CryoEM Structure with ATP Synthase Enables Late-Stage Diversification of Cruentaren†A.

Cruentaren A is a natural product that exhibits potent antiproliferative activity against various cancer cell lines, yet its binding site within ATP synthase remained unknown, thus limiting the development of improved analogues as anticancer agents. Herein, we report the cryogenic electron microscopy (cryoEM) structure of cruentaren A bound to ATP synthase, which allowed the design of new inhibitors through semisynthetic modification. Examples of cruentaren A derivatives include a trans-alkene isomer, which was found to exhibit similar activity to cruentaren A against three cancer cell lines as well as several other analogues that retained potent inhibitory activity. Together, these studies provide a foundation for the generation of cruentaren A derivatives as potential therapeutics for the treatment of cancer.

Manassantin A inhibits tumour growth under hypoxia through the activation of chaperone-mediated autophagy by modulating Hsp90 activity.

BACKGROUND: Chaperon-mediated autophagy (CMA) has taken on a new emphasis in cancer biology. However, the roles of CMA in hypoxic tumours are poorly understood. We investigated the anti-tumour effects of the natural product ManA through the activation of CMA in tumour progression under hypoxia. METHODS: The effect of ManA on CMA activation was assessed in mouse xenograft models and cells. The gene expressions of HIF-1α, HSP90AA1, and transcription factor EB (TFEB) were analysed using The Cancer Genome Atlas (TCGA) datasets to assess the clinical relevance of CMA. RESULTS: ManA activates photoswitchable CMA reporter activity and inhibits Hsp90 chaperone function by disrupting the Hsp90/F1F0-ATP synthase complex. Hsp90 inhibition enhances the interaction between CMA substrates and LAMP-2A and TFEB nuclear localisation, suggesting CMA activation by ManA. ManA-activated CMA retards tumour growth and displays cooperative anti-tumour activity with anti-PD-1 antibody. TCGA datasets show that a combined expression of HSP90AA1High/HIF1AHigh or TFEBLow/HIF1AHigh is strongly correlated with poor prognosis in patients with lung cancer. CONCLUSIONS: ManA-induced CMA activation by modulating Hsp90 under hypoxia induces HIF-1α degradation and reduces tumour growth. Thus, inducing CMA activity by targeting Hsp90 may be a promising therapeutic strategy against hypoxic tumours.

Synthesis and Evaluation of Simplified Cruentaren A Analogues.

The 90 kDa heat shock protein (Hsp90) belongs to a group of molecular chaperones that regulate homeostasis via the folding of nascent polypeptides into their biologically active proteins, many of which are involved in cancer development and progression. As a result, inhibition of Hsp90 is an exciting area of research for the treatment of cancer. However, most of the 18 Hsp90 N-terminal inhibitors evaluated in clinical trials exhibited deleterious side effects and toxicities. Cruentaren A is a natural product that manifests potent anticancer activity against various human cancer cell lines via disruption of interactions between Hsp90α and F1FO ATP synthase, which does not induce the pro-survival, heat shock response, a major limitation associated with current Hsp90 inhibitors. However, the development of cruentaren A as a new anticancer agent has been hindered by its complex structure. Herein, we systematically removed the functionalities present in fragment 2 of cruentaren A and incorporated some key structural modifications from previous work, which produced 12 simplified analogues. Our studies determined that all functional groups present in fragment 2 are essential for cruentaren A's anticancer activity.

Recent development in the discovery of PARP inhibitors as anticancer agents: a patent update (2016-2020).

