Identifying Novel Drug Targets by iDTPnd: A Case Study of Kinase Inhibitors

Current FDA-approved kinase inhibitors cause diverse adverse effects, some of which are due to the mechanism-independent effects of these drugs. Identifying these mechanism-independent interactions could improve drug safety and support drug repurposing. Here, we develop iDTPnd (integrated Drug Target Predictor with negative dataset), a computational approach for large-scale discovery of novel targets for known drugs. For a given drug, we construct a positive structural signature as well as a negative structural signature that captures the weakly conserved structural features of drug-binding sites. To facilitate assessment of unintended targets, iDTPnd also provides a docking-based interaction score and its statistical significance. We confirm the interactions of sorafenib, imatinib, dasatinib, sunitinib, and pazopanib with their known targets at a sensitivity of 52% and a specificity of 55%. We also validate 10 predicted novel targets by using in vitro experiments. Our results suggest that proteins other than kinases, such as nuclear receptors, cytochrome P450, and MHC class I molecules, can also be physiologically relevant targets of kinase inhibitors. Our method is general and broadly applicable for the identification of protein–small molecule interactions, when sufficient drug–target 3D data are available. The code for constructing the structural signatures is available at https://sfb.kaust.edu.sa/Documents/iDTP.zip.     

Development of a High-Throughput Three-Dimensional Invasion Assay for Anti-Cancer Drug Discovery

The lack of three-dimensional (3-D) high-throughput (HT) screening assays designed to identify anti-cancer invasion drugs is a major hurdle in reducing cancer-related mortality, with the key challenge being assay standardization. Presented is the development of a novel 3-D invasion assay with HT potential that involves surrounding cell-collagen spheres within collagen to create a 3-D environment through which cells can invade. Standardization was achieved by designing a tooled 96-well plate to create a precisely designated location for the cell-collagen spheres and by using dialdehyde dextran to inhibit collagen contraction, maintaining uniform size and shape. This permits automated readout for determination of the effect of inhibitory compounds on cancer cell invasion. Sensitivity was demonstrated by the ability to distinguish varying levels of invasiveness of cancer cell lines, and robustness was determined by calculating the Z-factor. A Z-factor of 0.65 was obtained by comparing the effects of DMSO and anti-β1-integrin antibody, an inhibitory reagent, on the invasion of Du145 cancer cells, suggesting this novel assay is suitable for large scale drug discovery. As proof of principle, the NCI Diversity Compound Library was screened against human invasive cancer cells. Nine compounds exhibiting high potency and low toxicity were identified, including DX-52-1, a compound previously reported to inhibit cell migration, a critical determinant of cancer invasion. The results indicate that this innovative HT platform is a simple, precise, and easy to replicate 3-D invasion assay for anti-cancer drug discovery.     

Human Lipocalin-Type Prostaglandin D Synthase-Based Drug Delivery System for Poorly Water-Soluble Anti-Cancer Drug SN-38

Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily, which is composed of secretory transporter proteins, and binds a wide variety of small hydrophobic molecules. Using this function, we have reported the feasibility of using L-PGDS as a novel drug delivery vehicle for poorly water-soluble drugs. In this study, we show the development of a drug delivery system using L-PGDS, one that enables the direct clinical use of 7-ethyl-10-hydroxy-camptothecin (SN-38), a poorly water-soluble anti-cancer drug. In the presence of 2 mM L-PGDS, the concentration of SN-38 in PBS increased 1,130-fold as compared with that in PBS. Calorimetric experiments revealed that L-PGDS bound SN-38 at a molecular ratio of 1:3 with a dissociation constant value of 60 μM. The results of an in vitro growth inhibition assay revealed that the SN-38/L-PGDS complexes showed high anti-tumor activity against 3 human cancer cell lines, i.e., Colo201, MDA-MB-231, and PC-3 with a potency similar to that of SN-38 used alone. The intravenous administration of SN-38/L-PGDS complexes to mice bearing Colo201 tumors showed a pronounced anti-tumor effect. Intestinal mucositis, which is one of the side effects of this drug, was not observed in mice administered SN-38/L-PGDS complexes. Taken together, L-PGDS enables the direct usage of SN-38 with reduced side effects.     

