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Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays an important role in one-carbon metabolism. The MTHFD2 gene is upregulated in various cancers but very low or undetectable in normal proliferating cells, and therefore a potential target for cancer treatment. In this study, we present the structure of MTHFD2 in complex with xanthine derivative 15, which allosterically binds to MTHFD2 and coexists with the substrate analogue. A kinetic study demonstrated the uncompetitive inhibition of MTHFD2 by 15. Allosteric inhibitors often provide good selectivity and, indeed, xanthine derivatives are highly selective for MTHFD2. Moreover, several conformational changes were observed upon the binding of 15, which impeded the binding of the cofactor and phosphate to MTHFD2. To the best of our knowledge, this is the first study to identify allosteric inhibitors targeting the MTHFD family and our results would provide insights on the inhibition mechanism of MTHFD proteins and the development of novel inhibitors.

Indoleamine 2,3-dioxygenase (IDO1) inhibitors are speculated to be useful in cancer immunotherapy, but a phase III clinical trial of the most advanced IDO1 inhibitor, epacadostat, did not meet its primary end point and was abandoned. In previous work, we identified the novel IDO1 inhibitor

Gastrointestinal stromal tumors (GISTs) are prototypes of stem cell factor receptor (c-KIT)-driven cancer. Two receptor tyrosine kinases, c-KIT and fms-tyrosine kinase (FLT3), are frequently mutated in acute myeloid leukemia (AML) patients, and these mutations are associated with poor prognosis. In this study, we discovered a multitargeted tyrosine kinase inhibitor, compound 15a, with potent inhibition against single or double mutations of c-KIT developed in GISTs. Moreover, crystal structure analysis revealed the unique binding mode of 15a with c-KIT and may elucidate its high potency in inhibiting c-KIT kinase activity. Compound 15a inhibited cell proliferation and induced apoptosis by targeting c-KIT in c-KIT-mutant GIST cell lines. The antitumor effects of 15a were also demonstrated in GIST430 and GIST patient-derived xenograft models. Further studies demonstrated that 15a inhibited the proliferation of c-KIT- and FLT3-driven AML cells in vitro and in vivo. The results of this study suggest that 15a may be a potential anticancer drug for the treatment of GISTs and AML.

Drug resistance due to acquired mutations that constitutively activate c-KIT is a significant challenge in the treatment of patients with gastrointestinal stromal tumors (GISTs). Herein, we identified 1-(5-ethyl-isoxazol-3-yl)-3-(4-{2-[6-(4-ethylpiperazin-1-yl)pyrimidin-4-ylamino]-thiazol-5-yl}phenyl)urea (10a) as a potent inhibitor against unactivated and activated c-KIT. The binding of 10a induced rearrangements of the DFG motif, αC-helix, juxtamembrane domain, and the activation loop to switch the activated c-KIT back to its structurally inactive state. To the best of our knowledge, it is the first structural evidence demonstrating how a compound can inhibit the activated c-KIT by switching back to its inactive state through a sequence of conformational changes. Moreover, 10a can effectively inhibit various c-KIT mutants and the proliferation of several GIST cell lines. The distinct binding features and superior inhibitory potency of 10a, together with its excellent efficacy in the xenograft model, establish 10a as worthy of further clinical evaluation in the advanced GISTs.

Epidermal growth factor receptor (EGFR)-targeted therapy in non-small cell lung cancer represents a breakthrough in the field of precision medicine. Previously, we have identified a lead compound, furanopyrimidine 2, which contains a (S)-2-phenylglycinol structure as a key fragment to inhibit EGFR. However, compound 2 showed high clearance and poor oral bioavailability in its pharmacokinetics studies. In this work, we optimized compound 2 by scaffold hopping and exploiting the potent inhibitory activity of various warhead groups to obtain a clinical candidate, 78 (DBPR112), which not only displayed a potent inhibitory activity against EGFRL858R/T790M double mutations but also exhibited tenfold potency better than the third-generation inhibitor, osimertinib, against EGFR and HER2 exon 20 insertion mutations. Overall, pharmacokinetic improvement through lead-to-candidate optimization yielded fourfold oral AUC better that afatinib along with F = 41.5%, an encouraging safety profile, and significant antitumor efficacy in in vivo xenograft models. DBPR112 is currently undergoing phase 1 clinical trial in Taiwan.

