H3B-5942 suppresses co-activator recruitment and shows potency in endocrine sensitive and resistant lines

H3B-5942 in combination with palbocyclib shows synergy in vitro and in vivo

ESR1 mutants are resistant to endocrine therapies in vitro and in vivo and H3B-5942 can suppress ER pathway activity.

H3B-5942 exhibits dose-dependent inhibition of ER target genes and shows significant efficacy ER Wt and mutant in vivo models

Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity. To this end, we developed a multiplexed high-throughput screening assay that monitors exonuclease, ATPase, and helicase activities of full-length WRN. This screening campaign led to the discovery of 2-sulfonyl/sulfonamide pyrimidine derivatives as novel covalent inhibitors of WRN helicase activity. The compounds are specific for WRN versus other human RecQ family members and show competitive behavior with ATP. Examination of these novel chemical probes established the sulfonamide NH group as a key driver of compound potency. One of the leading compounds, H3B-960, showed consistent activities in a range of assays (IC50 = 22 nM, KD = 40 nM, KI = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC50 ∼ 10 nM. These kinetic properties trend toward other known covalent druglike molecules. Our work provides a new avenue for screening WRN for inhibitors that may be adaptable to different therapeutic modalities such as targeted protein degradation, as well as a proof of concept for the inhibition of WRN helicase activity by covalent molecules.

Crystallography and amino acid coordinates

C530 residue is critical for H3B-5942 potency

Supplementary Data from Covalent ERα Antagonist H3B-6545 Demonstrates Encouraging Preclinical Activity in Therapy-Resistant Breast Cancer

Abstract Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy–resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089). Summary: H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors.

Abstract Mutations in the ligand-binding domain of estrogen receptor alpha (ERα) are detected in up to 30% of patients (pts) who have relapsed or progressed during endocrine therapy. By favoring the agonistic conformation in ERα, these hotspot mutations promote ligand-independent activation of ERα and confer partial resistance to ER-directed therapies. Of the various hotspot mutations, Y537S is the most constitutively active, promotes the greatest resistance phenotype to current endocrine therapies, and is associated with the worst prognosis relative to other ERα mutations. The fact that current ER-directed therapies have limited activity in the ERα mutant setting emphasizes the critical need to develop the next generation of high affinity ER antagonists that can overcome the aberrant activity of mutant ERα. H3B-6545 is a first-in-class selective ERα covalent antagonist (SERCA) which inactivates both wild-type and mutant ERα by irreversibly engaging cysteine-530. Biophysical and biochemical analyses confirm the long residence time achieved by covalent binding, and cellular analyses confirm the selectivity and single-digit nanomolar potency of H3B-6545 across a panel of ERαWT and ERαMUT breast cancer cell lines. H3B-6545 as a monotherapy demonstrates superior anti-tumor activity relative to fulvestrant across a set of CDK4/6 inhibitor naïve ERαWT and ERαY537S cell line-derived xenograft (CDX)/patient-derived xenograft (PDX) models, with regressions being noted in both the ERαWT and ERαMUT settings. Furthermore, H3B-6545 continues to demonstrate single agent activity in CDK4/6 inhibitor-resistant ERαWT and ERαY537S PDX models, in which fulvestrant fails to demonstrate significant anti-tumor activity. Lastly, improved activity and duration of response are noted when H3B-6545 is combined with several targeted therapies, including CDK4/6 inhibitors palbociclib and abemaciclib across a range of ERαWT and ERαY537S CDX/PDX models. The phase I-II trial (NCT03250676) enrolled 130 heavily pretreated pts with ER+, HER2- metastatic breast cancer, including 12 pts harboring high allele frequency clonal ESR1 Y537S circulating tumor DNA (ctDNA). Median number of prior therapy in the metastatic setting was 3 (range: 1-10). Consistent with the preclinical data, H3B-6545 demonstrated promising clinical activity among these pts with clonal Y537S mutations, with a median progression free survival of 7.3 months and an overall response rate of 25% (3 confirmed partial responses). In summary, these compelling preclinical data coupled with emerging clinical activity in heavily pretreated poor prognosis pts support further development of H3B-6545 as monotherapy or combination treatment. Citation Format: Manav Korpal, Craig Furman, Xiaoling Puyang, Zhaojie Zhang, Zhenhua Wu, Deepti Banka, Subhasree Das, Benoit Destenaves, Lei Gao, Erika Hamilton, Ming-Hong Hao, Sean Irwin, Stephen Johnston, Jaya J Joshi, Dejan Juric, Amy Kim, Tuong-Vi Nguyen, Marc Pipas, Timothy Pluard, Victoria Rimkunas, Nathalie Rioux, Joanne Schindler, Peter Smith, Michael Thomas, John Wang, Judy S Wang, Markus Warmuth, Huilan Yao, Shihua Yao, Lihua Yu, Frédéric H Vaillancourt, David M Bolduc, Nicholas A Larsen, GuoZhu Zheng, Sudeep Prajapati, Tarek Sahmoud, Antonio Gualberto, Ping Zhu. Development of H3B-6545, a first-in-class oral selective ER covalent antagonist (SERCA), for the treatment of ERaWT and ERaMUT breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-23.