New Targets for Drug Discovery against Malaria

A mathematical model which predicts the intraerythrocytic stages of Plasmodium falciparum infection was developed using data from malaria-infected mice. Variables selected accounted for levels of healthy red blood cells, merozoite (Plasmodium asexual phase) infected red blood cells, gametocyte (Plasmodium sexual phase) infected red blood cells and a phenomenological variable which accounts for the mean activity of the immune system of the host. The model built was able to reproduce the behavior of three different scenarios of malaria. It predicts the later dynamics of malaria-infected humans well after the first peak of parasitemia, the qualitative response of malaria-infected monkeys to vaccination and the changes observed in malaria-infected mice when they are treated with antimalarial drugs. The mathematical model was used to identify new targets to be focused on drug design. Optimization methodologies were applied to identify five targets for minimizing the parasite load; four of the targets thus identified have never before been taken into account in drug design. The potential targets include: 1) increasing the death rate of the gametocytes, 2) decreasing the invasion rate of the red blood cells by the merozoites, 3) increasing the transformation of merozoites into gametocytes, 4) decreasing the activation of the immune system by the gametocytes, and finally 5) a combination of the previous target with decreasing the recycling rate of the red blood cells. The first target is already used in current therapies, whereas the remainders are proposals for potential new targets. Furthermore, the combined target (the simultaneous decrease of the activation of IS by gRBC and the decrease of the influence of IS on the recycling of hRBC) is interesting, since this combination does not affect the parasite directly. Thus, it is not expected to generate selective pressure on the parasites, which means that it would not produce resistance in Plasmodium.     

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described. We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient''s tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.     

Mycobacterium Sulfur Metabolism and Implications for Novel Drug Targets

New antibiotic targets are urgently needed to tackle the multidrug resistant and latent Mycobacterium tuberculosis, the causative agent of the most formidable infectious disease tuberculosis. Sulfur metabolism is essential for the survival and virulence of many pathogens including M. tuberculosis. The absence of most genes involved in microbial sulfur metabolism in human beings suggests abundant novel potential antibiotic targets in pathogen sulfur metabolism. In this article, a comparative genomic landscape of Mycobacterium sulfur metabolism, such as the uptake, activation, and reduction of sulfate and allied enzymes, the biosynthesis pathway of some sulfated metabolites, and the enzymes involved in these pathways were presented. Novel clues for antibiotic targets are put forward.     

Uncovering potential Drug Targets for Tuberculosis using Protein Networks

The emergence of HIV-TB co-infection and multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) drive the need for new therapeutics against the infectious disease tuberculosis. Among the reported putative TB targets in the literature, the identification and characterization of the most probable therapeutic targets that influence the complex infectious disease, primarily through interactions with other influenced proteins, remains a statistical and computational challenge in proteomic epidemiology. Protein interaction network analysis provides an effective way to understand the relationships between protein products of genes by interconnecting networks of essential genes and its protein-protein interactions for 5 broad functional categories in Mtb. We also investigated the substructure of the protein interaction network and focused on highly connected nodes known as cliques by giving weight to the edges using data mining algorithms. Cliques containing Sulphate assimilation and Shikimate pathway enzymes appeared continuously inspite of increasing constraints applied by the K-Core algorithm during Network Decomposition. The potential target narrowed down through Systems approaches was Prephanate Dehydratase present in the Shikimate pathway this gives an insight to develop novel potential inhibitors through Structure Based Drug Design with natural compounds.     