A structure-based virtual screening strategy, comprising homology modeling, ligand-support binding site optimization, virtual screening, and structure clustering analysis, was developed and used to identify novel tryptophan 2,3-dioxygenase (TDO) inhibitors. Compound 1 (IC50 = 711 nM), selected by virtual screening, showed inhibitory activity toward TDO and was subjected to structural modifications and molecular docking studies. This resulted in the identification of a potent TDO selective inhibitor (11e, IC50 = 30 nM), making it a potential compound for further investigation as a cancer therapeutic and other TDO-related targeted therapy.

Preliminary biological data on 7-anilino-6-azaindoles (8-11) suggested that hydrophobic substituents at C7 contribute to enhancement of antiproliferative activity. A novel series of 7-aryl-6-azaindole-1-benzenesulfonamides (12-22) were developed and showed improved cytotoxicity compared to ABT751 (5). The conversion of C7 phenyl rings into C7 heterocycles led to a remarkable improvement of antiproliferative activity. Among all the synthetic products, 7-(2-furanyl)-1-(4-methoxybenzenesulfonyl)-6-azaindole (21) exhibited the most potent anticancer activity against KB, HT29, MKN45, and H460 cancer cell lines with IC50 values of 21.1, 32.0, 27.5, and 40.0 nM, respectively. Bioassays indicated that 21 not only inhibits tubulin polymerization by binding to tubulin at the colchicine binding site but also arrests the cell cycle at the G2/M phase with slight arrest at the sub-G1 phase. Compound 21 also functions as a vascular disrupting agent and dose-dependently inhibits tumor growth without significant change of body weight in an HT29 xenograft mouse model. Taken together, compound 21 has potential for further development as a novel class of anticancer agents.

Most anaplastic lymphoma kinase (ALK) inhibitors adopt a type I binding mode, but only limited type II ALK structural studies are available. Herein, we present the structure of ALK in complex with N1-(3-4-[([5-(tert-butyl)-3-isoxazolyl]aminocarbonyl)amino]-3-methylphenyl-1H-5-pyrazolyl)-4-[(4-methylpiperazino)methyl]benzamide (5a), a novel ALK inhibitor adopting a type II binding mode. It revealed binding of 5a resulted in the conformational change and reposition of the activation loop, αC-helix, and juxtamembrane domain, which are all important domains for the autoinhibition mechanism and downstream signal pathway regulation of ALK. A structure-activity relationship study revealed that modifications to the structure of 5a led to significant differences in the ALK potency and altered the protein structure of ALK. To the best of our knowledge, this is the first structural biology study to directly observe how changes in the structure of a small molecule can regulate the switch between the type I and type II binding modes and induce dramatic conformational changes.

Tryptophan metabolism has been recognized as an important mechanism in immune tolerance. Indoleamine 2,3-dioxygenase plays a key role in local tryptophan metabolism via the kynurenine pathway and has emerged as a therapeutic target for cancer immunotherapy. Our prior study identified phenyl benzenesulfonyl hydrazide 2 as a potent in vitro (though not in vivo) inhibitor of indoleamine 2,3-dioxygenase. Further lead optimization to improve in vitro potencies and pharmacokinetic profiles resulted in N′-(4-bromophenyl)-2-oxo-2,3-dihydro-1H-indole-5-sulfonyl hydrazide 40, which demonstrated 59% oral bioavailability and 73% of tumor growth delay without apparent body weight loss in the murine CT26 syngeneic model, after oral administration of 400 mg/kg. Accordingly, 40, is proposed as a potential drug lead worthy of advanced preclinical evaluation.

Indoleamine 2,3-dioxygenase 1 (IDO1), promoting immune escape of tumors, is a therapeutic target for the cancer immunotherapy. A number of IDO1 inhibitors have been identified, but only limited structural biology studies of IDO1 inhibitors are available to provide insights on the binding mechanism of IDO1. In this study, we present the structure of IDO1 in complex with 24, a NLG919 analogue with potent activity. The complex structure revealed the imidazole nitrogen atom of 24 to coordinate with the heme iron, and the imidazoleisoindole core situated in pocket A with the 1-cyclohexylethanol moiety extended to pocket B to interact with the surrounding residues. Most interestingly, 24 formed an extensive hydrogen bond network with IDO1, which is a distinct feature of IDO1/24 complex structure and is not observed in the other IDO1 complex structures. Further structure-activity relationship, UV spectra, and structural biology studies of several analogues of 24 demonstrated that extensive hydrophobic interactions and the unique hydrogen bonding network contribute to the great potency of imidazoleisoindole derivatives. These results are expected to facilitate the structure-based drug design of new IDO inhibitors.

