Discovery of ASP6918, a KRAS G12C inhibitor: Synthesis and structure-activity relationships of 1-{2,7-diazaspiro[3.5]non-2-yl}prop-2-en-1-one derivatives as covalent inhibitors with good potency and oral activity for the treatment of solid tumors.

Although KRAS protein had been classified as an undruggable target, inhibitors of KRAS G12C mutant protein were recently reported to show clinical efficacy in solid tumors. In our previous report, we identified 1-{2,7-diazaspiro[3.5]non-2-yl}prop-2-en-1-one derivative (1) as a KRAS G12C inhibitor that covalently binds to Cys12 of KRAS G12C protein. Compound 1 exhibited potent cellular pERK inhibition and cell growth inhibition against a KRAS G12C mutation-positive cell line and showed an antitumor effect on subcutaneous administration in an NCI-H1373 (KRAS G12C mutation-positive cell line) xenograft mouse model in a dose-dependent manner. In this report, we further optimized the substituents on the quinazoline scaffold based on the structure-based drug design from the co-crystal structure analysis of compound 1 and KRAS G12C to enhance in vitro activity. As a result, ASP6918 was found to exhibit extremely potent in vitro activity and induce dose-dependent tumor regression in an NCI-H1373 xenograft mouse model after oral administration.

Discovery of a Hidden Trypanosoma cruzi Spermidine Synthase Binding Site and Inhibitors through In Silico, In Vitro, and X-ray Crystallography.

In drug discovery research, the selection of promising binding sites and understanding the binding mode of compounds are crucial fundamental studies. The current understanding of the proteins-ligand binding model extends beyond the simple lock and key model to include the induced-fit model, which alters the conformation to match the shape of the ligand, and the pre-existing equilibrium model, selectively binding structures with high binding affinity from a diverse ensemble of proteins. Although methods for detecting target protein binding sites and virtual screening techniques using docking simulation are well-established, with numerous studies reported, they only consider a very limited number of structures in the diverse ensemble of proteins, as these methods are applied to a single structure. Molecular dynamics (MD) simulation is a method for predicting protein dynamics and can detect potential ensembles of protein binding sites and hidden sites unobservable in a single-point structure. In this study, to demonstrate the utility of virtual screening with protein dynamics, MD simulations were performed on Trypanosoma cruzi spermidine synthase to obtain an ensemble of dominant binding sites with a high probability of existence. The structure of the binding site obtained through MD simulation revealed pockets in addition to the active site that was present in the initial structure. Using the obtained binding site structures, virtual screening of 4.8 million compounds by docking simulation, in vitro assays, and X-ray analysis was conducted, successfully identifying two hit compounds.

Discovery and biological evaluation of 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one derivatives as covalent inhibitors of KRAS G12C with favorable metabolic stability and anti-tumor activity.

RAS protein plays a key role in cellular proliferation and differentiation. RAS gene mutation is a known driver of oncogenic alternation in human cancer. RAS inhibition is an effective therapeutic treatment for solid tumors, but RAS protein has been classified as an undruggable target. Recent reports have demonstrated that a covalent binder to KRAS protein at a mutated cysteine residue (G12C) is effective for the treatment of solid tumors. Here, we report a series of 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one derivatives as potent covalent inhibitors against KRAS G12C identified throughout structural optimization of an acryloyl amine moiety to improve in vitro inhibitory activity. From an X-ray complex structural analysis, the 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one moiety binds in the switch-II pocket of KRAS G12C. Further optimization of the lead compound (5c) led to the successful identification of 1-[7-[6-chloro-8-fluoro-7-(5-methyl-1H-indazol-4-yl)-2-[(1-methylpiperidin-4-yl)amino]quinazolin-4-yl]-2,7-diazaspiro[3.5]nonan-2-yl]prop-2-en-1-one (7b), a potent compound with high metabolic stabilities in human and mouse liver microsomes. Compound 7b showed a dose-dependent antitumor effect on subcutaneous administration in an NCI-H1373 xenograft mouse model.

Synthesis and structure-activity relationships of pyrimidine derivatives as potent and orally active FGFR3 inhibitors with both increased systemic exposure and enhanced in vitro potency.

Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of patients with bladder cancer harboring genetic alterations in FGFR3. We identified pyrimidine derivative 20b, which induced tumor regression following oral administration to a bladder cancer xenograft mouse model. Compound 20b was discovered by optimizing lead compound 1, which we reported previously. Specifically, reducing the molecular size of the substituent at the 4-position and replacing the linker of the 5-position in the pyrimidine scaffold resulted in an increase in systemic exposure. Furthermore, introduction of two fluorine atoms into the 3,5-dimethoxyphenyl ring enhanced FGFR3 inhibitory activity. Molecular dynamics (MD) simulation of 20b suggested that the fluorine atom interacts with the main chain NH moiety of Asp635 via a hydrogen bond.

Structure-based drug design of 1,3,5-triazine and pyrimidine derivatives as novel FGFR3 inhibitors with high selectivity over VEGFR2.

Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of bladder cancer. We identified 1,3,5-triazine derivative 18b and pyrimidine derivative 40a as novel structures with potent and highly selective FGFR3 inhibitory activity over vascular endothelial growth factor receptor 2 (VEGFR2) using a structure-based drug design (SBDD) approach. X-ray crystal structure analysis suggests that interactions between 18b and amino acid residues located in the solvent region (Lys476 and Met488), and between 40a and Met529 located in the back pocket of FGFR3 may underlie the potent FGFR3 inhibitory activity and high kinase selectivity over VEGFR2.

Effect of Fms-like tyrosine kinase 3 (FLT3) ligand (FL) on antitumor activity of gilteritinib, a FLT3 inhibitor, in mice xenografted with FL-overexpressing cells.

Therapeutic effects of FLT3 inhibitors have been reported in acute myeloid leukemia (AML) with constitutively activating FLT3 mutations, including internal tandem duplication (ITD) and point mutation, which are found in approximately one-third of AML patients. One of the critical issues of treatment with FLT3 inhibitors in FLT3-mutated AML is drug resistance. FLT3 ligand (FL) represents a mechanism of resistance to FLT3 inhibitors, including quizartinib, midostaurin, and sorafenib, in AML cells harboring both wild-type and mutant FLT3 (FLT3 wt/FLT3 mut). Here, we investigated the effect of FL on the efficacy of gilteritinib, a FLT3 inhibitor, in AML-derived cells in vitro and in mice. In contrast to other FLT3 inhibitors, FL stimulation had little effect on growth inhibition or apoptosis induction by gilteritinib. The antitumor activity of gilteritinib was also comparable between xenograft mouse models injected with FL-expressing and mock MOLM-13 cells. In the FLT3 signaling analyses, gilteritinib inhibited FLT3wt and FLT3-ITD to a similar degree in HEK293 and Ba/F3 cells, and similarly suppressed FLT3 downstream signaling molecules (including ERK1/2 and STAT5) in both the presence and absence of FL in MOLM-13 cells. Co-crystal structure analysis showed that gilteritinib bound to the ATP-binding pocket of FLT3. These results suggest that gilteritinib has therapeutic potential in FLT3-mutated AML patients with FL overexpression.

Synthesis, SAR study, and biological evaluation of novel 2,3-dihydro-1H-imidazo[1,2-a]benzimidazole derivatives as phosphodiesterase 10A inhibitors.

Phosphodiesterase 10A (PDE10A) inhibitors were designed and synthesized based on the dihydro-imidazobenzimidazole scaffold. Compound 5a showed moderate inhibitory activity and good permeability, but unfavorable high P-glycoprotein (P-gp) liability for brain penetration. We performed an optimization study to improve both the P-gp efflux ratio and PDE10A inhibitory activity. As a result, 6d was identified with improved P-gp liability and high PDE10A inhibitory activity. Compound 6d also showed satisfactory brain penetration, suppressed phencyclidine-induced hyperlocomotion and improved MK-801-induced working memory deficit.

Discovery and structural characterization of peficitinib (ASP015K) as a novel and potent JAK inhibitor.