Molecular Approaches to Receptors as Targets for Drug Discovery

Abstract

The cloning of a great number of receptors and channels has revealed that many of these targets for drug discovery can be grouped into superfamilies based on sequence and structural similarities. This review presents an overview of how molecular biological approaches have revealed a plethora of receptor subtypes, led to new definitions of subtypes and isoforms, and played a role in the development of highly selective drugs. Moreover, the diversity of subtypes has molded current views of the structure and function of receptor families. Practical difficulties and limitations inherent in the characterization of the ligand binding and signaling properties of expressed recombinant receptors are discussed. The importance of evaluating drug-receptor interactions that differ with temporally transient and distinct receptor conformational states is emphasized. Structural motifs and signal transduction features are presented for the following major receptor superfamilies: ligand-gated ion channel, voltage-dependent ion channel, G-protein coupled, receptor tyrosine-kinase, receptor protein tyrosine-phosphatase, cytokine and nuclear hormone. In addition, a prototypic receptor is analyzed to illustrate functional properties of a given family. The review concludes with a discussion of future directions in receptor research that will impact drug discovery, with a specific focus on orphan receptors as targets for drug discovery. Methods for classifying orphan receptors based upon homologies with members of existing superfamilies are presented together with molecular approaches to the greater challenge of defining their physiological roles. Besides revealing new orphan receptors, the human genome sequencing project will result in the identification of an abundance of novel receptors that will be molecular targets for the development of highly selective drugs. These findings will spur the discovery and development of an exciting new generation of receptor-subtype specific drugs with enhanced therapeutic specificity.     

DDTRP: Database of Drug Targets for Resistant Pathogens

Emergence of drug resistance is a major threat to public health. Many pathogens have developed resistance to most of the existing antibiotics, and multidrug-resistant and extensively drug resistant strains are extremely difficult to treat. This has resulted in an urgent need for novel drugs. We describe a database called 'Database of Drug Targets for Resistant Pathogens' (DDTRP). The database contains information on drugs with reported resistance, their respective targets, metabolic pathways involving these targets, and a list of potential alternate targets for seven pathogens. The database can be accessed freely at http://bmi.icmr.org.in/DDTRP.     

High-Content,Image-Based Screening for Drug Targets in Yeast

Background

Drug discovery and development are predicated on elucidation of the potential mechanisms of action and cellular targets of candidate chemical compounds. Recent advances in high-content imaging techniques allow simultaneous analysis of a range of cellular events. In this study, we propose a novel strategy to identify drug targets by combining genetic screening and high-content imaging in yeast.

Methodology

In this approach, we infer the cellular functions affected by candidate drugs by comparing morphologic changes induced by the compounds with the phenotypes of yeast mutants.

Conclusions

Using this method and four well-characterized reagents, we successfully identified previously known target genes of the compounds as well as other genes involved with functionally related cellular pathways. This is the first demonstration of a genetic high-content assay that can be used to identify drug targets based on morphologic phenotypes of a reference mutant panel.     

基于亲和探针的药物靶点鉴定技术研究进展

药物靶点的鉴定和相关研究在药学研究领域具有重要的理论指导意义和实用价值。利用亲和探针偶联靶分子的方法是目前发现药 物靶点的主要手段之一。该方法可从分子水平发现药物的作用靶点,从而对药物的分子作用机制提供细胞水平的直接证据。从 DNA 和小 分子药物探针的构筑和应用入手,对近些年鉴定 DNA 损伤识别蛋白的研究进展进行了较为详尽的讨论,并简要介绍目前探索小分子药物 作用靶点的主流技术。作为亲和偶联鉴定药物作用靶点方法的重要组成部分,亲和探针设计的合理性关系到方法本身的可操作性以及鉴 定结果的可靠性。从多个角度对 DNA 探针和小分子药物探针的设计经验进行了较为系统的总结,例如经典的亲和纯化分离方法,以及更 为高效的光激发共价偶联技术等。这些方法和思路为探索 DNA 损伤相关蛋白质的功能以及小分子药物的细胞作用机制提供了丰富的研究 工具,有助于从分子水平理解药物的作用机制。     

Current Status of Targets and Assays for Anti-HIV Drug Screening

HIV/AIDS is one of the most serious public health challenges globally. Despite the great efforts that are being devoted to prevent,treat and to better understand the disease,it is one of the main causes of morbidity and mortality worldwide. Currently,there are 30 drugs or combinations of drugs approved by FDA. Because of the side-effects,price and drug resistance,it is essential to discover new targets,to develop new technology and to find new anti-HIV drugs. This review summarizes the major targets and assays currently used in anti-HIV drug screening.     

