Discovery and structure-activity-relationship study of novel imidazole cyclopropyl amine analogues for mutant isocitric dehydrogenase 1 (IDH1) inhibitors

The discovery and optimization of imidazole cyclopropyl amime analogues as mutant IDH1 inhibitors via structure-based rational design were reported. The optimal compounds demonstrated both potent inhibition in IDH1^R132H enzymatic activity and 2HG production in IDH1 mutant HT1080 cell line, moderate liver microsome stability and PK properties.     

Synthesis and evaluation of radiolabeled AGI-5198 analogues as candidate radiotracers for imaging mutant IDH1 expression in tumors

Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are commonly found in gliomas. AGI-5198, a potent and selective inhibitor of the mutant IDH1 enzyme, was radiolabeled with radioiodine and fluorine-18. These radiotracers were evaluated as potential probes for imaging mutant IDH1 expression in tumors with positron emission tomography (PET). Radioiodination of AGI-5198 was achieved using a tin precursor in 79?±?6% yield (n?=?9), and ¹⁸F-labeling was accomplished by the Ugi reaction in a decay-corrected radiochemical yield of 2.6?±?1.6% (n?=?5). The inhibitory potency of the analogous nonradioactive compounds against mutant IDH1 (IDH1-R132H) was determined in enzymatic assays. Cell uptake studies using radiolabeled AGI-5198 analogues revealed somewhat higher uptake in IDH1-mutated cells than that in wild-type IDH1 cells. The radiolabeled compounds displayed favorable tissue distribution characteristics in vivo, and good initial uptake in IDH1-mutated tumor xenografts; however, tumor uptake decreased with time. Radioiodinated AGI-5198 exhibited higher tumor-to-background ratios compared with ¹⁸F-labeled AGI-5198; unfortunately, similar results were observed in wild-type IDH1 tumor xenografts as well, indicating lack of selectivity for mutant IDH1 for this tracer. These results suggest that AGI-5198 analogues are not a promising platform for radiotracer development. Nonetheless, insights gained from this study may help in design and optimization of novel chemical scaffolds for developing radiotracers for imaging the mutant IDH1 enzyme.     

Design and synthesis of cyclopropane-based conformationally restricted GABA analogues as selective inhibitors for betaine/GABA transporter 1

We previously designed and synthesized a series of cyclopropane-based conformationally restricted analogues of γ-aminobutyric acid (GABA). The study demonstrated that the critical conformation of the analogues that selectively active to betaine/GABA transporter 1 (BGT1) subtype is the trans-syn-form, in which the amino and carboxyl groups are in trans-configuration and the cyclopropane ring and the carboxyl group are in syn-arrangement. In this study, we designed and synthesized cyclopropane-based GABA analogues, which were conformationally restricted in the trans-syn-form by cyclopropylic strain based on the stereochemistry of the carbon adjacent to cyclopropane. Their conformation was confirmed as the syn-form by calculations and NMR studies, and their pharmacological evaluation clarified that compounds 11a and 11d had the BGT1 selectivity, although their inhibitory effects were insufficient.     

Discovery of DC_H31 as potential mutant IDH1 inhibitor through NADPH-based high throughput screening

IDH1 mutations are early events in the development of IDH-mutant gliomas and leukemias and are associated with various regulation of molecular process. Mutations of active site in IDH1 could lead to high levels of 2-HG and the suppression of cellular differentiation, while these changes can be reversed by molecule inhibitors target mutant IDH1. Here, through in-house developed enzymatic assay-based high throughput screening platform, we discovered DC_H31 as a novel IDH1-R132H/C inhibitor, with the IC₅₀ value of 0.41 μmol/L and 2.7 μmol/L respectively. In addition, saturable SPR binding assay indicated that DC_H31 bound to IDH1-R132H/C due to specific interaction. Further computational docking studies and structure-activity relationship (SAR) suggest that DC_H31 could occupy the allosteric pocket between the two monomers of IDH1-R132H homodimer, which accounts for its inhibitory ability. And it is possible to conclude that DC_H31 acts via an allosteric mechanism of inhibition. At the cellular level, DC_H31 could inhibit cell proliferation, promote cell differentiation and reduce the production of 2-HG with a dose-dependent manner in HT1080 cells. Taken together, DC_H31 is a potent selective inhibitor of IDH1-R132H/C both in vitro and in vivo, which can promote the development of more potent pan-inhibitors against IDH1-R132H/C through further structural decoration and provide a new insight for the pharmacological treatment of gliomas.     