Utilizing scaffold-hopping drug-design strategy, we sought to identify a backup drug candidate for BPR0L075 (1), an indole-based anticancer agent. For this purpose, 5,6-fused bicyclic heteroaromatic scaffolds were designed and synthesized through shuffling of the nitrogen from the N-1 position or by insertion of one or two nitrogen atoms into the indole core of 1. Among these, 7-azaindole core 12 showed potent in vitro anticancer activity and improved oral bioavailability (F = 35%) compared with 1 (F < 10%).

A series of aroylquinoline derivatives were synthesized and evaluated for anticancer activity. 5-Amino-6-methoxy-2-aroylquinoline 15 showed more potent antiproliferative activity (IC(50) values ranging from 0.2 to 0.4 nM) as compared to 1a (combretastatin A-4) (IC(50) = 1.9-835 nM) against various human cancer cell lines and a MDR-resistant cancer cell line. Compound 15 (IC(50) = 1.6 microM) exhibited more potent inhibition of tubulin polymerization than 1a (IC(50) = 2.1 microM) and showed strong binding property to the colchicine binding site of microtubules.

A pharmacophore model, Hypo1, was built on the basis of 21 training-set indole compounds with varying levels of antiproliferative activity. Hypo1 possessed important chemical features required for the inhibitors and demonstrated good predictive ability for biological activity, with high correlation coefficients of 0.96 and 0.89 for the training-set and test-set compounds, respectively. Further utilization of the Hypo1 pharmacophore model to screen chemical database in silico led to the identification of four compounds with antiproliferative activity. Among these four compounds, 43 showed potent antiproliferative activity against various cancer cell lines with the strongest inhibition on the proliferation of KB cells (IC(50) = 187 nM). Further biological characterization revealed that 43 effectively inhibited tubulin polymerization and significantly induced cell cycle arrest in G(2)-M phase. In addition, 43 also showed the in vivo-like anticancer effects. To our knowledge, 43 is the most potent antiproliferative compound with antitubulin activity discovered by computer-aided drug design. The chemical novelty of 43 and its anticancer activities make this compound worthy of further lead optimization.

Utilizing medicinal chemistry design strategies such as benzo splitting and ring expansion, we converted PPARalpha/gamma dual agonist 1 to selective PPARgamma agonists 19 and 20. Compounds 19 and 20 were 2- to 4-fold better than rosiglitazone at PPARgamma receptor, with 80- to 100-fold PPARgamma selectivity over PPARalpha receptor. X-ray cocrystal studies in PPARgamma and modeling studies in PPARalpha give molecular insights for the improved PPARgamma potency and selectivity for 19 when compared to 1.

Aurora kinases have emerged as attractive targets for the design of anticancer drugs. Through structure-based virtual screening, novel pyrazole hit 8a was identified as Aurora kinase A inhibitor (IC(50) = 15.1 microM). X-ray cocrystal structure of 8a in complex with Aurora A protein revealed the C-4 position ethyl carboxylate side chain as a possible modification site for improving the potency. On the basis of this insight, bioisosteric replacement of the ester with amide linkage and changing the ethyl substituent to hydrophobic 3-acetamidophenyl ring led to the identification of 12w with a approximately 450-fold improved Aurora kinase A inhibition potency (IC(50) = 33 nM), compared to 8a. Compound 12w showed selective inhibition of Aurora A kinase over Aurora B/C, which might be due to the presence of a unique H-bond interaction between the 3-acetamido group and the Aurora A nonconserved Thr217 residue, which in Aurora B/C is Glu and found to sterically clash with the 3-acetamido group in modeling studies.

A series of novel indole-based PPAR agonists is described leading to discovery of 10k, a highly potent PPAR pan-agonist. The structural biology and molecular docking studies revealed that the distances between the acidic group and the linker, when a ligand was complexed with PPARgamma protein, were important for the potent activity. The hydrophobic tail part of 10k makes intensive hydrophobic interaction with the PPARgamma protein resulting in potent activity.

