Therapeutic Targeting the Allosteric Cysteinome of RAS and Kinase Families

Allosteric mechanisms are pervasive in nature, but human-designed allosteric perturbagens are rare. The history of KRAS^G12C inhibitor development suggests that covalent chemistry may be a key to expanding the armamentarium of allosteric inhibitors. In that effort, irreversible targeting of a cysteine converted a non-deal allosteric binding pocket and low affinity ligands into a tractable drugging strategy. Here we examine the feasibility of expanding this approach to other allosteric pockets of RAS and kinase family members, given that both protein families are regulators of vital cellular processes that are often dysregulated in cancer and other human diseases. Moreover, these heavily studied families are the subject of numerous drug development campaigns that have resulted, sometimes serendipitously, in the discovery of allosteric inhibitors. We consequently conducted a comprehensive search for cysteines, a commonly targeted amino acid for covalent drugs, using AlphaFold-generated structures of those families. This new analysis presents potential opportunities for allosteric targeting of validated and understudied drug targets, with an emphasis on cancer therapy.     

Structure-activity relationships in a new class of non-substrate-like covalent inhibitors of the bacterial glycosyltransferase LgtC

Lipooligosaccharide (LOS) structures in the outer core of Gram-negative mucosal pathogens such as Neisseria meningitidis and Haemophilus influenzae contain characteristic glycoepitopes that contribute significantly to bacterial virulence. An important example is the digalactoside epitope generated by the retaining α-1,4-galactosyltransferase LgtC. These digalactosides camouflage the pathogen from the host immune system and increase its serum resistance. Small molecular inhibitors of LgtC are therefore sought after as chemical tools to study bacterial virulence, and as potential candidates for anti-virulence drug discovery. We have recently discovered a new class of non-substrate-like inhibitors of LgtC. The new inhibitors act via a covalent mode of action, targeting a non-catalytic cysteine residue in the LgtC active site. Here, we describe, for the first time, structure-activity relationships for this new class of glycosyltransferase inhibitors. We have carried out a detailed analysis of the inhibition kinetics to establish the relative contribution of the non-covalent binding and the covalent inactivation steps for overall inhibitory activity. Selected inhibitors were also evaluated against a serum-resistant strain of Haemophilus influenzae, but did not enhance the killing effect of human serum.     

基于小分子化合物库筛选协同多腺苷二磷酸核糖聚合酶抑制剂杀伤卵巢癌细胞的药物

多腺苷二磷酸核糖聚合酶(poly(ADP-ribose) polymerase, PARP)抑制剂是一类靶向 DNA 修复缺陷癌细胞的新型药物。早期研究表明 PARP 抑制剂取得了令人满意的结果,然而药物治疗后出现的耐药机制尚未完全揭露。因此,有必要寻找更多的靶向药物与PARP 抑制剂联用,以达到杀伤肿瘤细胞的目的。本文基于379种小分子化合物和PARP抑制剂尼拉帕尼(Niraparib)的联合用药筛选,通过细胞增殖实验、克隆存活实验和免疫荧光染色等方法筛选潜在的具有协同PARP抑制剂杀伤卵巢癌细胞的药物。结果表明,其中有8种小分子化合物具有较好的联合用药效果,包括2种已经报道的与PARP抑制剂具有联用效果的小分子化合物STF-118804和Disulfiram。我们从中选取原肌球蛋白受体激酶 A (tropomyosin receptor kinase A,TrKA)的抑制剂GW441756,进行了多种肿瘤细胞的验证以及初步机制的探究。Niraparib和TrKA抑制剂的联合用药显著增加肿瘤细胞对PARP抑制剂的敏感性(P<0.05)。从机制上分析,联合用药组细胞内γH2AX foci的数目显著增加(P<0.05),说明TrKA抑制剂阻碍损伤后细胞的DNA损伤修复能力;同时,联合用药显著降低细胞内同源重组修复(homologous recombination repair,HRR)标志物RAD51 foci(P<0.05)的形成,说明TrKA抑制剂可能通过抑制细胞的HRR效率阻碍细胞的DNA损伤修复。本研究的结果提示,TrKA抑制剂可以作为一种与PARP抑制剂联用杀伤卵巢癌细胞的潜在药物。     

NSAID-induced gut inflammation and vasoconstriction: Causes and potential reversal with beta-CGRP - A hypothesis

