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Supplementary Tables S1-S7

USP1 and PARP inhibition are synergistic in a BRCA1/2-dependent manner - related to Figure 4

CRISPR-UMI analysis and characterization of PCNA dynamics - related to Figure 3

Supplementary methods

I-138 inhibits USP1-UAF1 - related to Figure 1

I-138 activity in BRCA1/2 WT and mutant cells - related to Figure 2

CRISPR-UMI analysis of screens performed in NCI-H1693 cells - related to Figure 5

CRISPR Cas9-based screening is a powerful approach for identifying and characterizing novel drug targets. Here, we elucidate the synthetic lethal mechanism of deubiquitinating enzyme USP1 in cancers with underlying DNA damage vulnerabilities, specifically BRCA1/2 mutant tumors and a subset of BRCA1/2 wild-type (WT) tumors. In sensitive cells, pharmacologic inhibition of USP1 leads to decreased DNA synthesis concomitant with S-phase–specific DNA damage. Genome-wide CRISPR-Cas9 screens identify RAD18 and UBE2K, which promote PCNA mono- and polyubiquitination respectively, as mediators of USP1 dependency. The accumulation of mono- and polyubiquitinated PCNA following USP1 inhibition is associated with reduced PCNA protein levels. Ectopic expression of WT or ubiquitin-dead K164R PCNA reverses USP1 inhibitor sensitivity. Our results show, for the first time, that USP1 dependency hinges on the aberrant processing of mono- and polyubiquitinated PCNA. Moreover, this mechanism of USP1 dependency extends beyond BRCA1/2 mutant tumors to selected BRCA1/2 WT cancer cell lines enriched in ovarian and lung lineages. We further show PARP and USP1 inhibition are strongly synergistic in BRCA1/2 mutant tumors. We postulate USP1 dependency unveils a previously uncharacterized vulnerability linked to posttranslational modifications of PCNA. Taken together, USP1 inhibition may represent a novel therapeutic strategy for BRCA1/2 mutant tumors and a subset of BRCA1/2 WT tumors.

TNG348 is a selective and potent inhibitor of the deubiquitinating enzyme USP1 specifically designed to target BRCA1/2mut vulnerabilities in breast and ovarian tumors. Here we present the biochemical, mechanistic, and in vitro and in vivo characterization of TNG348, an oral, allosteric and highly potent inhibitor of USP1. Upon treatment, TNG348 causes loss of viability in a panel of breast and ovarian BRCA1/2mut cancer cell lines and displays dose-dependent tumor growth inhibition in BRCA1/2mut xenograft models. Furthermore, TNG348 activity extends beyond BRCA1/2mut models with PARP inhibitor (PARPi) sensitivity and oncogene-induced replication stress being additional features correlating with USP1 inhibitor sensitivity based on cell line panel and CRISPR screening results. We show that TNG348 induces cell death through a pathway that is distinct from PARPi and TNG348 demonstrates robust synergy when combined with first- or second-generation PARPi. The clinical development plan intends to evaluate TNG348 in patients with BRCA1/2 mutations as single agent and in combination with PARP1i in patients naïve to PARPi and with prior PARPi treatment history. Citation Format: Antoine Simoneau, Hsin-Jung Wu, Madhavi Bandi, Katherine Lazarides, Sining Sun, Shangtao Liu, Samuel Meier, Ashley Choi, Hongxiang Zhang, Binzhang Shen, Douglas Whittington, Sirimas Sudsakorn, Wenhai Zhang, Yi Yu, Yong Liu, Colin Liang, Michael Palmieri, Yingnan Chen, Brian Haines, Alice Tsai, Minjie Zhang, Alan Huang, Jannik Andersen, Tianshu Feng, Scott Throner, John Maxwell. Characterization of the clinical development candidate TNG348 as a potent and selective inhibitor of USP1 for the treatment of BRCA1/2mut cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4968.

JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of...

