Receptor-ligand interaction-based virtual screening for novel Eg5/kinesin spindle protein inhibitors

Eg5/KSP is a promising mitotic spindle target for drug discovery in cancer chemotherapy and the development of agents against fungal diseases. A range of Eg5 targeting compounds identified by in vitro or cell-based screening is currently in development. We employed structure-based virtual screening of a database of 700,?000 compounds to identify three novel Eg5 inhibitors bearing quinazoline (24) or thioxoimidazolidine (30 and 37) scaffolds. The new compounds inhibit Eg5 ATPase activity, show growth inhibition in proliferation assays, and induce monoastral spindles in cells, the characteristic phenotype for Eg5 inhibiting agents. This is the first successful reported procedure for the identification of Eg5 inhibitors via receptor-ligand interaction-based virtual screening.     

Allosteric mechanism of an oximino‐piperidino‐piperidine antagonist for the CCR5 chemokine receptor

The first step for the HIV‐1 virus infecting host cell is bound with the CCR5 chemokine receptor. A set of allosteric inhibitors of oximino‐piperidino‐piperidine antagonists for CCR5 chemokine receptor was discovered. However, the allosteric mechanism of these inhibitors is still unsolved. Therefore, residue‐level dynamics correlation network combining with on molecular dynamics simulation was used to investigate the allosteric mechanism. The dynamics correlation network of bound CCR5 is significantly different from that of free CCR5. The community of the most active complex suggests that the allosteric information can freely transfer from the allosteric site to the effector site of the second extracellular loop, while the information transfers bottleneck for the less active one. Here, a hypothesis was proposed that “binding‐induced allosteric mechanism” was used to reveal the allosteric regulation of antagonists and the network perturbation confirmed it. Finally, the shortest path algorithm was used to identify the possible allosteric pathway with Gly173‐Lys171‐Thr177‐Tyr89‐LIG which was evaluated by the network perturbation of key residue. Furthermore, the efficiency of allostery for the most active system is the highest among these antagonist complexes. The strategy targeting the allosteric pathway can be used to design novel inhibitors of HIV‐1 virus.     

STLC-resistant cell lines as tools to classify chemically divergent Eg5 targeting agents according to their mode of action and target specificity

Determining the mechanism of action of drugs and their target specificity in cells remains a major challenge. Here we describe the use of cell lines expressing two point mutations in the allosteric inhibitor binding pocket of the mitotic kinesin Eg5 (D130A, in the loop L5 region and L214A in helix α3), which following transfection, were selected for their ability to proliferate normally in the presence of STLC, a well known Eg5 inhibitor. The cell lines were used to discriminate the mechanism of action of other chemically distinct small molecule inhibitors of Eg5 that differ in their mode of action. The STLC resistant cells were capable of continuous proliferation in the presence of ATP uncompetitive inhibitors, such as K858 and dimethylenastron, but were still sensitive to ATP competitive inhibitors that are thought to bind to a distinct site on Eg5 than the allosteric binding pocket. The STLC resistant cell lines can therefore be used as a filter to distinguish Eg5 loop L5 binding drugs from drugs binding to other pockets without prior structural information. Additionally, the cells can be used to analyze whether inhibitors of Eg5 are specific to this potential drug target or whether they have additional targets in dividing cells.     

Structure-activity relationship and multidrug resistance study of new S-trityl-L-cysteine derivatives as inhibitors of Eg5

The mitotic spindle is a validated target for cancer chemotherapy. Drugs such as taxanes and vinca alkaloids specifically target microtubules and cause the mitotic spindle to collapse. However, toxicity and resistance are problems associated with these drugs. Thus, alternative approaches to inhibiting the mitotic spindle are being pursued. These include targeting Eg5, a human kinesin involved in the formation of the bipolar spindle. We previously identified S-trityl-L-cysteine (STLC) as a potent allosteric inhibitor of Eg5. Here, we report the synthesis of a new series of STLC-like compounds with in vitro inhibition in the low nanomolar range. We also performed a multidrug resistance study in cell lines overexpressing P-glycoprotein and showed that some of these inhibitors may have the potential to overcome susceptibility to this efflux pump. Finally, we performed molecular docking of the compounds and determined the structures of two Eg5-inhibitor complexes to explain the structure-activity relationship of these compounds.     