Dipeptidyl peptidase IV (DPP-IV) inhibitors are expected to become a new type of antidiabetic drugs. Most known DPP-IV inhibitors often resemble the dipeptide cleavage products, with a proline mimic at the P1 site. As off-target inhibitions of DPP8 and/or DPP9 have shown profound toxicities in the in vivo studies, it is important to develop selective DPP-IV inhibitors for clinical usage. To achieve this, a new class of 2-[3-[[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethyl]amino]-1-oxopropyl]-based DPP-IV inhibitors was synthesized. SAR studies resulted in a number of DPP-IV inhibitors, having IC(50) values of50 nM with excellent selectivity over both DPP8 (IC(50)100 microM) and DPP-II (IC(50)30 microM). Compound 21a suppressed the blood glucose elevation after an oral glucose challenge in Wistar rats and also inhibited plasma DPP-IV activity for up to 4 h in BALB/c mice. The results show that compound 21a possesses in vitro and in vivo activities comparable to those of NVP-LAF237 (4), which is in clinical development.

The synthesis and structure-activity relationship studies of novel indole derivatives as peroxisome proliferator-activated receptor (PPAR) agonists are reported. Indole, a drug-like scaffold, was studied as a core skeleton for the acidic head part of PPAR agonists. The structural features (acidic head, substitution on indole, and linker) were optimized first, by keeping benzisoxazole as the tail part, based on binding and functional activity at PPARgamma protein. The variations in the tail part, by introducing various heteroaromatic ring systems, were then studied. In vitro evaluation led to identification of a novel series of indole compounds with a benzisoxazole tail as potent PPAR agonists with the lead compound 14 (BPR1H036) displaying an excellent pharmacokinetic profile in BALB/c mice and an efficacious glucose lowering activity in KKA(y) mice. Structural biology studies of 14 showed that the indole ring contributes strong hydrophobic interactions with PPARgamma and could be an important moiety for the binding to the protein.

The Asp-Phe-Gly (DFG) motif plays an important role in the regulation of kinase activity. Structure-based drug design was performed to design compounds able to interact with the DFG motif; epidermal growth factor receptor (EGFR) was selected as an example. Structural insights obtained from the EGFR/2a complex suggested that an extension from the meta-position on the phenyl group (ring-5) would improve interactions with the DFG motif. Indeed, introduction of an N,N-dimethylamino tail resulted in 4b, which showed almost 50-fold improvement in inhibition compared to 2a. Structural studies confirmed this N,N-dimethylamino tail moved toward the DFG motif to form a salt bridge with the side chain of Asp831. That the interactions with the DFG motif greatly contribute to the potency of 4b is strongly evidenced by synthesizing and testing compounds 2a, 3g, and 4f: when the charge interactions are absent, the inhibitory activity decreased significantly.

A ligand-based virtual screening strategy (a combination of pharmacophore model generation, shape-based scoring, and structure clustering analysis) was developed to discover novel SGLT2 inhibitors. The best pharmacophore model, generated from eight glycoside inhibitors, was utilized to virtually screen three chemical databases that led to the identification of three non-glycoside SGLT2 inhibitors. This is the first report of the generation of a pharmacophore model from glycosides that has then been used to discover novel non-glycosides hits.

A focused library of furanopyrimidine (350 compounds) was rapidly synthesized in parallel reactors and in situ screened for Aurora and epidermal growth factor receptor (EGFR) kinase activity, leading to the identification of some interesting hits. On the basis of structural biology observations, the hit 1a was modified to better fit the back pocket, producing the potent Aurora inhibitor 3 with submicromolar antiproliferative activity in HCT-116 colon cancer cell line. On the basis of docking studies with EGFR hit 1s, introduction of acrylamide Michael acceptor group led to 8, which inhibited both the wild and mutant EGFR kinase and also showed antiproliferative activity in HCC827 lung cancer cell line. Furthermore, the X-ray cocrystal study of 3 and 8 in complex with Aurora and EGFR, respectively, confirmed their hypothesized binding modes. Library construction, in situ screening, and structure-based drug design (SBDD) strategy described here could be applied for the lead identification of other kinases.

Shiraiachrome-A and -B have been isolated from the mycelium of the Chinese bamboo fungus Shiraia bambusicola as the cytotoxic principles. A series of new perylene derivatives (7-27) related to Shiraiachrome-A and -B as well as Calphostin-C have been synthesized and evaluated for their cytotoxicity, antiviral activity, and inhibitory activity against protein kinase C. The results indicated that 11 and 12 are potent cytotoxic agents against HCT-8, RPMI-7951, and TE-671 solid tumor cells, whereas 24 and 26 demonstrated strong antiviral activity against HSV-1 and HSV-2. Compound 10 is an inhibitor of protein kinase C.

