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PI3Ks are frequently hyper-activated in breast cancer and targeting PI3Kα has exhibited promising but variable response in preclinical and clinical settings. CYH33 is a novel PI3Kα-selective inhibitor in phase I clinical trial. We investigated the efficacy of CYH33 against breast cancer and explored potential predictive biomarkers. CYH33 potently restrained tumor growth in mice bearing human breast cancer cell xenografts and in R26-Pik3caH1047R;MMTV-Cre transgenic mice. CYH33 significantly inhibited proliferation of a panel of human breast cancer cells, while diversity in sensitivity has been observed. Cells harboring activating PIK3CA mutation, amplified HER2 were more responsive to CYH33 than their counterparts. Besides, cells in HER2-enriched or luminal subtype were more sensitive to CYH33 than basal-like breast cancer. Sensitivity to CYH33 has been further revealed to be associated with induction of G1 phase arrest and simultaneous inhibition of Akt and ERK. Sensitivity of patient-derived xenograft to CYH33 was also positively correlated with decrease in phosphorylated ERK. Taken together, CYH33 is a promising PI3Kα inhibitor for breast cancer treatment and decrease in ERK phosphorylation may indicate its efficacy, which provides useful clues for rational design of the ongoing clinical trials.

// Jin-Xue He 1, * , Meng Wang 2, * , Xia-Juan Huan 1 , Chuan-Huizi Chen 1 , Shan-Shan Song 1 , Ying-Qing Wang 1 , Xue-Mei Liao 1 , Cun Tan 2 , Qian He 2 , Lin-Jiang Tong 1 , Yu-Ting Wang 1 , Xiao-Hua Li 1 , Yi Su 1 , Yan-Yan Shen 1 , Yi-Ming Sun 1 , Xin-Ying Yang 1 , Yi Chen 1 , Zhi-Wei Gao 3 , Xiao-Yan Chen 3 , Bing Xiong 2 , Xiu-Lian Lu 4 , Jian Ding 1 , Chun-Hao Yang 2 , Ze-Hong Miao 1 1 Division of Anti-Tumor Pharmacology and State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China, University of Chinese Academy of Sciences, Beijing 100049, China 2 Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China, University of Chinese Academy of Sciences, Beijing 100049, China 3 Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China 4 Cisen Pharmaceutical Co., LTD, Jining 272073, Shandong, China * These authors contributed equally to this work Correspondence to: Ze-Hong Miao, email: zhmiao@simm.ac.cn Chun-Hao Yang, email: chyang@simm.ac.cn Keywords: MPH, PARP inhibitor, homologous recombination, antitumor activity, synthetic lethality Received: April 01, 2016 Accepted: November 23, 2016 Published: December 01, 2016 ABSTRACT The approval of poly(ADP-ribose) polymerase (PARP) inhibitor AZD2281 in 2014 marked the successful establishment of the therapeutic strategy targeting homologous recombination repair defects of cancers in the clinic. However, AZD2281 has poor water solubility, low tissue distribution and relatively weak in vivo anticancer activity, which appears to become limiting factors for its clinical use. In this study, we found that mefuparib hydrochloride (MPH) was a potent PARP inhibitor, possessing prominent in vitro and in vivo anticancer activity. Notably, MPH displayed high water solubility (> 35 mg/ml) and potent PARP1/2 inhibition in a substrate-competitive manner. It reduced poly(ADP-ribose) (PAR) formation, enhanced γH2AX levels, induced G2/M arrest and subsequent apoptosis in homologous recombination repair (HR)-deficient cells. Proof-of-concept studies confirmed the MPH-caused synthetic lethality. MPH showed potent in vitro and in vivo proliferation and growth inhibition against HR-deficient cancer cells and synergistic sensitization of HR-proficient xenografts to the anticancer drug temozolomide. A good relationship between the anticancer activity and the PARP inhibition of MPH suggested that PAR formation and γH2AX accumulation could serve as its pharmacodynamic biomarkers. Its high bioavailability (40%~100%) and high tissue distribution in both monkeys and rats were its most important pharmacokinetic features. Its average concentrations were 33-fold higher in the tissues than in the plasma in rats. Our work supports the further clinical development of MPH as a novel PARP1/2 inhibitor for cancer therapy.

In various human cancers, PI3Ks pathway is ubiquitously dysregulated and thus become a promising anti-cancer target. To discover new potent and selective PI3K inhibitors as potential anticancer drugs, new pyrrolo[2,1-f][1,2,4]triazines were designed, leading to the discovery of compound 37 (CYH33), a selective PI3Kα inhibitor (IC50 = 5.9 nM, β/α, δ/α,γ/α = 101-, 13-, 38-fold). Western blot analysis confirmed that compound 37 could inhibit phosphorylation of AKT in human cancer cells to modulate the cellular PI3K/AKT/mTOR pathway. And further evaluation in vivo against SKOV-3 xenograft models demonstrated that a dose-dependent antitumor efficacy was achieved.