Licochalcone A suppresses the proliferation of sarcoma HT-1080 cells,as a selective R132C mutant IDH1 inhibitor

IDH1 mutations are closely related to the development and progression of various human cancers, such as glioblastoma, sarcoma, and acute myeloid leukemia. By screening dozens of reported natural compounds using both wild-type and mutant IDH1 enzymatic assays, we discovered Licochalcone A is a selective inhibitor to the R132C-mutant IDH1 with an IC₅₀ value of 5.176 μM, and inhibits the proliferation of sarcoma HT-1080 cells with an IC₅₀ value of 10.75 μM. Suggested by the molecular docking results, Licochalcone A might occupy the allosteric pocket between the two monomers of IDH1 homodimer, and the R132H mutation was unfavorable for the binding of Licochalcone A with the IDH1 protein, as compared to the R132C mutation. Revealed by the RNA-Seq data analysis, the Cell Cycle pathway was the most over-represented pathway for HT-1080 cells treated with Licochalcone A. Consistent with these results, Licochalcone A induced apoptosis and cell cycle arrest of HT-1080 cells, while it showed minimal effect against the proliferation of normal RCTEC cells. The discovery of Licochalcone A as a mutation-selective IDH1 inhibitor can serve as a promising starting point for the development of mutation-selective anti-tumor lead compounds targeting IDH1.     

On the structure-based design of novel inhibitors of H5N1 influenza A virus neuraminidase (NA)

The structure-based design of novel H5N1 neuraminidase inhibitors is currently a research topic of vital importance owing to both a recent pandemic threat by the worldwide spread of H5N1 avian influenza and the high resistance of H5N1 virus to the most widely used commercial drug, oseltamivir-OTV (Tamiflu). A specific criterion used in this work for determining fully acceptable conformations of potential inhibitors is a previous experimental proposal of exploiting potential benefits for drug design offered by the ‘150-cavity’ adjacent to the NA active site. Using the crystal structure of H5N1 NA (PDB ID: 2hty) as the starting point, in a set of 54 inhibitors previously proposed by modifying the side chains of oseltamivir, 4 inhibitors were identified using two different computational strategies (ArgusLab4.0.1, FlexX-E3.0.1) both to lower the binding free energy (BFE) of oseltamivir and to have partially acceptable conformations. These 4 oseltamivr structure-based analogues were found to adopt the most promising conformations by identifying the guanidinium side chain of Arg156 as a prospective partner for making polar contacts, but none of the modified 4-amino groups of oseltamivir in the 4 favorable conformations was found to make polar contacts with the guanidinium side chain of Arg156. Hence, the structures of two additional inhibitors were designed and shown to further lower the binding free energy of OTV relative to the previous 54 inhibitors. These two novel structures clearly suggest that it may be possible for a new substituent to be developed by functional modifications at position of the 4-amino group of oseltamivir in order to make polar contacts with the guanidinium side chain of Arg156, and thereby enhance the binding of a more potent inhibitor. Several standpoints of vital importance for designing novel structures of potentially more effective H5N1 NA inhibitors are established.     

Identification of novel allosteric inhibitors of mutant isocitrate dehydrogenase 1 by cross docking-based virtual screening

IDH1 mutation (mIDH1) occurs in 20–30% of gliomas and is a promising target for the cancer therapy. In this article, a cross docking-based virtual screening was employed to identify seven small molecules for the allosteric site of mIDH1. Compounds ZX01, ZX05 and ZX06 exhibited the potent inhibitory activity and the high selectivity against WT-IDH1, providing a good starting point for the further development of highly selective mIDH1 inhibitors. Importantly, the parallel artificial membrane permeation assay of the blood-brain barrier (PAMPA-BBB) identified ZX06 with a good ability to penetrate BBB. These findings indicate that ZX06 deserves further optimization as a lead compound for the treatment of patients with IDH1 mutated brain cancers.     

Structure-based design and SAR development of 5,6-dihydroimidazolo[1,5-f]pteridine derivatives as novel Polo-like kinase-1 inhibitors

Using structure-based drug design, we identified a novel series of 5,6-dihydroimidazolo[1,5-f]pteridine PLK1 inhibitors. Rational improvements to compounds of this class resulted in single-digit nanomolar enzyme and cellular activity against PLK1, and oral bioavailability. Compound 1 exhibits >7 fold induction of phosphorylated Histone H3 and is efficacious in an in vivo HT-29 tumor xenograft model.     

Discovery of the 1,7-diazacarbazole class of inhibitors of checkpoint kinase 1

Checkpoint kinase 1 (ChK1) is activated in response to DNA damage, acting to temporarily block cell cycle progression and allow for DNA repair. It is envisaged that inhibition of ChK1 will sensitize tumor cells to treatment with DNA-damaging therapies, and may enhance the therapeutic window. High throughput screening identified carboxylate-containing diarylpyrazines as a prominent hit series, but with limited biochemical potency and no cellular activity. Through a series of SAR investigations and X-ray crystallographic analysis the critical role of polar contacts with conserved waters in the kinase back pocket was established. Structure-based design, guided by in silico modeling, transformed the series to better satisfy these contacts and the novel 1,7-diazacarbazole class of inhibitors was discovered. Here we present the genesis of this novel series and the identification of GNE-783, a potent, selective and orally bioavailable inhibitor of ChK1.     