INTRODUCTION: Discovery of small molecules that impede the activity of single-strand DNA repair enzyme, PARP1, has led to four marketed drugs for the treatment of advanced-stage cancers. Hence, there is a renewed enthusiasm in the PARP inhibitor discovery arena. To reduce nonspecific interactions or potential toxicities, and to understand the role of other minimally explored PARP enzymes, exciting new findings have emerged toward the development of selective inhibitors and targeted chemical biology probes. Importantly, the conventional PARP inhibitor design has evolved in a way that could potentially lead to multienzyme-targeting - a polypharmacological approach against aggressive cancers. AREAS COVERED: This review comprises recent progress made in the development of PARP inhibitors, primarily focused on human cancers. Discovery of novel PARP inhibitors with pan, selective, and multi-target inhibition using in vitro and in vivo cancer models is summarized and critically evaluated. Emphasis is given to patents published during 2016-2020, excluding TNKS 1/2 inhibitors. EXPERT OPINION: The outstanding success demonstrated by the FDA approved PARP inhibitors has fueled further clinical evaluations for expansion of their clinical utilities. The current clinical investigations include new candidates as well as marketed PARP-targeted drugs, both as single agents and in combination with other chemotherapeutics. Recent advances have also unveiled critical roles of other PARPs in oncogenic signal transduction, in addition to those of the well-documented PARP1/2 and TNKS1/2 enzymes. Further studies on lesser-known PARP members are urgently needed for functional annotations and for understanding their roles in cancer progression and other human diseases.

Natural products and other inhibitors of F1FO ATP synthase.

F1FO ATP synthase is responsible for the production of >95% of all ATP synthesis within the cell. Dysregulation of its expression, activity or localization is linked to various human diseases including cancer, diabetes, and Alzheimer's and Parkinson's disease. In addition, ATP synthase is a novel and viable drug target for the development of antimicrobials as evidenced by bedaquiline, which was approved in 2012 for the treatment of tuberculosis. Historically, natural products have been a rich source of ATP synthase inhibitors that help unravel the role of F1FO ATP synthase in cellular bioenergetics. During the last decade, new modulators of ATP synthase have been discovered through the isolation of novel natural products as well as through a ligand-based drug design process. In addition, new data has been obtained with regards to the structure and function of ATP synthase under physiological and pathological conditions. Crystal structure studies have provided a significant insight into the rotary function of the enzyme and may provide additional opportunities to design a new generation of inhibitors. This review provides an update on recently discovered ATP synthase modulators as well as an update on existing scaffolds.

Design and Synthesis of Poly(ADP-ribose) Polymerase Inhibitors: Impact of Adenosine Pocket-Binding Motif Appendage to the 3-Oxo-2,3-dihydrobenzofuran-7-carboxamide on Potency and Selectivity.

Poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors are a class of anticancer drugs that block the catalytic activity of PARP proteins. Optimization of our lead compound 1 (( Z)-2-benzylidene-3-oxo-2,3-dihydrobenzofuran-7-carboxamide; PARP-1 IC50 = 434 nM) led to a tetrazolyl analogue (51, IC50 = 35 nM) with improved inhibition. Isosteric replacement of the tetrazole ring with a carboxyl group (60, IC50 = 68 nM) gave a promising new lead, which was subsequently optimized to obtain analogues with potent PARP-1 IC50 values (4-197 nM). PARP enzyme profiling revealed that the majority of compounds are selective toward PARP-2 with IC50 values comparable to clinical inhibitors. X-ray crystal structures of the key inhibitors bound to PARP-1 illustrated the mode of interaction with analogue appendages extending toward the PARP-1 adenosine-binding pocket. Compound 81, an isoform-selective PARP-1/-2 (IC50 = 30 nM/2 nM) inhibitor, demonstrated selective cytotoxic effect toward breast cancer gene 1 ( BRCA1)-deficient cells compared to isogenic BRCA1-proficient cells.

Comprehensive Synthesis of Amino Acid-Derived Thiazole Peptidomimetic Analogues to Understand the Enigmatic Drug/Substrate-Binding Site of P-Glycoprotein.