Triphenylbutanamines: kinesin spindle protein inhibitors with in vivo antitumor activity

The human mitotic kinesin Eg5 represents a novel mitotic spindle target for cancer chemotherapy. We previously identified S-trityl-l-cysteine (STLC) and related analogues as selective potent inhibitors of Eg5. We herein report on the development of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from the STLC scaffold. This new generation systematically improves on potency: the most potent C-trityl analogues exhibit K(i)(app) ≤ 10 nM and GI(50) ≈ 50 nM, comparable to results from the phase II clinical benchmark ispinesib. Crystallographic studies reveal that they adopt the same overall binding configuration as S-trityl analogues at an allosteric site formed by loop L5 of Eg5. Evaluation of their druglike properties reveals favorable profiles for future development and, in the clinical candidate ispinesib, moderate hERG and CYP inhibition. One triphenylbutanamine analogue and ispinesib possess very good bioavailability (51% and 45%, respectively), with the former showing in vivo antitumor growth activity in nude mice xenograft studies.     

Type III or allosteric kinase inhibitors for the treatment of non-small cell lung cancer

Introduction: In recent times, there has been much interest in the development of pharmacological kinase inhibitors that treat NSCLC. Furthermore, treatment options have been guided by the development of a wide panel of synthetic small molecule kinase inhibitors. Most of the molecules developed belong to the type I class of inhibitors that target the ATP-binding site in its active conformation. The high sequence similarity in the ATP-binding site among members of the kinase families often results in low selectivity and additional toxicities. Also, second mutations in the ATP-binding site, such as threonine to methionine at position 790, have been described as a mechanism of resistance to ATP-competitive kinase inhibitors. For these reasons, alternative drug development approaches targeting sites other than the ATP cleft are being pursued. The class III or allosteric inhibitors, which bind outside the ATP-binding site, have been shown to negatively modulate kinase activity.

Areas covered: In this review, the authors discuss the most well-characterised allosteric inhibitors that have reached clinical development in NSCLC.

Expert opinion: Great progress has made in developing inhibitors with entirely new modes of action. That being said, it is important to highlight that despite their apparent simplicity, biochemical assays will remain at the core of drug discovery activities to better explore these new opportunities.     

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-l-cysteine in tumor derived cell lines

The kinesin Eg5 plays an essential role in bipolar spindle formation. A variety of structurally diverse inhibitors of the human kinesin Eg5, including monastrol and STLC, share the same binding pocket on Eg5, composed by helix α2/loop L5, and helix α3 of the Eg5 motor domain. Previous biochemical analysis in the inhibitor binding pocket of Eg5 identified key residues in the inhibitor binding pocket of Eg5 that in the presence of either monastrol or STLC exhibited ATPase activities similar to the untreated wild type Eg5. Here we evaluated the ability of full-length human Eg5 carrying point mutations in the drug binding pocket to confer resistance in HeLa and U2OS cells to either monastrol or STLC, as measured by the formation of bipolar spindles. Both transfected cells expressing wild type Eg5 and untransfected cells were equally sensitive to both inhibitors. Expression of Eg5 single point mutants R119A, D130A, L132A, I136A, L214A and E215A conferred significant resistance to monastrol. Certain mutations inducing monastrol resistance such as R119A, D130A and L214A also conferred significant resistance to STLC. For the first time at a cellular level, the propensity of selected Eg5 point mutants to confer drug resistance confirms the target specificity of monastrol and STLC for Eg5. These data also suggest a possible mechanism by which drug resistance may occur in tumors treated with agents targeting Eg5.     