Discovery of 5-(methylthio)pyrimidine derivatives as L858R/T790M mutant selective epidermal growth factor receptor (EGFR) inhibitors

To overcome the drug-resistance of first generation EGFR inhibitors and the nonselective toxicities of second generation inhibitors among NSCLC patients, a series of 5-(methylthio)pyrimidine derivatives were discovered as novel EGFR inhibitors, which harbored not only potent enzymatic and antiproliferative activities against EGFR^L858R/T790M mutants, but good selectivity over wide-type form of the receptor. This goal was achieved by employing structure-based drug design and traditional optimization strategies, based on WZ4002 and CO1686. These derivatives inhibited the enzymatic activity of EGFR^L858R/T790M mutants with IC₅₀ values in subnanomolar ranges, while exhibiting hundreds of fold less potency on EGFR^WT. These compounds also strongly inhibited the proliferation of H1975 non-small cell lung cancer cells bearing EGFR^L858R/T790M, while being significantly less toxic to A431 human epithelial carcinoma cells with overexpressed EGFR^WT. The EGFR kinase inhibitory and antiproliferative activities were further validated by Western blot analysis for activation of EGFR and the downstream signaling in cancer cells.     

Identification and functional characterization of isocitrate dehydrogenase 1 (IDH1) mutations in thyroid cancer

Mutations in the genes for isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) have been recently identified in glioblastoma. In the present study, we investigated IDH1 and IDH2 mutations in follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC), with the latter, like glioblastoma, having a rapidly aggressive and lethal clinical course. By direct genomic DNA sequencing, we analyzed exon 4 of the IDH1 and IDH2 genes that harbored the mutation hot spots codon 132 and 172 of the two genes in glioblastoma, respectively, in 12 thyroid cancer cell lines, 20 FTC, and 18 ATC tumor samples. A novel homozygous G367A IDH1 mutation, resulting in a G123R amino acid change in codon 123, was identified in a case of ATC. A previously described IDH1 V71I mutation was found in a case of FTC and a case of ATC and no mutations were found in the cell lines. The overall prevalence of mutations was thus 1/20 (5%) in FTC and 2/18 (11%) in ATC. We did not find mutation in the IDH2 gene in these thyroid cancer cell lines and tumor samples. Sequence alignment analysis of 16 species revealed that the novel IDH1 G123R mutation was located in a highly conserved region, raising the possibility of a serious functional consequence as could also be predicted by the occurrence of a positively charged amino acid from this mutation. To test this, we created a G123R mutant by site-directed mutagenesis and demonstrated a decreased enzymatic activity of IDH1, similar to the expected reduction in the enzymatic activity of the previously described R132H IDH1 mutant measured as a control. Thus, functionally relevant IDH1 mutations can also occur in thyroid cancer, particularly ATC, suggesting a potential tumorigenic role of the IDH1 system that could represent a new therapeutic target for thyroid cancer.     

Discovery of novel benzo[b][1,4]oxazin-3(4H)-ones as poly(ADP-ribose)polymerase inhibitors

Structure based drug design of a series of novel 1,4-benzoxazin-3-one derived PARP-1 inhibitors are described. The synthesis, enzymatic & cellular activities and pharmacodynamic effects are described. Optimized analogs demonstrated inhibition of poly-ADP-ribosylation in SW620 tumor bearing nude mice through 24 h following a single dose.     

Design and synthesis of 2-amino-6-(1H,3H-benzo[de]isochromen-6-yl)-1,3,5-triazines as novel Hsp90 inhibitors

A novel series of 2-amino-1,3,5-triazines bearing a tricyclic moiety as heat shock protein 90 (Hsp90) inhibitors is described. Molecular design was performed using X-ray cocrystal structures of the lead compound CH5015765 and natural Hsp90 inhibitor geldanamycin with Hsp90. We optimized affinity to Hsp90, in vitro cell growth inhibitory activity, water solubility, and liver microsomal stability of inhibitors and identified CH5138303. This compound showed high binding affinity for N-terminal Hsp90α (K_d = 0.52 nM) and strong in vitro cell growth inhibition against human cancer cell lines (HCT116 IC₅₀ = 0.098 μM, NCI-N87 IC₅₀ = 0.066 μM) and also displayed high oral bioavailability in mice (F = 44.0%) and potent antitumor efficacy in a human NCI-N87 gastric cancer xenograft model (tumor growth inhibition = 136%).     