Traditional non-steroidal anti-inflammatory drugs, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitors control inflammation. While these drugs are formulated to reduce one of the cardinal signs of inflammation by reducing prostaglandin levels at the site of inflammation, COX-1 inhibitors induce inflammation in the stomach as well as the small bowel. The COX-2 inhibitors, a large portion of the non-steroidal anti-inflammatory drug market, provide a gastro-intestinally safer class of drugs. However, COX-2 inhibitors induce vasoconstriction via actions in renal and cardiovascular tissues. Since COX-2 inhibitors also have anticancer potential, it is worthwhile to design drug formulations that will not cause hypertension or cardiovascular damage. An attempt has thus been made in this article to formulate a hypothesis to circumvent the COX inhibitors induced inflammation and vasoconstriction through COX independent activation of calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide found throughout the vascular and sensory nervous system.     

Selective immunoproteasome inhibitors with non-peptide scaffolds identified from structure-based virtual screening

As a major component of the crucial nonlysosomal protein degradation pathway in the cells, the proteasome has been implicated in many diseases such as Alzheimer’s disease, Huntington’s disease, inflammatory bowel diseases, autoimmune diseases, multiple myeloma (MM) and other cancers. There are two main proteasome subtypes: the constitutive proteasome which is expressed in all eukaryotic cells and the immunoproteasome which is expressed in immune cells and can be induced in other cell types. Majority of currently available proteasome inhibitors are peptide backbone-based, having short half-lives in the body. It is highly desirable to identify novel, immunoproteasome-selective inhibitors with non-peptide scaffolds for development of novel therapeutics. Through combined virtual screening and experimental studies targeting the immunoproteasome, we have identified a set of novel immunoproteasome inhibitors with diverse non-peptide scaffolds. Some of the identified inhibitors have significant selectivity for the immunoproteasome over the constitutive proteasome. Unlike most of the currently available proteasome inhibitors, these new inhibitors lacking electrophilic pharmacophores are not expected to form a covalent bond with proteasome after the binding. These non-peptide scaffolds may provide a new platform for future rational drug design and discovery targeting the immunoproteasome.     

Covalent vs. Non‐Covalent Inhibition: Tackling Drug Resistance in EGFR – A Thorough Dynamic Perspective

A persistent challenge in the treatment of non‐small cell lung cancer (NSCLC) with EGFR is the emergence of drug‐resistant caused by somatic mutations. The EGFR L858R/T790 M double mutant (EGFR^DM) was found to be the most alarming variant. Despite the development of a wide range of inhibitors, none of them could inhibit EGFR^DM effectively. Recently, 11h and 45a , have been found to be potent inhibitors against EGFR^DM through two distinctive mechanisms, non‐covalent and covalent binding, respectively. However, the structural and dynamic implications of the two modes of inhibitions remain unexplored. Herein, two molecular dynamics simulation protocols, coupled with free‐energy calculations, were applied to gain insight into the atomistic nature of each binding mode. The comparative analysis confirmed that there is a significant difference in the binding free energy between 11h and 45a (ΔΔG_bind=?21.17 kcal/mol). The main binding force that governs the binding of both inhibitors is vdW, with a higher contribution for 45a . Two residues ARG841 and THR854 were found to have curtailed role in the binding of 45a to EGFR^DM by stabilizing its flexible alcohol chain. The 45a binding to EGFR^DM induces structural rearrangement in the active site to allow easier accessibility of 45a to target residue CYS797. The findings of this work can substantially shed light on new strategies for developing novel classes of covalent and non‐covalent inhibitors with increased specificity and potency.     

Road Map for the Structure-Based Design of Selective Covalent HCV NS3/4A Protease Inhibitors

Over the last 2 decades, covalent inhibitors have gained much popularity and is living up to its reputation as a powerful tool in drug discovery. Covalent inhibitors possess many significant advantages including increased biochemical efficiency, prolonged duration and the ability to target shallow, solvent exposed substrate-binding domains. However, rapidly mounting concerns over the potential toxicity, highly reactive nature and general lack of selectivity have negatively impacted covalent inhibitor development. Recently, a great deal of emphasis by the pharmaceutical industry has been placed toward the development of novel approaches to alleviate the major challenges experienced through covalent inhibition. This has unexpectedly led to the emergence of “selective” covalent inhibitors. The purpose of this review is not only to provide an overview from literature but to introduce a technical guidance as to how to initiate a systematic “road map” for the design of selective covalent inhibitors which we believe may assist in the design and development of optimized potential selective covalent HCV NS3/4A viral protease inhibitors.     