Analysis of c‐Met Kinase Domain Complexes: A New Specific Catalytic Site Receptor Model for Defining Binding Modes of ATP‐Competitive Ligands

The receptor tyrosine kinase c‐Met have multiple roles during cancer development and is currently considered as an important target for molecularly targeted therapies. Structural knowledge of how compounds interact on c‐Met catalytic site could guide structure‐based drug design strategies towards more effective and selective anticancer drug candidates. However, although 17 crystal structures of c‐Met complexed with adenosine triphosphate (ATP)‐competitive kinase inhibitors are publicly available (August 2009), there are still open questions regarding the prediction of ligand binding modes. We have applied molecular modeling and molecular mechanics to analyze the distribution of ligands interaction energy on c‐Met residues, and deduced a new model of the active site allowing for an unambiguous identification of ligand binding modes. We demonstrate that the binding of known ligands on the c‐Met catalytic site involves seven identified structurally‐distinct areas. Five of these match the generic kinase ATP binding site model built by Novartis scientists in the 1990s, while the two others are distinct allosteric regions that can be exploited by second generation kinase inhibitors such as Gleevec. We show here that c‐Met can accept both such kinds of allosteric inhibitors, a very unusual feature in the kinase family that opens new grounds for highly specific drug design.     

Kinesin spindle protein inhibitors in cancer: a patent review (2008 – present)

Introduction: Inhibition of kinesin spindle protein (KSP) has emerged as a novel and validated therapeutic strategy against cancers. A lot of new KSP inhibitors have been identified in recent years and some of them have entered clinical trials. This may provide more selections in future cancer therapy.

Areas covered: In the present review, the authors will describe the most recent classes of KSP inhibitors by reviewing about 96 literatures in which 24 patent applications were included from 2008 to now.

Expert opinion: Many new KSP inhibitors have been discovered that act either by binding in an allosteric site of KSP or by ATP competitive inhibition. There are several ATP non-competitive KSP inhibitors entering clinical investigation. Although they were both well tolerated and showed acceptable pharmacokinetic profiles, limited clinical response was always the problem. Mutation of the binding pocket was also a hindrance in the development of these allosteric inhibitors. The appearance of ATP competitive KSP inhibitors was considered to be able to overcome mutation-mediated resistance to the allosteric inhibitors, which could be a new approach for the development of novel KSP inhibitors.     

Site‐directed Mutagenesis of Key Residues Unveiled a Novel Allosteric Site on Human Adenosine Kinase for Pyrrolobenzoxa(thia)zepinone Non‐Nucleoside Inhibitors

Most nucleoside kinases, besides the catalytic domain, feature an allosteric domain which modulates their activity. Generally, non‐substrate analogs, interacting with allosteric sites, represent a major opportunity for developing more selective and safer therapeutics. We recently developed a series of non‐nucleoside non‐competitive inhibitors of human adenosine kinase (hAK), based on a pyrrolobenzoxa(thia)zepinone scaffold. Based on computational analysis, we hypothesized the existence of a novel allosteric site on hAK, topographically distinct from the catalytic site. In this study, we have adopted a multidisciplinary approach including molecular modeling, biochemical studies, and site‐directed mutagenesis to validate our hypothesis. Based on a three‐dimensional model of interaction between hAK and our molecules, we designed, cloned, and expressed specific, single and double point mutants of hAK (Q74A, Q78A, H107A, K341A, F338A, and Q74A‐F338A). Kinetic characterization of recombinant enzymes indicated that these mutations did not affect enzyme functioning; conversely, mutated enzymes are endowed of reduced susceptibility to our non‐nucleoside inhibitors, while maintaining comparable affinity for nucleoside inhibitors to the wild‐type enzyme. This study represents the first characterization and validation of a novel allosteric site in hAK and may pave the way to the development of novel selective and potent non‐nucleoside inhibitors of hAK endowed with therapeutic potential.     