Abstract Carbamoyl Phosphate Synthetase 1 (CPS1) catalyzes the first and rate-limiting step in ammonia detoxification through the urea cycle, generating carbamoyl phosphate from ammonia, bicarbonate and ATP. CPS1 is overexpressed in several cancers, including LKB1-deficient non-small cell lung carcinoma (NSCLC), where its activity has been proposed to support tumor growth by generating carbamoyl phosphate for use in pyrimidine synthesis. In other cancers, CPS1 has been reported to remove toxic ammonia from growing tumors to allow for sustained growth. Currently, no small molecule inhibitors of CPS1 have been identified, limiting researcher's ability to dissect the involvement of CPS1 function in cancer biology. We describe here the discovery of the first known small molecule inhibitors of CPS1. From a high throughput screen, we identify two chemical series, which achieve inhibition of CPS1 by blocking bicarbonate phosphorylation in the first step of carbamoyl phosphate synthesis. Biochemical experiments reveal inhibition of bicarbonate phosphorylation occurs through competition between inhibitor and ATP. High-resolution co-crystal structures demonstrate that these novel inhibitors bind to a previously unidentified allosteric pocket located between the integrating domain and carbamate synthetase domain of CPS1. This allosteric mechanism of action allows for highly selective CPS1 inhibition, with no inhibition of CPS2 observed. Analogs with increased potency were developed, which are able to inhibit CPS1 cellular activity, blocking both urea production and the CPS1-mediated pyrimidine biosynthetic pathway in cultured cells. These novel CPS1 inhibitors are valuable, first-in-class tools for probing CPS1 biology. We will also discuss the opportunity to utilize these novel inhibitors of CPS1, along with genetic approaches, to probe the functional roles of CPS1 in LKB1-deficient NSCLC. Citation Format: Shihua Yao, Tuong-Vi Nguyen, Alan Rolfe, Anant A. Agrawal, Jiyuan Ke, Shouyong Peng, Federico Colombo, Sean Yu, Patricia Bouchard, Jiayi Wu, Kuan-Chun Huang, Xingfeng Bao, Kiyoyuki Omoto, Anand Selvaraj, Lihua Yu, Stephanos Ioannidis, Frédéric H. Vaillancourt, Ping Zhu, Nicholas A. Larsen, David M. Bolduc. Discovery of selective inhibitors of carbamoyl phosphate synthetase I (CPS1) to modulate cancer relevant metabolic pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2334.

Phosphatidylinositol 3,4,5-triphosphate (PIP3), a lipid second messenger, signals the initiation of cascades that result in, among other processes, cell proliferation, migration, and survival. PIP3 is generated by the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-3-kinase-α (PI3Kα) in response to activation by receptor tyrosine kinases (RTKs) or their substrates. PI3Kα has a residual constitutive activity that is counteracted by the action of PTEN, a phosphatase that is subjected to a large number of post-translational modifications. Thus, levels of PIP3 are controlled by a balance between the opposite actions of PI3Kα, a kinase, and PTEN, a phosphatase. Both enzymes are highly mutated in tumors. Oncogenic mutations have opposite effects on the activities of the two proteins: mutations activate PI3Kα but suppress the activity of PTEN. Both of these effects result in increased levels of PIP3 and provide selective advantage to the tumor cells carrying the mutations. In the physiologic activation of PI3Kα, the increase in activity is caused by the phosphotyrosine-containing region of the RTK dislodging the nSH2, a PI3Kα inhibitory domain from the regulatory subunit p85α, from its inhibitory interaction with the catalytic subunit p110α. A similar mechanism is used by some oncogenic mutations: they activate the enzyme by reducing the inhibitory action of the nSH2. Importantly, this effect is not a consequence of a transition to a new structure that can be identified as a new ground state. Instead, the mutations affect the dynamics of the protein in such a way that the new landscape favors catalytically competent conformations. Analysis of the interdomain interactions between the p110α and the p85α of the wild-type and the mutants suggests that the tumor-associated mutations effectively weaken the interactions between p110α and p85α by disrupting key stabilizing interactions. These findings support the notion that oncogenic mutations increase the enzymatic activity by enhancing the dynamics of the protein. Other oncogenic PI3Kα mutations increase the kinase activity by increasing the interaction of the enzyme with the membrane, and therefore augmenting accessibility to the substrate. In the case of PTEN, activity is controlled, at least in part, by the phosphorylation of four Ser/Thr residues (380, 382, 383, 385) in its 52-residue long C-terminal tail. This multiple phosphorylation results in an inhibition of the membrane-associated phosphatase activity. Using a variety of biochemical and biophysical techniques, we determined structural and physical chemical details of the mechanism controlling this inhibition. Briefly, in contrast to the unphosphorylated form, the tetra phosphorylated PTEN adopts a more compact conformation in which its carboxy terminal tail interacts with the C2 domain in a fashion that reduces its affinity for the cell membrane and diminishes its catalytic activity. Citation Format: Ignacia Echeverria, Yunlong Liu, Sweta Maheshwari, Mayukh Chakrabarti, Michelle Miller, David Bolduc, Philip Cole, Sandra B. Gabelli, L. Mario Amzel. Control of PIP3 levels by PI3Kα and PTEN [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA09.