A Functional Variomics Tool for Discovering Drug-Resistance Genes and Drug Targets

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Highlights? A functional variomics tool for drug-target identification is described ? This tool compares favorably with other target-identification tools ? This tool will greatly facilitate the linking of phenotypes to point mutations ? This work discovered Pmp3 as a target of amphotericin B     

Recursive organizer (ROR): an analytic framework for sequence-based association analysis

The advent of next-generation sequencing technologies affords the ability to sequence thousands of subjects cost-effectively, and is revolutionizing the landscape of genetic research. With the evolving genotyping/sequencing technologies, it is not unrealistic to expect that we will soon obtain a pair of diploidic fully phased genome sequences from each subject in the near future. Here, in light of this potential, we propose an analytic framework called, recursive organizer (ROR), which recursively groups sequence variants based upon sequence similarities and their empirical disease associations, into fewer and potentially more interpretable super sequence variants (SSV). As an illustration, we applied ROR to assess an association between HLA-DRB1 and type 1 diabetes (T1D), discovering SSVs of HLA-DRB1 with sequence data from the Wellcome Trust Case Control Consortium. Specifically, ROR reduces 36 observed unique HLA-DRB1 sequences into 8 SSVs that empirically associate with T1D, a fourfold reduction of sequence complexity. Using HLA-DRB1 data from Type 1 Diabetes Genetics Consortium as cases and data from Fred Hutchinson Cancer Research Center as controls, we are able to validate associations of these SSVs with T1D. Further, SSVs consist of nine nucleotides, and each associates with its corresponding amino acids. Detailed examination of these selected amino acids reveals their potential functional roles in protein structures and possible implication to the mechanism of T1D.     

Drug Targets for Cell Cycle Dysregulators in Leukemogenesis: In Silico Docking Studies

Alterations in cell cycle regulating proteins are a key characteristic in neoplastic proliferation of lymphoblast cells in patients with Acute Lymphoblastic Leukemia (ALL). The aim of our study was to investigate whether the routinely administered ALL chemotherapeutic agents would be able to bind and inhibit the key deregulated cell cycle proteins such as - Cyclins E1, D1, D3, A1 and Cyclin Dependent Kinases (CDK) 2 and 6. We used Schrödinger Glide docking protocol to dock the chemotherapeutic drugs such as Doxorubicin and Daunorubicin and others which are not very common including Clofarabine, Nelarabine and Flavopiridol, to the crystal structures of these proteins. We observed that the drugs were able to bind and interact with cyclins E1 and A1 and CDKs 2 and 6 while their docking to cyclins D1 and D3 were not successful. This binding proved favorable to interact with the G1/S cell cycle phase proteins that were examined in this study and may lead to the interruption of the growth of leukemic cells. Our observations therefore suggest that these drugs could be explored for use as inhibitors for these cell cycle proteins. Further, we have also highlighted residues which could be important in the designing of pharmacophores against these cell cycle proteins. This is the first report in understanding the mechanism of action of the drugs targeting these cell cycle proteins in leukemia through the visualization of drug-target binding and molecular docking using computational methods.     

Apelin/APJ：血管功能调节因子和药物治疗新靶点

Apelin(APJendogenousligand)是血管紧张素Ⅱ1型受体相关蛋白(angiotensin receptor-like 1,APJ)的内源性配体.Apelin/APJ系统在机体内广泛分布,在众多血管系统表达水平较高,如心血管系统、肺血管系统等.研究发现,apelin可调节血管张力,促进血管平滑肌细胞增殖、视网膜血管新生以及单核细胞向内皮细胞黏附,促进肝门静脉和冠状动脉侧枝形成等.本文就apelin调节血管功能及其相关疾病(高血压、肺动脉高压、动脉粥样硬化、胶质瘤、肺癌、门静脉高压、糖尿病血管并发症等)进行综述,揭示了apelin与血管及其相关疾病的内在联系,表明apelin/APJ可作为血管疾病的治疗靶点.