Drug screening for influenza neuraminidase inhibitors

Neuraminidase (NA) is one of the most important targets to screen the drugs of anti-influenza virus A and B. After virtual screening approaches were applied to a compound database which possesses more than 10000 compound structures, 160 compounds were selected for bioactivity assay, then a High Throughput Screening (HTS) model established for influenza virus NA inhibitors was applied to detect these compounds. Finally, three compounds among them displayed higher inhibitory activities, the range of their IC50 was from 0.1 μmol/L to 3μmol/L. Their structural scaffolds are novel and different from those of NA inhibitors approved for influenza treatment, and will be useful for the design and research of new NA inhibitors. The resuit indicated that the combination of virtual screening with HTS was very significant to drug screening and drug discovery.     

Modification of Enzyme Properties by the use of Inhibitors During Their Stabilisation by Multipoint Covalent Attachment

The effect of a number of inhibitors adsorbed at the active site of penicillin acylase during immobilisation/stabilisation is reported. Each inhibitor, when it is adsorbed at the active centre of penicillin acylase promotes a specific enzymatic conformation which remains fixed after the stabilisation process by multipoint covalent attachment to pre-existing supports. A number of inhibitors: penicillin sulfoxide, phenylacetic acid, mandelic acid, and phenylglycine were employed to induce conformational changes. The activity towards different substrates of the enzyme derivative (in hydrolysis and in synthesis) was determined. The stability of the derivatives was also measured. This technique provides a broad spectrum of enzymatic derivatives with a range of activity/stability depending on the inhibitors used in their stabilisation. The resulting choice offers a considerably increased potential for the use of the enzyme since one can select a derivative which will specifically catalyse the reaction of interest.     

Joint effects of acetochlor and urea on germinating characteristics of crop seeds

Neuraminidase (NA) is one of the most important targets to screen the drugs of anti-influenza virus A and B. After virtual screening approaches were applied to a compound database which possesses more than 10000 compound structures, 160 compounds were selected for bioactivity assay, then a High Throughput Screening (HTS) model established for influenza virus NA inhibitors was applied to detect these compounds. Finally, three compounds among them displayed higher inhibitory activities, the range of their IC5o was from 0.1 μmol/L to 3 μmol/L. Their structural scaffolds are novel and different from those of NA inhibitors approved for influenza treatment, and will be useful for the design and research of new NA inhibitors. The result indicated that the combination of virtual screening with HTS was very significant to drug screening and drug discovery.     

Discovery of potent,highly selective covalent irreversible BTK inhibitors from a fragment hit

Bruton's Tyrosine Kinase (BTK) is a member of the TEC kinase family that is expressed in cells of hematopoietic lineage (e.g., in B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible BTK inhibitor targeting Cys481 within the ATP-binding pocket, for example ibrutinib, has been applied in the treatment of B-cell malignancies. Starting from a fragment hit, we discovered a novel series of potent covalent irreversible BTK inhibitors that occupy selectivity pocket of the active site of the BTK kinase domain. Guided by X-ray structures and a fragment-based drug design (FBDD) approach, we generated molecules showing comparable cellular potency to ibrutinib and higher kinome selectivity against undesirable off-targets like EGFR.     

Insight into binding mechanisms of EGFR allosteric inhibitors using molecular dynamics simulations and free energy calculations

Abstract

Lung cancer is the leading cause of cancer death, and epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small-cell lung cancer (NSCLC), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the EGFR inhibitors Gefitinib and Erlotinib initially, but soon develop resistance to them due to the emergence of the gatekeeper mutation T790M. The new-generation inhibitors such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to Cys 797, but ultimately lose their efficacy upon the emergence of the C797S mutation that abolishes the covalent bonding. Allosteric inhibitors EAI001 and EAI045 are a new type of EGFR inhibitors that bind to EGFR away from the ATP-binding site and not relying on Cys 797. In this study, molecular dynamics simulations and free energy calculations were carried out on EAI001 and EAI045 in complex with EGFR, revealing the detailed inhibitory mechanism of EAI001 and EAI045 as EGFR allosteric inhibitor, which was expected to provide a basis for rational drug design of the EGFR allosteric inhibitors.