Fibroblast growth factor receptors (FGFR) 2 and 3 have been established as drivers of numerous types of cancer with multiple drugs approved or entering late stage clinical trials. A limitation of current inhibitors is vulnerability to gatekeeper resistance mutations. Using a combination of targeted high-throughput screening and structure-based drug design, we have developed a series of aminopyrazole based FGFR inhibitors that covalently target a cysteine residue on the P-loop of the kinase. The inhibitors show excellent activity against the wild-type and gatekeeper mutant versions of the enzymes. Further optimization using SAR analysis and structure-based drug design led to analogues with improved potency and drug metabolism and pharmacokinetics properties.

Carbamoyl phosphate synthetase 1 (CPS1) is a potential synthetic lethal target in LKB1-deficient nonsmall cell lung cancer, where its overexpression supports the production of pyrimidine synthesis. In other cancer types, CPS1 overexpression and activity may prevent the accumulation of toxic levels of intratumoral ammonia to support tumor growth. Herein we report the discovery of a novel series of potent and selective small-molecule inhibitors of CPS1. Piperazine 2 was initially identified as a promising CPS1 inhibitor through a high-throughput screening effort. Subsequent structure-activity relationship optimization and structure-based drug design led to the discovery of piperazine H3B-616 (25), a potent allosteric inhibitor of CPS1 (IC50 = 66 nM).

Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.

DNMT3A (DNA methyltransferase 3A) is a de novo DNA methyltransferase responsible for establishing CpG methylation patterns within the genome. DNMT3A activity is essential for normal development, and its dysfunction has been linked to developmental disorders and cancer. DNMT3A is frequently mutated in myeloid malignancies with the majority of mutations occurring at Arg-882, where R882H mutations are most frequent. The R882H mutation causes a reduction in DNA methyltransferase activity and hypomethylation at differentially-methylated regions within the genome, ultimately preventing hematopoietic stem cell differentiation and leading to leukemogenesis. Although the means by which the R882H DNMT3A mutation reduces enzymatic activity has been the subject of several studies, the precise mechanism by which this occurs has been elusive. Herein, we demonstrate that in the context of the full-length DNMT3A protein, the R882H mutation stabilizes the formation of large oligomeric DNMT3A species to reduce the overall DNA methyltransferase activity of the mutant protein as well as the WT-R882H complex in a dominant-negative manner. This shift in the DNMT3A oligomeric equilibrium and the resulting reduced enzymatic activity can be partially rescued in the presence of oligomer-disrupting DNMT3L, as well as DNMT3A point mutations along the oligomer-forming interface of the catalytic domain. In addition to modulating the oligomeric state of DNMT3A, the R882H mutation also leads to a DNA-binding defect, which may further reduce enzymatic activity. These findings provide a mechanistic explanation for the observed loss of DNMT3A activity associated with the R882H hot spot mutation in cancer.

Abstract Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs. Significance: Nearly 30% of endocrine therapy–resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176–93. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1047

PTEN is a tumor suppressor that negatively regulates the PI3K/AKT signaling pathway by functioning as the lipid phosphatase for phosphatidyl inositol 3,4,5‐triphosphate (PIP3). Alterations in the PI3K/AKT pathway have tremendous influence on the oncogenesis of cancer, whereby loss of PTEN function leads to aberrant changes in cell proliferation, growth, survival, and protein synthesis. There are multiple modes of altering the cellular phosphatase activity of PTEN, including decrease or loss of expression, mutation, or post‐translational modification. A cluster of post‐translational phosphorylation sites are located on the C‐terminal tail at residues 380, 382, 383, and 385, which serve to modulate the lipid phosphatase function of PTEN by driving a conformational shift from an open active to closed inhibited state, resulting in a decrease in enzymatic activity and reduction in lipid membrane binding. Many questions centered on the mechanism of PTEN's C‐terminal tail dependent autoinhibition still remain unanswered. In order to provide a detailed mechanism on the molecular features that contribute to its autoinhibitory state, we employed expressed protein ligation (EPL) to install site‐specific and stoichiometric phosphorylation at these residues. We used photo‐crosslinking and designed mutants, which revealed tail interactions between the C‐tail and both the C2 and catalytic domain. These data have enhanced our understanding of the autoinhibitory conformation of PTEN and may pave the way for the rational design of new therapies for the treatment of cancer. Support or Funding Information NIH 1F32GM120855