Janus kinases (JAKs) are considered promising targets for the treatment of autoimmune diseases including rheumatoid arthritis (RA) due to their important role in multiple cytokine receptor signaling pathways. Recently, several JAK inhibitors have been developed for the treatment of RA. Here, we describe the identification of the novel orally bioavailable JAK inhibitor 18, peficitinib (also known as ASP015K), which showed moderate selectivity for JAK3 over JAK1, JAK2, and TYK2 in enzyme assays. Chemical modification at the C4-position of lead compound 5 led to a large increase in JAK inhibitory activity and metabolic stability in liver microsomes. Furthermore, we determined the crystal structures of JAK1, JAK2, JAK3, and TYK2 in a complex with peficitinib, and revealed that the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide scaffold of peficitinib forms triple hydrogen bonds with the hinge region. Interestingly, the binding modes of peficitinib in the ATP-binding pockets differed among JAK1, JAK2, JAK3, and TYK2. WaterMap analysis of the crystal structures suggests that unfavorable water molecules are the likely reason for the difference in orientation of the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide scaffold to the hinge region among JAKs.

Fragment-Based Discovery of Pyrimido[1,2-b]indazole PDE10A Inhibitors.

In this study, we report the identification of potent pyrimidoindazoles as phosphodiesterase10A (PDE10A) inhibitors by using the method of fragment-based drug discovery (FBDD). The pyrazolopyridine derivative 2 was found to be a fragment hit compound which could occupy a part of the binding site of PDE10A enzyme by using the method of the X-ray co-crystal structure analysis. On the basis of the crystal structure of compound 2 and PDE10A protein, a number of compounds were synthesized and evaluated, by means of structure-activity relationship (SAR) studies, which culminated in the discovery of a novel pyrimidoindazole derivative 13 having good physicochemical properties.

In silico, in vitro, X-ray crystallography, and integrated strategies for discovering spermidine synthase inhibitors for Chagas disease.

Chagas disease results from infection by Trypanosoma cruzi and is a neglected tropical disease (NTD). Although some treatment drugs are available, their use is associated with severe problems, including adverse effects and limited effectiveness during the chronic disease phase. To develop a novel anti-Chagas drug, we virtually screened 4.8 million small molecules against spermidine synthase (SpdSyn) as the target protein using our super computer "TSUBAME2.5" and conducted in vitro enzyme assays to determine the half-maximal inhibitory concentration values. We identified four hit compounds that inhibit T. cruzi SpdSyn (TcSpdSyn) by in silico and in vitro screening. We also determined the TcSpdSyn-hit compound complex structure using X-ray crystallography, which shows that the hit compound binds to the putrescine-binding site and interacts with Asp171 through a salt bridge.

Structural insights into the novel inhibition mechanism of Trypanosoma cruzi spermidine synthase.

Trypanosoma cruzi causes Chagas disease, a severe disease affecting 8-10 million people in Latin America. While nifurtimox and benznidazole are used to treat this disease, their efficacy is limited and adverse effects are observed. New therapeutic targets and novel drugs are therefore urgently required. Enzymes in the polyamine-trypanothione pathway are promising targets for the treatment of Chagas disease. Spermidine synthase is a key enzyme in this pathway that catalyzes the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. Fragment-based drug discovery was therefore conducted to identify novel, potent inhibitors of spermidine synthase from T. cruzi (TcSpdSyn). Here, crystal structures of TcSpdSyn in complex with dcSAM, trans-4-methylcyclohexylamine and hit compounds from fragment screening are reported. The structure of dcSAM complexed with TcSpdSyn indicates that dcSAM stabilizes the conformation of the `gatekeeping' loop to form the putrescine-binding pocket. The structures of fragments bound to TcSpdSyn revealed two fragment-binding sites: the putrescine-binding pocket and the dimer interface. The putrescine-binding pocket was extended by an induced-fit mechanism. The crystal structures indicate that the conformation of the dimer interface is required to stabilize the gatekeeping loop and that fragments binding to this interface inhibit TcSpdSyn by disrupting its conformation. These results suggest that utilizing the dynamic structural changes in TcSpdSyn that occur upon inhibitor binding will facilitate the development of more selective and potent inhibitors.

Addressing phototoxicity observed in a novel series of biaryl derivatives: discovery of potent, selective and orally active phosphodiesterase 10A inhibitor ASP9436.