Communicated by Ramaswamy H. Sarma     

Drug discovery in leishmaniasis using protein lipidation as a target

The leishmaniases are infectious diseases caused by a number of species of obligate intracellular protozoa of the genus Leishmania with disease manifesting as cutaneous, mucocutaneous and visceral forms. Despite being endemic in more than 80 countries and its being the cause of high morbidity and mortality, leishmaniasis remains a neglected tropical disease. Chemotherapy is the frontline treatment, but drugs in current use suffer from toxic side effects, difficulties in administration and extended treatment times — moreover, resistance is emerging. New anti-leishmanial drugs are a recognised international priority. Here, we review investigations into N-myristoyltransferase (NMT) as a potential drug target. NMT catalyses the co-translational transfer of a C₁₄ fatty acid from myristoyl-CoA onto the N-terminal glycine residue of a significant subset of proteins in eukaryotic cells. This covalent modification influences the stability and interactions of substrate proteins with lipids and partner proteins. Structure-guided development of new lead compounds emerging from high-throughput screening campaigns targeting Leishmania donovani NMT has led to the discovery of potent inhibitors which have been used to gain insights into the role of protein myristoylation in these parasites and to validate NMT as a drug target.     

Pyridine derivatives as anticancer lead compounds with Fatty Acid Synthase as the target: An in silico-guided in vitro study

For the past few decades, structure-based drug discovery (SBDD) has become an inevitable technique in the drug development process for screening hit compounds against therapeutic targets. Here, we have successfully used the SBDD approach viz. virtual high-throughput screening to identify potential inhibitors against the Ketoacyl synthase (KS) domain of Fatty acid synthase (FASN). Overexpression of FASN, and subsequent enhancement of de novo lipogenesis is a key survival strategy of cancer cells. Hence, targeting lipid metabolism using FASN inhibitors has been considered as a promising method to induce metabolic stress, thereby posing a survival disadvantage to cancer cells. In the present study, we have successfully identified eight FASN inhibitors from Asinex Elite database by implementing in silico tools. Five of the hit compounds share a common ring structure, which enables characteristic binding interactions with FASN-KS. Among them, in vitro validation showed that SFA 22637550 possesses significant FASN inhibitory activity and antiproliferative effect in human cancer cells of various origins. The maximum sensitivity was exhibited towards HepG2 hepatocellular carcinoma cells (IC50 = 28 µM). The mode of cell death was found to be apoptosis with a significant increase in SubG0 population without affecting any other phases of the cell cycle. The current study puts forward an excellent core structure for the development of potent FASN inhibitors for successfully targeting cancer cell metabolism, thereby causing selective cell death.     

Structural basis for producing selective MAP2K7 inhibitors

Mitogen-activated protein kinase kinase 7 (MAP2K7) in the c-Jun N-terminal kinase signal cascade is an attractive drug target for a variety of diseases. The selectivity of MAP2K7 inhibitors against off-target kinases is a major barrier in drug development. We report a crystal structure of MAP2K7 complexed with a potent covalent inhibitor bearing an acrylamide moiety as an electrophile, which discloses a structural basis for producing selective and potent MAP2K7 inhibitors.     

An Insight into the Structural Requirements and Pharmacophore Identification of Carbonic Anhydrase Inhibitors to Combat Oxidative Stress at High Altitudes: An In-Silico Approach