A novel set of 64 analogues based on our lead compound 1 was designed and synthesized with an initial objective of understanding the structural requirements of ligands binding to a highly perplexing substrate-binding site of P-glycoprotein (P-gp) and their effect on modulating the ATPase function of the efflux pump. Compound 1, a stimulator of P-gp ATPase activity, was transformed to ATPase inhibitory compounds 39, 53, and 109. The ATPase inhibition by these compounds was predominantly contributed by the presence of a cyclohexyl group in lieu of the 2-aminobenzophenone moiety of 1. The 4,4-difluorocyclohexyl analogues, 53 and 109, inhibited the photolabeling by [125I]-IAAP, with IC50 values of 0.1 and 0.76 µM, respectively. Selected compounds were shown to reverse paclitaxel resistance in HEK293 cells overexpressing P-gp and were selective toward P-gp over CYP3A4. Induced-fit docking highlighted a plausible binding pattern of inhibitory compounds in the putative-binding pocket of P-gp. The current study underscores the stringent requirement by P-gp to bind to chemically similar molecules.

The BTK Inhibitor Ibrutinib (PCI-32765) Overcomes Paclitaxel Resistance in ABCB1- and ABCC10-Overexpressing Cells and Tumors.

Paclitaxel is one of the most widely used antineoplastic drugs in the clinic. Unfortunately, the occurrence of cellular resistance has limited its efficacy and application. The ATP-binding cassette subfamily B member 1 (ABCB1/P-glycoprotein) and subfamily C member 10 (ABCC10/MRP7) are the major membrane protein transporters responsible for the efflux of paclitaxel, constituting one of the most important mechanisms of paclitaxel resistance. Here, we demonstrated that the Bruton tyrosine kinase inhibitor, ibrutinib, significantly enhanced the antitumor activity of paclitaxel by antagonizing the efflux function of ABCB1 and ABCC10 in cells overexpressing these transporters. Furthermore, we demonstrated that the ABCB1 or ABCC10 protein expression was not altered after treatment with ibrutinib for up to 72 hours using Western blot analysis. However, the ATPase activity of ABCB1 was significantly stimulated by treatment with ibrutinib. Molecular docking analysis suggested the binding conformation of ibrutinib within the large cavity of the transmembrane region of ABCB1. Importantly, ibrutinib could effectively enhance paclitaxel-induced inhibition on the growth of ABCB1- and ABCC10-overexpressing tumors in nude athymic mice. These results demonstrate that the combination of ibrutinib and paclitaxel can effectively antagonize ABCB1- or ABCC10-mediated paclitaxel resistance that could be of great clinical interest. Mol Cancer Ther; 16(6); 1021-30. ©2017 AACR.

Thiazole-valine peptidomimetic (TTT-28) antagonizes multidrug resistance in vitro and in vivo by selectively inhibiting the efflux activity of ABCB1.

Multidrug resistance (MDR) attenuates the chemotherapy efficacy and increases the probability of cancer recurrence. The accelerated drug efflux mediated by ATP-binding cassette (ABC) transporters is one of the major MDR mechanisms. This study investigated if TTT-28, a newly synthesized thiazole-valine peptidomimetic, could reverse ABCB1-mediated MDR in vitro and in vivo. TTT-28 reversed the ABCB1-mediated MDR and increased the accumulation of [3H]-paclitaxel in ABCB1 overexpressing cells by selectively blocking the efflux function of ABCB1, but not interfering with the expression level and localization of ABCB1. Animal study revealed that TTT-28 enhanced the intratumoral concentration of paclitaxel and promoted apoptosis, thereby potently inhibiting the growth of ABCB1 overexpressing tumors. But TTT-28 did not induce the toxicity (cardiotoxicity/myelosuppression) of paclitaxel in mice. In this study, we synthesized and evaluated a novel selective inhibitor of ABCB1 (TTT-28) with high efficacy and low toxicity. The identification and characterization of this new thiazole-valine peptidomimetic will facilitate design and synthesis of a new generation of ABCB1 inhibitors, leading to further research on multidrug resistance and combination chemotherapy. Furthermore, the strategy that co-administer MDR-ABCB1 inhibitor to overcome the resistance of one FDA approved, widely used chemotherapeutic paclitaxel, may be promising direction for the field of adjuvant chemotherapy.

In Vitro Antibacterial Activity of Rhodanine Derivatives against Pathogenic Clinical Isolates.