BPR0L075 (2) is a potential anticancer drug candidate designed from Combretastatin A-4 (1) based on the bioisosterism principle. Metabolites of 2, proposed from in vitro human microsome studies, were synthesized, leading to the identification of metabolite-derived analogue 10 with 40-350 pM potency against various cancer cell lines. Insights gained from the major inactive metabolite of 2 led to the development of 29, with better pharmacokinetics and improved potency in the tumor xenograft model than 2.

A series of aroylnaphthalene derivatives were prepared as bioisosteres of combrestatin A-4 and evaluated for anticancer activity. 2-Amino-1-aroylnaphthalene and 2-hydroxy-1-aroylnaphthalene, 9 and 8, respectively, showed strong antiproliferative activity with IC(50) values of 2.1-26.3 nM against a panel of human cancer cell lines including multiple-drug resistant cell line. Compound 9 demonstrated better antiproliferative activity and has a comparable tubulin binding efficacy as that of colchicine.

A series of C-4 hydroxylated and halogenated anilino derivatives of epipodophyllotoxin or 4'-demethylepipodophyllotoxin have been synthesized and evaluated for their inhibitory activity against the human DNA topoisomerase II as well as for their activity in causing cellular protein-linked DNA breakage. Compounds 11-17 and 22 are more potent than etoposide in causing DNA breakage, while compounds 11-13, 15, 16, and 20 are as active or more active than etoposide in their inhibition of the human DNA topoisomerase II. The cytotoxicity in KB cells appears to have no direct correlation with their ability to inhibit DNA topoisomerase II and to cause protein-linked DNA breaks in cells.

Type 2 diabetes has rapidly reached an epidemic proportion becoming a major threat to global public health. PPAR agonists have emerged as a leading class of oral antidiabetic drugs. We report a structure biology analysis of novel indole-based PPAR agonists to explain the structure-activity relationships and present a critical analysis of reasons for change in selectivity with change in the orientation of the same scaffolds. The results would be helpful in designing novel PPAR agonists.

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3C-like protease (3CL(pro) or M(pro)) is an attractive target for the development of anti-SARS drugs because of its crucial role in the viral life cycle. In this study, a compound database was screened by the structure-based virtual screening approach to identify initial hits as inhibitors of SARS-CoV 3CL(pro). Out of the 59,363 compounds docked, 93 were selected for the inhibition assay, and 21 showed inhibition against SARS-CoV 3CL(pro) (IC(50)

Peroxisome proliferator-activated receptor gamma (PPARgamma) is well-known as the receptor of thiazolidinedione antidiabetic drugs. In this paper, we present a successful example of employing structure-based virtual screening, a method that combines shape-based database search with a docking study and analogue search, to discover a novel family of PPARgamma agonists based upon pyrazol-5-ylbenzenesulfonamide. Two analogues in the family show high affinity for, and specificity to, PPARgamma and act as partial agonists. They also demonstrate glucose-lowering efficacy in vivo. A structural biology study reveals that they both adopt a distinct binding mode and have no H-bonding interactions with PPARgamma. The absence of H-bonding interaction with the protein provides an explanation why both function as partial agonists since most full agonists form conserved H-bonds with the activation function helix (AF-2 helix) which, in turn, enhances the recruitment of coactivators. Moreover, the structural biology and computer docking studies reveal the specificity of the compounds for PPARgamma could be due to the restricted access to the binding pocket of other PPAR subtypes, i.e., PPARalpha and PPARdelta, and steric hindrance upon the ligand binding.

Following the identification through virtual screening of 4-(2,4-dimethyl-thiazol-5-yl)pyrimidin-2-ylamines as moderately potent inhibitors of cyclin-dependent kinase-2 (CDK2), a CDK inhibitor analogue program was initiated. The first aims were to optimize potency and to evaluate the cellular mode of action of lead candidate molecules. Here the synthetic chemistry, the structure-guided design approach, and the structure-activity relationships (SARs) that led to the discovery of 2-anilino-4-(thiazol-5-yl)pyrimidine ATP-antagonistic CDK2 inhibitors, many with very low nM K(i)s against CDK2, are reported. Furthermore, X-ray crystal structures of four representative analogues from our chemical series in complex with CDK2 are presented, and these structures are used to rationalize the observed biochemical SARs. Finally results are reported that show, using the most potent CDK2 inhibitor compound from the current series, that the observed antiproliferative and proapoptotic effects are consistent with cellular CDK2 and CDK9 inhibition.