Structure-based design and synthesis of novel furan-diketopiperazine-type derivatives as potent microtubule inhibitors for treating cancer

Plinabulin, a synthetic analog of the marine natural product “diketopiperazine phenylahistin,” displayed depolymerization effects on microtubules and targeted the colchicine site, which has been moved into phase III clinical trials for the treatment of non-small cell lung cancer (NSCLC) and the prevention of chemotherapy-induced neutropenia (CIN). To develop more potent anti-microtubule and cytotoxic derivatives, the co-crystal complexes of plinabulin derivatives were summarized and analyzed. We performed further modifications of the tert-butyl moiety or C-ring of imidazole-type derivatives to build a library of molecules through the introduction of different groups for novel skeletons. Our structure–activity relationship study indicated that compounds 17o (IC₅₀ = 14.0 nM, NCI-H460) and 17p (IC₅₀ = 2.9 nM, NCI-H460) with furan groups exhibited potent cytotoxic activities at the nanomolar level against various human cancer cell lines. In particular, the 5-methyl or methoxymethyl substituent of furan group could replace the alkyl group of imidazole at the 5-position to maintain cytotoxic activity, contradicting previous reports that the tert-butyl moiety at the 5-position of imidazole was essential for the activity of such compounds. Immunofluorescence assay indicated that compounds 17o and 17p could efficiently inhibit microtubule polymerization. Overall, the novel furan-diketopiperazine-type derivatives could be considered as a potential scaffold for the development of anti-cancer drugs.     

Discovery of novel quinoline carboxylic acid series as DGAT1 inhibitors

Herein we report the design and synthesis of a series of novel bicyclic DGAT1 inhibitors with a carboxylic acid moiety. The optimization of the initial lead compound 7 based on in vitro and in vivo activity led to the discovery of potent indoline and quinoline classes of DGAT1 inhibitors. The structure–activity relationship studies of these novel series of bicyclic carboxylic acid derivatives as DGAT1 inhibitors are described.     

Fragment-based screening of programmed death ligand 1 (PD-L1)

The PD-1 immune checkpoint pathway is a highly validated target for cancer immunotherapy. Despite the potential advantages of small molecule inhibitors over antibodies, the discovery of small molecule checkpoint inhibitors has lagged behind. To discover small molecule inhibitors of the PD-1 pathway, we have utilized a fragment-based approach. Small molecules were identified that bind to PD-L1 and crystal structures of these compounds bound to PD-L1 were obtained.     

Structure-based design of novel HCV NS5B thumb pocket 2 allosteric inhibitors with submicromolar gt1 replicon potency: Discovery of a quinazolinone chemotype

We describe the structure-based design of a novel lead chemotype that binds to thumb pocket 2 of HCV NS5B polymerase and inhibits cell-based gt1 subgenomic reporter replicons at sub-micromolar concentrations (EC₅₀ <200 nM). This new class of potent thumb pocket 2 inhibitors features a 1H-quinazolin-4-one scaffold derived from hybridization of a previously reported, low affinity thiazolone chemotype with our recently described anthranilic acid series. Guided by X-ray structural information, a key NS5B–ligand interaction involving the carboxylate group of anthranilic acid based inhibitors was replaced by a neutral two-point hydrogen bonding interaction between the quinazolinone scaffold and the protein backbone. The in vitro ADME and in vivo rat PK profile of representative analogs are also presented and provide areas for future optimization of this new class of HCV polymerase inhibitors.     

Design and synthesis of novel 2-arylbenzimidazoles as selective mutant isocitrate dehydrogenase 2 R140Q inhibitors

A series of novel 2-arylbenzimidazoles have been designed, synthesized and evaluated for their inhibitory activity against IDH2 R140Q mutant. The preliminary results indicated that four compounds 7b, 7c, 7m and 7r displayed the potent inhibitory activity against IDH2 R140Q mutant. Among them, compound 7c showed the highest inhibitory activity, with the IC₅₀ value of 0.26 μM, which was more active than positive control enasidenib. The exquisite selectivity of 7c for IDH2 R140Q mutant isoform was demonstrated by the poor activity against the IDH1 R132C mutant, IDH1 R132H mutant, wild-type IDH1, IDH2 R172K mutant and the wild-type IDH2.     

Discovery of 2-arylquinazoline derivatives as a new class of ASK1 inhibitors

The development of a new series of apoptosis signal-regulating kinase 1 (ASK1) inhibitors is described. Starting from purine, pyrimidine and quinazoline scaffolds identified by high throughput screening, we used tools of structure-based drug design to develop a series of potent kinase inhibitors, including 2-arylquinazoline derivatives 12 and 23, with submicromolar inhibitory activities against ASK1. Kinetic analysis demonstrated that the 2-arylquinazoline scaffold ASK1 inhibitors described herein are ATP competitive.