Carbonic anhydrases (CA) inhibitory action could be linked to the treatment of a number of ailments, including cancer, osteoporosis, glaucoma, and several neurological problems. For the development of effective CA inhibitors, a variety of heterocyclic rings have been investigated. Furthermore, at high altitudes, oxygen pressure drops, resulting in the formation of reactive oxygen and nitrogen species, and CA inhibitors having role in combating this oxidative stress. Acetazolamide contains thiadiazole ring, which has aroused researchers’ interest because of its CA inhibitory action. In the present study, we used a number of drug design tools, such as pharmacophore modeling, 3D QSAR, docking, and virtual screening on twenty-seven 1,3,4-thiadiazole derivatives that have been described as potential CA inhibitors in the literature. An atom-based 3D-QSAR analysis was carried out to determine the contribution of individual atoms to model generation, while a pharmacophore mapping investigation was carried out to find the common unique pharmacophoric properties required for biological activity. The coefficient of determination for both the training and test sets were statistically significant in the generated model. The best QSAR model was chosen based on the values of R² (0.8757) and Q² (0.7888). A molecular docking study was also conducted against the most potent analogue 4m, which has the highest SP docking score (−5.217) (PDB ID: 6g3v). The virtual screening revealed a number of promising compounds. The screened compound ZINC77699643 interacted with the amino acid residues, Pro201 and Thr199, in the virtual screening study (PDB ID: 6g3v). These interactions demonstrated the significance of the CA inhibitory activity of the compound. Furthermore, ADME study revealed useful information regarding compound’s drug-like properties. Therefore, the findings of the present investigation could aid in the development of more potent CA inhibitors, which could benefit the treatment of oxidative stress at high altitudes.     

The AutoDock suite at 30

The AutoDock suite provides a comprehensive toolset for computational ligand docking and drug design and development. The suite builds on 30 years of methods development, including empirical free energy force fields, docking engines, methods for site prediction, and interactive tools for visualization and analysis. Specialized tools are available for challenging systems, including covalent inhibitors, peptides, compounds with macrocycles, systems where ordered hydration plays a key role, and systems with substantial receptor flexibility. All methods in the AutoDock suite are freely available for use and reuse, which has engendered the continued growth of a diverse community of primary users and third‐party developers.     

Discovery of benzo[g]indoles as a novel class of non-covalent Keap1-Nrf2 protein-protein interaction inhibitor

The Keap1-Nrf2 system is an attractive target for drug discovery regarding various unmet medical needs. Only covalent inhibitors for protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 have been approved or are under clinical trials, but such electrophilic compounds lack selectivity. Therefore, specific non-covalent Keap1-Nrf2 PPI inhibitors are expected to be safer Nrf2 activators. We found a novel class of non-covalent Keap1-Nrf2 PPI inhibitor that has a benzo[g]indole skeleton and an indole-3-hydroxamic acid moiety and that exhibits significant PPI inhibitory activity. Additionally, the benzo[g]indole-3-carbohydrazide derivatives were newly prepared. The benzo[g]indole derivatives showed a stronger Keap1-Nrf2 PPI inhibitory activity than Cpd16, a previously reported non-covalent PPI inhibitor. Moreover, most of the PPI inhibitors showed a high metabolic stability in a human microsome system with a low cytotoxicity against HepG2 cell lines, which suggests that novel benzo[g]indole-type Keap1-Nrf2 PPI inhibitors are expected to be biological tools or lead compounds for Nrf2 activators.     

Piperidine and piperazine inhibitors of fatty acid amide hydrolase targeting excitotoxic pathology

FAAH inhibitors offer safety advantages by augmenting the anandamide levels “on demand” to promote neuroprotective mechanisms without the adverse psychotropic effects usually seen with direct and chronic activation of the CB1 receptor. FAAH is an enzyme implicated in the hydrolysis of the endocannabinoid N-arachidonoylethanolamine (AEA), which is a partial agonist of the CB1 receptor. Herein, we report the discovery of a new series of highly potent and selective carbamate FAAH inhibitors and their evaluation for neuroprotection. The new inhibitors showed potent nanomolar inhibitory activity against human recombinant and purified rat FAAH, were selective (>1000-fold) against serine hydrolases MGL and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Evaluation of FAAH inhibitors 9 and 31 using the in vitro competitive activity-based protein profiling (ABPP) assay confirmed that both inhibitors were highly selective for FAAH in the brain, since none of the other FP-reactive serine hydrolases in this tissue were inhibited by these agents. Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on known FAAH cocrystal structures. To rationally design new molecules that are irreversibly bound to FAAH, we have constructed “precovalent” FAAH-ligand complexes to identify good binding geometries of the ligands within the binding pocket of FAAH and then calculated covalent docking poses to select compounds for synthesis. FAAH inhibitors 9 and 31 were evaluated for neuroprotection in rat hippocampal slice cultures. In the brain tissue, both inhibitors displayed protection against synaptic deterioration produced by kainic acid-induced excitotoxicity. Thus, the resultant compounds produced through rational design are providing early leads for developing therapeutics against seizure-related damage associated with a variety of disorders.