Recent progress in the discovery of Akt inhibitors as anticancer agents

Akt, also referred to as protein kinase B (PKB) or Related to A and C (RAC), is one of the major direct downstream targets of phosphoinositide 3-kinase (PI3K). As it plays a central role in promoting cancer cell proliferation and survival through a growing list of key substrates, intense efforts are underway to find inhibitors of Akt for the treatment of cancer. Discovery of potent and novel inhibitors of Akt has been facilitated greatly by the availability of the X-ray structure of the active form of Akt and by its structural similarity with other serine/threonine kinases. In this review, new Akt inhibitors for the treatment of cancer are comprehensively reviewed, with emphasis on small molecule inhibitors that bind to the ATP-binding site, allosteric sites and the PH domains. Inhibitors of pseudosubstrates and antisense oligonucleotides, as well as Akt inhibitors with unknown mechanism of actions, are also reviewed. Results of clinical trials of several Akt drug candidates are briefly discussed. A brief summary of Akt structure and regulation and the evidences supporting Akt as a cancer target is provided as well. The patent literature is surveyed through July 2007.     

Peptide Inhibitors Against Dengue Virus Infection

Dengue virus (DENV) infection has become a public health problem worldwide. The development of anti‐DENV drug is urgently needed because neither licensed vaccine nor specific drug is currently available. Inhibition of DENV attachment and entry to host cells by blocking DENV envelope (E) protein is an attractive strategy for anti‐DENV drug development. A hydrophobic pocket on the DENV E protein is essential for structural transition in the membrane fusion, and inhibition of this process is able to inhibit DENV infection. To search for a safe anti‐DENV drug, we identified short peptides targeting the hydrophobic pocket by molecular docking. In addition, the information of predicted ligand‐binding site of reported active compounds of DENV2 hydrophobic pocket was also used for peptide inhibitors selection. The di‐peptide, EF, was the most effective on DENV2 infection inhibition in vitro with a half maximal inhibition concentration (IC50) of 96 μm . Treatment of DENV2 with EF at the concentration of 200 μm resulted in 83.47% and 84.15% reduction in viral genome and intracellular E protein, respectively. Among four DENV serotypes, DENV2 was the most effective for the inhibition. Our results provide the proof of concept for the development of therapeutic peptide inhibitors against DENV infection by the computer‐aided molecular design.     

SHP2及其变构抑制剂在肿瘤靶向治疗中的研究进展

含 Src 同源 2 结构域蛋白酪氨酸磷酸酶2（Src homology 2 region-containing protein tyrosine phosphatase 2, SHP2）是由PTPN11编码的胞内非受体蛋白酪氨酸磷酸酶,已被证实参与多种癌症的发生发展,主要通过RAS/MAPK、PI3K/AKT、JAK/STAT、PD-L1/PD-1等信号通路调控肿瘤细胞生长、增殖、侵袭、凋亡和免疫逃逸。自2016年诺华团队发现了SHP2的变构位点开始,对其抑制剂的研究已从催化位点抑制剂转向新型的变构位点抑制剂,克服了催化位点抑制剂高极性和特异性差的缺点,目前已有许多小分子化合物被报道,且具有成药潜力的SHP2变构抑制剂相继被开发进入临床试验阶段。本文将重点介绍近五年文献报道的SHP2靶点的结构与功能、在肿瘤靶向治疗中的价值及其变构抑制剂的研究进展。     

Overcoming vincristine resistance in cancer: Computational design and discovery of piperine‐inspired P‐glycoprotein inhibitors