Bacterial infections present a serious challenge to healthcare practitioners due to the emergence of resistance to numerous conventional antibacterial drugs. Therefore, new bacterial targets and new antimicrobials are unmet medical needs. Rhodanine derivatives have been shown to possess potent antimicrobial activity via a novel mechanism. However, their potential use as antibacterials has not been fully examined. In this study, we determined the spectrum of activity of seven rhodanine derivatives (compounds Rh 1-7) against clinical isolates of Gram-positive and Gram-negative bacterial strains and Candida albicans. We also synthesized and tested three additional compounds, ethyl ester and amide of rhodanine 2 (Rh 8 and Rh 10, respectively) and ethyl ester of rhodanine 3 (Rh 9) to determine the significance of the carboxyl group modification towards antibacterial activity and human serum albumin binding. A broth microdilution assay confirmed Rh 1-7 exhibit bactericidal activity against Gram-positive pathogens. Rh 2 had significant activity against various vancomycin-resistant (MIC90 = 4 µM) and methicillin-resistant (MIC90 = 4 µM) Staphylococcus aureus (VRSA and MRSA), Staphylococcus epidermidis (MIC = 4 µM) and vancomycin-resistant Enterococcus (VRE) strains (MIC90 = 8 µM). The rhodanine compounds exhibited potent activity against Bacillus spp., including Bacillus anthracis, with MIC range of 2-8 µM. In addition, they had potent activity against Clostridium difficile. The most potent compound, Rh 2, at 4 and 8 times its MIC, significantly decreased S. epidermidis biofilm mass by more than 35% and 45%, respectively. None of the rhodanine compounds showed antimicrobial activity (MIC > 128 µM) against various 1) Gram-negative pathogens (Acinetobacter baumannii, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, and Salmonella Typhimurium) or 2) strains of Candida albicans (MIC > 64 µM). The MTS assay confirmed that rhodanines were not toxic to mouse murine macrophage (J774.1A) up to 64 µM, human keratinocytes (HaCat) up to 32 µM, and human ileocecal colorectal cell (HRT-18) up to 128 µM. Overall, these data suggest that certain rhodanine compounds may have potential use for the treatment of several multidrug-resistant Gram-positive bacterial infections.

Bafetinib (INNO-406) reverses multidrug resistance by inhibiting the efflux function of ABCB1 and ABCG2 transporters.

ATP-Binding Cassette transporters are involved in the efflux of xenobiotic compounds and are responsible for decreasing drug accumulation in multidrug resistant (MDR) cells. Discovered by structure-based virtual screening algorithms, bafetinib, a Bcr-Abl/Lyn tyrosine kinase inhibitor, was found to have inhibitory effects on both ABCB1- and ABCG2-mediated MDR in this in-vitro investigation. Bafetinib significantly sensitized ABCB1 and ABCG2 overexpressing MDR cells to their anticancer substrates and increased the intracellular accumulation of anticancer drugs, particularly doxorubicin and [(3)H]-paclitaxel in ABCB1 overexpressing cells; mitoxantrone and [(3)H]-mitoxantrone in ABCG2 overexpressing cells, respectively. Bafetinib stimulated ABCB1 ATPase activities while inhibited ABCG2 ATPase activities. There were no significant changes in the expression level or the subcellular distribution of ABCB1 and ABCG2 in the cells exposed to 3 µM of bafetinib. Overall, our study indicated that bafetinib reversed ABCB1- and ABCG2-mediated MDR by blocking the drug efflux function of these transporters. These findings might be useful in developing combination therapy for MDR cancer treatment.

Dual inhibition of Staphylococcus aureus DNA gyrase and topoisomerase IV activity by phenylalanine-derived (Z)-5-arylmethylidene rhodanines.

