-189659 has yet to enter clinical trials, but its preclinical profile indicates that this agent can produce substantial anti-inflammatory effects without producing class-associated side effects in animal models. A number of presentations were also given on the utility of PDE5 inhibitors in the treatment of male erectile dysfunction (MED). The widespread use of Viagra (sildenafil; Pfizer Inc) over the last year has reinforced the perception that PDE5 inhibitors are safe and effective agents for the treatment of MED. The overall tenor of the meeting was distinctly upbeat, with most participants believing that PDE isozymes are becoming ever more accessible as targets for drug discovery in a variety of therapeutic areas.     

The role of emerging genomics and proteomics technologies in cancer drug target discovery

Cancer drugs have traditionally been identified in screens designed to produce broad biological end points such as cell death. A serious undesired outcome of drugs discovered in these screens is that the mechanism of drug action is unknown and such drugs often have adverse side effects. Designing cancer drugs that act on specific targets offer the advantage that the mechanism of drug action can be understood and accurately monitored in clinical trials leading to development of better drugs. The pharmacological industry has recently shifted to a target directed drug discovery model. However, until recently potential cancer drug targets comprised of only a small fraction of the human genome. The human genome project and high-throughput structural and functional genomics have dramatically increased the number of cancer drug targets. Deciphering cancer drug targets requires the understanding of biochemical pathways that are affected in the cancer genome. It has been suggested that utilization of Single-nucleotide polymorphisms (SNPs) will aid in identifying individuals at high risk of developing certain cancers, and will also help in development of tailored medication or identify genetic profiles of specific drug action and toxicity. Achieving successful new cancer drug development schemes will require a merger of research disciplines that include pharmacology, genomics, comparative genomics, functional genomics, proteomics and bioinformatics. In this review the significance and challenges of these rapidly evolving technologies in cancer drug target discovery are discussed.     

Design of second generation HIV-1 integrase inhibitors

The prospect of HIV-1 integrase (IN) as a therapeutically viable retroviral drug target is on the verge of realization. The observed preclinical and clinical performance of beta-diketo containing and naphthyridine carboxamide compounds provides direct proof for the clinical application of IN inhibition. These validated lead compounds are useful in the design and development of second generation IN inhibitors. The results from preclinical and clinical studies on the first generation IN inhibitors reiterate a demand for novel second generation inhibitors with improved pharmacokinetic and metabolic properties. Pharmacophore-based drug design techniques facilitate the discovery of novel compounds on the basis of validated lead compounds specific for a drug target. In this article we have comprehensively reviewed the application of pharmacophore-based drug design methods in the field of IN inhibitor discovery.     

Aspartic proteases in drug discovery

Aspartic proteases are the smallest class of human proteases with only 15 members. Over the past years, they have received considerable attention as potential targets for pharmaceutical intervention since many have been shown to play important roles in physiological and pathological processes. Despite numerous efforts, however, the only inhibitors for aspartic proteases currently on the market are directed against the HIV protease, an aspartic protease of viral origin. Nevertheless, several inhibitors including those targeting renin, BACE1 and gamma-secretase are in clinical or preclinical development, and some other aspartic proteases are discussed as potential drug target. The crystal structures of seven human aspartic proteases have now been solved and, together with a detailed kinetic understanding of their catalytic mechanism, this has greatly contributed to the design and discovery of novel inhibitors for this protease class. This review describes current aspartic protease drug targets and summarizes the drug discovery efforts in this field. In addition, it highlights recent developments which may lead to a new generation of aspartic protease inhibitors.     

Targeting kinases for the treatment of inflammatory diseases

Importance of the field: Inflammatory diseases are one of the major health issues and have become a major focus in the pharmaceutical and biotech industries. To date, drugs prescribed for treatment of these diseases target enzymes that are not specific to the immune system resulting in adverse effects. The main challenge of this research field is, therefore, identifying targets that act specifically on the diseased tissue. Areas covered in this review: This review summarizes drug discovery efforts on kinases that have been identified as key players mediating inflammation and autoimmune disorders. In particular, we discuss recent developments on well-established targets such as mammalian target of rapamycin, JAK3, spleen tyrosine kinase, p38α and lymphocyte specific kinase but provide also a perspective on emerging targets. What the reader will gain: The reader will obtain an overview of drug discovery efforts on kinases in inflammation, recent clinical and preclinical data and developed inhibitor scaffolds. In addition, the reader will be updated on issues in target validation of current drug targets and the potential of selected novel kinase targets in this important disease area. Take home message: Cellular signaling networks that regulate inflammatory response are still poorly understood making rational selection of targets challenging. Recent data suggest that kinase targets that are specific to the immune system and mediate signals immediately downstream of surface receptors are most efficacious in the clinic.     

基于分子片段药物发现技术的β-分泌酶抑制剂研究进展

β-分泌酶抑制剂是开发新型阿尔茨海默病治疗药物的重要研究方向,基于分子片段的药物发现技术是近年来发展的一种基于靶标的药物研究策略。本文综述了近年来利用这一技术发现β-分泌酶抑制剂的研究进展。