P‐glycoprotein (P‐gp)/MDR‐1 plays a major role in the development of multidrug resistance (MDR) by pumping the chemotherapeutic drugs out of the cancer cells and reducing their efficacy. A number of P‐gp inhibitors were reported to reverse the MDR when co‐administered with chemotherapeutic drugs. Unfortunately, none has approved for clinical use due to toxicity issues. Some of the P‐gp inhibitors tested in the clinics are reported to have cross‐reactivity with CYP450 drug‐metabolizing enzymes, resulting in unpredictable pharmacokinetics and toxicity of co‐administered chemotherapeutic drugs. In this study, two piperine analogs ( 3 and 4 ) having lower cross‐reactivity with CYP3A4 drug‐metabolizing enzyme are identified as P‐glycoprotein (P‐gp) inhibitors through computational design, followed by synthesis and testing in MDR cancer cell lines over‐expressing P‐gp (KB Ch^R 8–5, SW480‐VCR, and HCT‐15). Both the analogs significantly increased the vincristine efficacy in MDR cancer cell lines at low micromole concentrations. Specifically, 3 caused complete reversal of vincristine resistance in KB Ch^R 8–5 cells and found to act as competitive inhibitor of P‐gp as well as potentiated the vincristine‐induced NF‐KB‐mediated apoptosis. Therefore, 3 ((2E,4E)‐1‐(6,7‐dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)‐5‐(4‐hydroxy‐3‐methoxyphenyl)penta‐2,4‐dien‐1‐one) can serve as a potential P‐gp inhibitor for in vivo investigations, to reverse multidrug resistance in cancer.     

Perspectives on the discovery of NOTCH2‐specific inhibitors

The Notch pathway is a cell‐cell communication system where membrane‐bound ligands interact with the extracellular region of Notch receptors to induce intracellular, downstream effects on gene expression. Aberrant Notch signaling promotes tumorigenesis, and the Notch pathway has tremendous potential for novel targeting strategies in cancer treatment. While γ‐secretase inhibitors as Notch‐inhibiting agents are already promising in clinical trials, they are highly non‐specific with adverse side‐effects. One of the underlying challenges is that two of the four known human Notch paralogs, NOTCH1 and 2, share very high structural similarity but play opposing roles in some tumorigenesis pathways. This perspective explores the feasibility of developing Notch‐specific small molecule inhibitors targeting the anti‐NOTCH2 antibody‐binding epitopes or the “S2‐Leu‐plug‐binding site” using a computer‐aided drug discovery approach.     

Targeting the Plasmodium falciparum plasmepsin V by ligand‐based virtual screening

Malaria is a devastating disease depending only on chemotherapy as treatment. However, medication is losing efficacy, and therefore, there is an urgent need for the discovery of novel pharmaceutics. Recently, plasmepsin V, an aspartic protease anchored in the endoplasmaic reticulum, was demonstrated as responsible for the trafficking of parasite‐derived proteins to the erythrocytic surface and further validated as a drug target. In this sense, ligand‐based virtual screening has been applied to design inhibitors that target plasmepsin V of P. falciparum (PMV). After screening 5.5 million compounds, four novel plasmepsin inhibitors have been identified which were subsequently analyzed for the potency at the cellular level. Since PMV is membrane‐anchored, the verification in vivo by using transgenic PMV overexpressing P. falciparum cells has been performed in order to evaluate drug efficacy. Two lead compounds, revealing IC₅₀ values were 44.2 and 19.1 μm , have been identified targeting plasmepsin V in vivo and do not significantly affect the cell viability of human cells up to 300 μm . We herein report the use of the consensus of individual virtual screening as a new technique to design new ligands, and we propose two new lead compounds as novel protease inhibitors to target malaria.     

Ensemble‐based ADME–Tox profiling and virtual screening for the discovery of new inhibitors of the Leishmania mexicana cysteine protease CPB2.8ΔCTE

In an effort to identify novel molecular warheads able to inhibit Leishmania mexicana cysteine protease CPB2.8ΔCTE, fused benzo[b]thiophenes and β,βʹ‐triketones emerged as covalent inhibitors binding the active site cysteine residue. Enzymatic screening showed a moderate‐to‐excellent activity (12%–90% inhibition of the target enzyme at 20 μm ). The most promising compounds were selected for further profiling including in vitro cell‐based assays and docking studies. Computational data suggest that benzo[b]thiophenes act immediately as non‐covalent inhibitors and then as irreversible covalent inhibitors, whereas a reversible covalent mechanism emerged for the 1,3,3ʹ‐triketones with a Y‐topology. Based on the predicted physicochemical and ADME–Tox properties, compound 2b has been identified as a new drug‐like, non‐mutagen, non‐carcinogen, and non‐neurotoxic lead candidate.     