Methicillin resistant Staphylococcus aureus (MRSA) is a major drug resistant bacteria that persists in both community and clinical settings due to growing resistance to current drug regimens. Thus, there is a continued need for novel compounds that are active against this organism. Previously, we reported that various rhodanine derivatives inhibited the supercoiling activity of DNA gyrase. In this study, we determined the effect of new phenylalanine-derived (Z)-5-arylmethylidene rhodanines (which are efficacious against MRSA) on the activity of the two type II bacterial topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Compounds 1 and 9 showed the greatest efficacy against DNA gyrase with a minimal inhibitory concentration (MIC) of 5 µM while compounds 2 and 3 were the most efficacious against Topo IV with MIC values of 0.75 µM and 0.5 µM, respectively. Induced fit docking, using the crystallographic structures of the target enzymes, indicated that these rhodanine derivatives bind to the ATPase domain of gyrB and ParE subunits on DNA gyrase and Topo IV, respectively. These new compounds were efficacious against both DNA gyrase and Topo IV. The increased efficacy of these new rhodanine compounds, as compared to other rhodanine derivatives, results from their dual inhibition of DNA gyrase and Topo IV, thereby making them good candidates for further drug design and development.

Semi-synthetic ocotillol analogues as selective ABCB1-mediated drug resistance reversal agents.

Overexpression of ATP-Binding Cassette transporters leads to multidrug resistance in cancer cells and results in the failure of chemotherapy. In this in-vitro study, we investigated whether or not (20S, 24R/S)-epoxy-12ß, 25-dihydroxy-dommarane-3ß-amine (ORA and OSA), a pair of semi-synthetic ocotillol analogue epimers, could inhibit the ABCB1 transporter. ORA (1 µM and 3 µM) significantly reversed the resistance to paclitaxel and vincristine in ABCB1-overexpressing SW620/Ad300 and HEK/ABCB1 cells, whereas OSA had no significant effects. In addition, ORA (3 µM) significantly increased the intracellular accumulation of [3H]-paclitaxel by suppressing the efflux function of ABCB1. Meanwhile, both ORA (3 µM) and OSA (3 µM) did not significantly alter the expression level or the subcellular location of ABCB1 protein. Moreover, the ABCB1 ATPase study suggested that ORA had a stronger stimulatory effect on the ATPase activity than OSA. ORA also exhibited a higher docking score as compared with OSA inside transmembrane domain of ABCB1. Overall, we concluded that ORA reverse ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function.

Novel 4-thiazolidinones as non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase.

In continuation of our efforts to develop new derivatives as hepatitis C virus (HCV) NS5B inhibitors, we synthesized novel 5-arylidene-4-thiazolidinones. The novel compounds 29-42, together with their synthetic precursors 22-28, were tested for HCV NS5B inhibitory activity; 12 of these compounds displayed IC50 values between 25.3 and 54.1 µM. Compound 33, an arylidene derivative, was found to be the most active compound in this series with an IC50 value of 25.3 µM. Molecular docking studies were performed on the thumb pocket-II of NS5B to postulate the binding mode for these compounds.

Design and synthesis of human ABCB1 (P-glycoprotein) inhibitors by peptide coupling of diverse chemical scaffolds on carboxyl and amino termini of (S)-valine-derived thiazole amino acid.

P-glycoprotein (P-gp) serves as a therapeutic target for the development of multidrug resistance reversal agents. In this study, we synthesized 21 novel compounds by peptide coupling at corresponding carboxyl and amino termini of (S)-valine-based bis-thiazole and monothiazole derivatives with diverse chemical scaffolds. Using calcein-AM efflux assay, we identified compound 28 (IC50 = 1.0 µM) carrying 3,4,5-trimethoxybenzoyl and 2-aminobenzophenone groups, respectively, at the amino and carboxyl termini of the monothiazole zwitter-ion. Compound 28 inhibited the photolabeling of P-gp with [(125)I]-iodoarylazidoprazosin with IC50 = 0.75 µM and stimulated the basal ATP hydrolysis of P-gp in a concentration-dependent manner (EC50 ATPase = 0.027 µM). Compound 28 at 3 µM reduced resistance in cytotoxicity assay to paclitaxel in P-gp-expressing SW620/Ad300 and HEK/ABCB1 cell lines. Biochemical and docking studies showed site-1 to be the preferable binding site for 28 within the drug-binding pocket of human P-gp.

Design, synthesis, and biological evaluation of (S)-valine thiazole-derived cyclic and noncyclic peptidomimetic oligomers as modulators of human P-glycoprotein (ABCB1).