We synthesized several biaryl derivatives as PDE10A inhibitors to prevent phototoxicity of 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (1) and found that the energy difference between the energy-minimized conformation and the coplanar conformation of the biaryl moiety helped facilitate prediction of the phototoxic potential of biaryl compounds. Replacement of the quinoline ring of 1 with N-methyl benzimidazole increased this energy difference and prevented phototoxicity in the 3T3 NRU test. Further optimization identified 1-methyl-5-(1-methyl-3-{[4-(1-methyl-1H-benzimidazol-4-yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one (38b). Compound 38b exhibited good selectivity against other PDEs, and oral administration of 38b improved visual-recognition memory deficit in mice at doses of 0.001 and 0.003mg/kg in the novel object recognition test. ASP9436 (sesquiphosphate of 38b) may therefore be used for the treatment of schizophrenia with a low risk of phototoxicity.

Identification of N-ethylmethylamine as a novel scaffold for inhibitors of soluble epoxide hydrolase by crystallographic fragment screening.

Soluble epoxide hydrolase (sEH) is a potential target for the treatment of inflammation and hypertension. X-ray crystallographic fragment screening was used to identify fragment hits and their binding modes. Eight fragment hits were identified via soaking of sEH crystals with fragment cocktails, and the co-crystal structures of these hits were determined via individual soaking. Based on the binding mode, N-ethylmethylamine was identified as a promising scaffold that forms hydrogen bonds with the catalytic residues of sEH, Asp335, Tyr383, and Tyr466. Compounds containing this scaffold were selected from an in-house chemical library and assayed. Although the starting fragment had a weak inhibitory activity (IC50: 800µM), we identified potent inhibitors including 2-({[2-(adamantan-1-yl)ethyl]amino}methyl)phenol exhibiting the highest inhibitory activity (IC50: 0.51µM). This corresponded to a more than 1500-fold increase in inhibitory activity compared to the starting fragment. Co-crystal structures of the hit compounds demonstrate that the binding of N-ethylmethylamine to catalytic residues is similar to that of the starting fragment. We therefore consider crystallographic fragment screening to be appropriate for the identification of weak but promising fragment hits.

Structures of complexes of type 5 17ß-hydroxysteroid dehydrogenase with structurally diverse inhibitors: insights into the conformational changes upon inhibitor binding.

Type 5 17ß-hydroxysteroid dehydrogenase (17ß-HSD5) is an aldo-keto reductase expressed in the human prostate which catalyzes the conversion of androstenedione to testosterone. Testosterone is converted to 5α-dihydrotestosterone, which is present at high concentrations in patients with castration-resistant prostate cancer (CRPC). Inhibition of 17ß-HSD5 is therefore considered to be a promising therapy for treating CRPC. In the present study, crystal structures of complexes of 17ß-HSD5 with structurally diverse inhibitors derived from high-throughput screening were determined. In the structures of the complexes, various functional groups, including amide, nitro, pyrazole and hydroxyl groups, form hydrogen bonds to the catalytic residues His117 and Tyr55. In addition, major conformational changes of 17ß-HSD5 were observed following the binding of the structurally diverse inhibitors. These results demonstrate interactions between 17ß-HSD5 and inhibitors at the atomic level and enable structure-based drug design for anti-CRPC therapy.

Synthesis, SAR study, and biological evaluation of novel quinoline derivatives as phosphodiesterase 10A inhibitors with reduced CYP3A4 inhibition.

A novel class of phosphodiesterase 10A inhibitors with potent PDE10A inhibitory activity and reduced CYP3A4 inhibition was designed and synthesized starting from 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (1). Replacement of pyridine ring of 1 with N-methyl pyridone ring drastically improved CYP3A4 inhibition, and further optimization of these quinoline analogues identified 1-methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one (42b), which showed potent PDE10A inhibitory activity and a good CYP3A4 inhibition profile. A PET study with (11)C-labeled 42b indicated that 42b exhibited good brain penetration and specifically accumulated in the rodent striatum. Further, oral administration of 42b dose-dependently attenuated phencyclidine-induced hyperlocomotion in mice with an ED50 value of 2.0mg/kg and improved visual-recognition memory impairment at 0.1 and 0.3mg/kg in mice novel object recognition test.

Novel benzimidazole derivatives as phosphodiesterase 10A (PDE10A) inhibitors with improved metabolic stability.