DNA methyltransferase inhibitors: an updated patent review (2012-2015)

Introduction: DNA methyltransferases (DNMTs), important enzymes involved in epigenetic regulation of gene expression, represent promising targets in cancer therapy. DNMT inhibitors (DNMTi), which can modulate the aberrant DNA methylation pattern in a reversible way via inhibiting DNMT activity, have attracted significant attention in recent years.

Areas covered: This review outlines the newly patented inhibitors targeting DNMTs, mainly incorporating small molecular inhibitors and oligonucleotide derivatives. The chemical structures, biological activity, and the encouraging clinical research in progress are delineated in detail.

Expert opinion: Two drugs, azacitidine and decitabine, have evidently shown efficacy in hematologic malignancies, yet do not work well on solid tumors, have low specificity, substantial toxicity, and poor bioavailability. With the rapid advancement in systems biology, drug combinations, such as DNMTi, in conjugation with histone deacetylase inhibitors (HDACi) or immunotherapy, probably serve as an efficient way of implementing epigenetic therapy. Meanwhile, the resolved autoinhibitory structures of DNMTs afford a novel strategy for targeting the protein-protein interface involved in the autoinhi-bitory interactions. The molecular mechanism underlying the conformational transitions would also shed new light on the design of allosteric inhibitors. Both strategies would produce inhibitors with more selectivity compared to nucleotide derivatives.     

Discovery of selective protein arginine methyltransferase 5 inhibitors and biological evaluations

Protein arginine methyltransferase 5 (PRMT5) is an important protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine resides on histones or non‐histone substrate proteins. It has been thought as a promising target for many diseases, particularly cancer. Despite the potential applications of PRMT5 inhibitors in cancer treatment, very few of PRMT5i have been publicly reported. In this investigation, virtual screening and structure–activity relationship studies were carried out to discover novel PRMT5i, which finally led to the identification of a number of new PRMT5i. The most active compound, P5i‐6 , exhibited a considerable inhibitory potency against PRMT5 with an IC₅₀ value of 0.57 μm , and a high selectivity for PRMT5 against other tested PRMTs. It displayed a very good antiviability activity against two colorectal cancer cell lines, HT‐29 and DLD‐1, and one hepatic cancer cell line, HepG2, in a sensitivity assay against 36 different cancer cell lines. Western blot assays indicated that P5i‐6 selectively inhibited the symmetric dimethylations of H4R3 and H3R8 in DLD‐1 cells. Overall, P5i‐6 could be used as a chemical probe to investigate new functions of PRMT5 in biology and also served as a good lead compound for the development of new PRMT5‐targeting therapeutic agents.     

A patent review of arginine methyltransferase inhibitors (2010–2018)

Introduction: Protein arginine methyltransferases (PRMTs) are fundamental enzymes that specifically modify the arginine residues of versatile substrates in cells. The aberrant expression and abnormal enzymatic activity of PRMTs are associated with many human diseases, especially cancer. PRMTs are emerging as promising drug targets in both academia and industry.

Areas covered: This review summarizes the updated patented inhibitors targeting PRMTs from 2010 to 2018. The authors illustrate the chemical structures, molecular mechanism of action, pharmacological activities as well as the potential clinical application including combination therapy and biomarker-guided therapy. PRMT inhibitors in clinical trials are also highlighted. The authors provide a future perspective for further development of potent and selective PRMT inhibitors.

Expert opinion: Although a number of small molecule inhibitors of PRMTs with sufficient potency have been developed, the selectivity of most PRMT inhibitors remains to be improved. Hence, novel approaches such as allosteric regulation need to be further studied to identify PRMT inhibitors. So far, three PRMT inhibitors have entered clinical trials, including PRMT5 inhibitor GSK3326595 and JNJ-64619178 as well as PRMT1 inhibitor GSK3368715. PRMT inhibitors with novel mechanism of action and good drug-like properties may shed new light on drug research and development progress.