The nuclear protein poly(ADP-ribose) polymerases-1/2 (PARP-1/2) are involved in DNA repair damaged by endogenous or exogenous process. And PARP-1/2 inhibitors have been proved to be clinically efficacious for DNA repair deficient tumors in the past decade. We have developed a series of 4,5,6,7-tetrahydrothienopyridin-2-yl benzimidazole carboxamides as novel and potent PARP-1/2 inhibitors. The best compound resulted from this series is compound 27 which displays excellent PARP-1 and PARP-2 inhibitory activity with IC50 of 18 nM and 42 nM, respectively. Furthermore, it can selectively kill BRCA2 deficient V-C8 cells with a CC50 of 920 nM. In the MDA-MB-436 (BRCA-1 mutant) xenograft model, this compound was well tolerated and showed single-agent activity. Based on the results above, compound 27 has been selected as a lead candidate targeting PARP-1/2 and its preclinical characterization is also underway.

Objective Activating KRAS mutations are the most common drivers in the development of non-small cell lung cancer (NSCLC). However, unsuccess of treatment by direct inhibition of KRAS has been proven. Deregulation of PI3K signaling plays an important role in tumorigenesis and drug resistance in NSCLC. The activity of PI3Kα-selective inhibition against KRAS-mutated NSCLC remains largely unknown.Methods Cell proliferation was detected by sulforhodamine B assay. Cell cycle distribution and apoptosis were measured by flow cytometry. Cell signaling was assessed by Western blot and immunohistochemistry. RNA interference was used to down-regulate the expression of cyclin D1. Human NSCLC xenografts were employed to detect therapeutic efficacy in vivo.Results CYH33 possessed variable activity against a panel of KRAS-mutated NSCLC cell lines. Although CYH33 blocked AKT phosphorylation in all tested cells, Rb phosphorylation decreased in CYH33-sensitive, but not in CYH33-resistant cells, which was consistent with G1 phase arrest in sensitive cells. Combined treatment with the CDK4/6 inhibitor, PD0332991, and CYH33 displayed synergistic activity against the proliferation of both CYH33-sensitive and CYH33-resistant cells, which was accompanied by enhanced G1-phase arrest. Moreover, down-regulation of cyclin D1 sensitized NSCLC cells to CYH33. Reciprocally, CYH33 abrogated the PD0332991-induced up-regulation of cyclin D1 and phosphorylation of AKT in A549 cells. Co-treatment with these two drugs demonstrated synergistic activity against A549 and H23 xenografts, with enhanced inhibition of Rb phosphorylation.Conclusions Simultaneous inhibition of PI3Kα and CDK4/6 displayed synergistic activity against KRAS-mutated NSCLC. These data provide a mechanistic rationale for the combination of a PI3Kα inhibitor and a CDK4/6 inhibitor for the treatment of KRAS-mutated NSCLC.

Phosphatidylinositol 3-kinaseα isoform (PI3Kα) is frequently deregulated in a wide variety of human solid tumors. A series of new pyrrolo[2,1-f][1,2,4]triazines has been prepared in an effort to discover new PI3Kα-selective inhibitors. Intensive structure-activity relationship (SAR) and structure-pharmacokinetic relationship (SPR) studies on this new series led to the discovery of compound 40 (CYH33), a potent and highly selective PI3Kα inhibitor. CYH33 displays the most potent activity against PI3Kα among the class I PI3K isoforms, with IC50s of 5.9 nM/598 nM/ 78.7 nM/225 nM against α/β/δ/γ isoform respectively. Kinome profiling revealed that CYH33 showed little activity among more than 300 kinases. The selectivity of CYH33 against PI3Kα was further confirmed with a panel of Rh30-Myr-p110s isogenic cells, which express constitutively active PI3K isoform respectively. Accordingly, CYH33 inhibited PI3K/AKT/mTOR signaling in glioblastoma U87MG and rhabdomyosarcoma Rh30 cells, which is associated with its potent anti-proliferative activity in against cell proliferation in a panel cancer cell lines originated from breast, lung, ovary and colon, prostate etc. It is noteworthy that most of sensitive cells were originated from breast cancer, which was consistent with the fact that PI3K is frequently hyper-activated in breast cancer. Oral administration of CYH33 to mice bearing ovarian cancer SKOV-3 xenografts resulted in decreased phosphorylation of AKT in tumor tissue, which is consistent with the significant efficacy of CYH33 to inhibit the growth of SKOV-3 xenograft. Moreover, CYH33 did not cause significant weight loss and toxic symptoms during the treatment period. We also detected the glucose tolerance after mice bearing SKOV-3 xenografts were administered with CYH33 for 18 days. Though CYH33 treatment delayed the restoration of blood glucose to normal level, blood glucose of the CYH33-treated groups were similar to that of control group 2 h after the injection of glucose. CYH33 displays a favorable pharmacokinetic profile with an oral bioavailability of 36.9% and little activity against the major cytochrome P450 isozymes. In summary, CYH33 emerged as a novel selective PI3Kα inhibitor with potent activity against human solid tumors and favorable pharmaceutical profile, which makes it a promising drug candidate and enter clinical trials in China. Citation Format: Haoyue Xiang, Xiang Wang, Yanhong Chen, Xi Zhang, Yi Chen, Cun Tan, Yi Wang, Jian Ding, Ling-Hua Meng, Chunhao Yang. Discovery of clinical candidate methyl (5-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-4-morpholinopyrrolo[2,1-f][1,2,4]triazin-2-yl)-4-(trifluoromethyl)pyridin-2-yl)carbamate (CYH33) : A highly potent and selective PI3K alpha inhibitor for the treatment of advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-268.