In this study, we report the identification of potent benzimidazoles as PDE10A inhibitors. We first identified imidazopyridine 1 as a high-throughput screening hit compound from an in-house library. Next, optimization of the imidazopyridine moiety to improve inhibitory activity gave imidazopyridinone 10b. Following further structure-activity relationship development by reducing lipophilicity and introducing substituents, we acquired 35, which exhibited both improved metabolic stability and reduced CYP3A4 time-dependent inhibition.

Structural insights into binding of inhibitors to soluble epoxide hydrolase gained by fragment screening and X-ray crystallography.

Soluble epoxide hydrolase (sEH) is a component of the arachidonic acid cascade and is a candidate target for therapies for hypertension or inflammation. Although many sEH inhibitors are available, their scaffolds are not structurally diverse, and knowledge of their specific interactions with sEH is limited. To obtain detailed structural information about protein-ligand interactions, we conducted fragment screening of sEH, analyzed the fragments using high-throughput X-ray crystallography, and determined 126 fragment-bound structures at high resolution. Aminothiazole and benzimidazole derivatives were identified as novel scaffolds that bind to the catalytic triad of sEH with good ligand efficiency. We further identified fragment hits that bound to subpockets of sEH called the short and long branches. The water molecule conserved in the structure plays an important role in binding to the long branch, whereas Asp496 and the main chain of Phe497 form hydrogen bonds with fragment hits in the short branch. Fragment hits and their crystal structures provide structural insights into ligand binding to sEH that will facilitate the discovery of novel and potent inhibitors of sEH.

Design and synthesis of novel benzimidazole derivatives as phosphodiesterase 10A inhibitors with reduced CYP1A2 inhibition.

A novel class of phosphodiesterase 10A (PDE10A) inhibitors with reduced CYP1A2 inhibition were designed and synthesized starting from 2-{[(1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}quinoline (1). Introduction of an isopropyl group at the 2-position and a methoxy group at the 5-position of the benzimidazole ring of lead compound 1 resulted in the identification of 2-{[(2-isopropyl-5-methoxy-1-phenyl-1H-benzimidazol-6-yl)oxy]methyl}quinoline (25b), which exhibited potent PDE10A inhibitory activity with reduced CYP1A2 inhibitory activity compared to compound 1.

Structural basis for telmisartan-mediated partial activation of PPAR gamma.

Telmisartan, a selective angiotensin II type 1 receptor blocker, has recently been shown to act as a partial agonist for peroxisome proliferator-activated receptor gamma (PPARγ). To understand how telmisartan partially activates PPARγ, we determined the ternary complex structure of PPARγ, telmisartan, and a coactivator peptide from steroid receptor coactivator-1 at a resolution of 2.18 Å. Crystallographic analysis revealed that telmisartan exhibits an unexpected binding mode in which the central benzimidazole ring is engaged in a non-canonical--and suboptimal--hydrogen-bonding network around helix 12 (H12). This network differs greatly from that observed when full-agonists bind with PPARγ and prompt high-coactivator recruitment through H12 stabilized by multiple hydrogen bonds. Binding with telmisartan results in a less stable H12 that in turn leads to attenuated coactivator binding, thus explaining the mechanism of partial activation.

Lead generation and examples opinion regarding how to follow up hits.

In fragment-based drug discovery (FBDD), not only identifying the starting fragment hit to be developed but also generating a drug lead from that starting fragment hit is important. Converting fragment hits to leads is generally similar to a high-throughput screening (HTS) hits-to-leads approach in that properties associated with activity for a target protein, such as selectivity against other targets and absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox), and physicochemical properties should be taken into account. However, enhancing the potency of the fragment hit is a key requirement in FBDD, unlike HTS, because initial fragment hits are generally weak. This enhancement is presently achieved by adding additional chemical groups which bind to additional parts of the target protein or by joining or combining two or more hit fragments; however, strategies for effecting greater improvements in effective activity are needed. X-ray analysis is a key technology attractive for converting fragments to drug leads. This method makes it clear whether a fragment hit can act as an anchor and provides insight regarding introduction of functional groups to improve fragment activity. Data on follow-up chemical synthesis of fragment hits has allowed for the differentiation of four different strategies: fragment optimization, fragment linking, fragment self-assembly, and fragment evolution. Here, we discuss our opinion regarding how to follow up on fragment hits, with a focus on the importance of fragment hits as an anchor moiety to so-called hot spots in the target protein using crystallographic data.