Multidrug resistance caused by ATP binding cassette transporter P-glycoprotein (P-gp) through extrusion of anticancer drugs from the cells is a major cause of failure in cancer chemotherapy. Previously, selenazole-containing cyclic peptides were reported as P-gp inhibitors and were also used for co-crystallization with mouse P-gp, which has 87 % homology to human P-gp. It has been reported that human P-gp can simultaneously accommodate two to three moderately sized molecules at the drug binding pocket. Our in silico analysis, based on the homology model of human P-gp, spurred our efforts to investigate the optimal size of (S)-valine-derived thiazole units that can be accommodated at the drug binding pocket. Towards this goal, we synthesized varying lengths of linear and cyclic derivatives of (S)-valine-derived thiazole units to investigate the optimal size, lipophilicity, and structural form (linear or cyclic) of valine-derived thiazole peptides that can be accommodated in the P-gp binding pocket and affects its activity, previously an unexplored concept. Among these oligomers, lipophilic linear (13) and cyclic trimer (17) derivatives of QZ59S-SSS were found to be the most and equally potent inhibitors of human P-gp (IC50 =1.5 µM). As the cyclic trimer and linear trimer compounds are equipotent, future studies should focus on noncyclic counterparts of cyclic peptides maintaining linear trimer length. A binding model of the linear trimer 13 within the drug binding site on the homology model of human P-gp represents an opportunity for future optimization, specifically replacing valine and thiazole groups in the noncyclic form.

The synthesis and SAR study of phenylalanine-derived (Z)-5-arylmethylidene rhodanines as anti-methicillin-resistant Staphylococcus aureus (MRSA) compounds.

A focused library of rhodanine compounds containing novel substituents at the C5-position was synthesized and tested in vitro against a panel of clinically relevant MRSA strains. The present SAR study was based on our lead compound 1 (MIC=1.95 µg/mL), with a focus on identifying optimal C5-arylidene substituents. In order to obtain this objective, we condensed several unique aromatic aldehydes with phenylalanine-derived rhodanine intermediates to obtain C5-substituted target rhodanine compounds for evaluation as anti-MRSA compounds. These efforts produced three compounds with significant efficacy: 23, 32 and 44, with MIC values ranging from 0.98 to 1.95 µg/mL against all tested MRSA strains as compared to the reference antibiotics penicillin G (MIC=15.60-250.0 µg/mL) and ciprofloxacin (MIC=7.80-62.50 µg/mL) and comparable to that of vancomycin (MIC=0.48 µg/mL). In addition, compounds 24, 28, 37, 41, 46 and 48 (MIC=1.95-3.90 µg/mL) were efficacious against all MRSA strains. The majority of the synthesized compounds had bactericidal activity at concentrations only two to fourfold higher than their MIC. Overall, the results suggest that compounds 23, 32 and 44 may be of potential use in the treatment of MRSA infections.

Design and synthesis of L- and D-phenylalanine derived rhodanines with novel C5-arylidenes as inhibitors of HCV NS5B polymerase.

Hepatitis C virus (HCV) NS5B polymerase is a key target for anti-HCV therapeutics development. Herein, we report the synthesis and in vitro evaluation of anti-NS5B polymerase activity of a molecular hybrid of our previously reported lead compounds 1 (IC50=7.7 µM) and 2 (IC50=10.6 µM) as represented by hybrid compound 27 (IC50=6.7 µM). We have explored the optimal substituents on the terminal phenyl ring of the 3-phenoxybenzylidene moiety in 27, by generating a set of six analogs. This resulted in the identification of compound 34 with an IC50 of 2.6 µM. To probe the role of stereochemistry towards the observed biological activity, we synthesized and evaluated the D-isomers 41 (IC50=19.3 µM) and 45 (IC50=5.4 µM) as enantiomers of the l-isomers 27 and 34, respectively. The binding site of compounds 32 and 34 was mapped to palm pocket-I (PP-I) of NS5B. The docking models of 34 and 45 within the PP-I of NS5B were investigated to envisage the molecular mechanism of inhibition.