CRISPR-based functional genomic screening is a powerful approach for identifying novel classes of synthetic lethal drug targets. Here, we define the deubiquitinase USP1 as a synthetic lethal target in cancers with underlying DNA repair vulnerabilities. A highly potent and selective small molecule USP1 inhibitor conferred a viability defect in BRCA1-mutant, but not WT cell lines by activating replication stress. Genome-wide CRISPR screening uncovered RAD18 and UBE2K, which promote PCNA mono- and poly-ubiquitination respectively, as key mediators of USP1-BRCA1 dependency. Increased cellular mono- and poly-ubiquitination reduced PCNA protein levels, and restoration of PCNA protein expression rescued USP1 inhibitor sensitivity. USP1 dependency is associated with upregulated RAD18 and UBE2K expression, suggesting that elevated PCNA ubiquitination in the context of BRCA1 deficiency mediates USP1 synthetic lethality. Interestingly, USP1, but not PARP1 inhibition, elicited a viability defect in a subset of BRCA1/2 WT lung cancer cell lines, indicative of novel synthetic lethal interactions unique to USP1. Moreover, dual inhibition of PARP1 and USP1 are strongly synergistic in PARP1 inhibitor-responsive cell line models. Strong in vivo anti-tumor activity across multiple tumor models was demonstrated with USP1 inhibition alone and in combination with the PARP1 inhibitor olaparib. Our studies suggest that USP1 and PARP1 inhibitors target BRCA1-mutant cancer though distinct yet synergistic mechanisms. As such, USP1 inhibitors may provide novel treatment strategies for PARP1 inhibitor-resistant and -naïve BRCA1-mutant cancer. Citation Format: Justin Engel, Madhavi Bandi, Antione Simoneau, Katherine Lazarides, Deepali Gotur, Truc Pham, Shangtao Liu, Samuel Meier, Ashley Choi, Hongxiang Zhang, Binzhang Shen, Fang Li, Douglas Whittington, Shanzhong Gong, Xuewen Pan, Yi Yu, Lina Gu, Scott Throner, John Maxwell, Yingnan Chen, Alan Huang, Jannik Andersen, Tianshu Feng. USP1 inhibitor synthetic lethality in BRCA1-mutant cancer is driven by PCNA ubiquitination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2603.

Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER+ breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of N-[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl]pyridin-3-amine (28). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that 28 had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.

JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit

Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-molecule inhibitors that are selective for a specific family member would represent highly desirable tools for deconvoluting the intricacies of JAK family biology. Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays ∼1000-fold selectivity over the other highly homologous JAK family members (determined by biochemical assays), while also possessing good selectivity over other kinases (determined by panel screening). Moreover, this compound was demonstrated to be orally bioavailable and possesses acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1 inhibition).

The preparation of a range of 1,5-disubstituted tetrazoles has been achieved through palladium-catalyzed Suzuki coupling. Using appropriately substituted 5-p-toluenesulfonyltetrazoles as substrates (obtained by cycloaddition of a substituted azide with p-toluenesulfonyl cyanide), this methodology provides access to a variety of highly substituted tetrazoles that would be difficult to access otherwise. The procedure is compatible with functional groups commonly found in drug-like molecules, and has been used to generate a number of compounds of potential biological interest.

Protein lysine methyltransferases (KMTs) have emerged as important regulators of epigenetic signaling. These enzymes catalyze the transfer of donor methyl groups from the cofactor S-adenosylmethionine to specific acceptor lysine residues on histones, leading to changes in chromatin structure and transcriptional regulation. These enzymes also methylate an array of nonhistone proteins, suggesting additional mechanisms by which they influence cellular physiology. SMYD2 is reported to be an oncogenic methyltransferase that represses the functional activity of the tumor suppressor proteins p53 and RB. HTS screening led to identification of five distinct substrate-competitive chemical series. Determination of liganded crystal structures of SMYD2 contributed significantly to "hit-to-lead" design efforts, culminating in the creation of potent and selective inhibitors that were used to understand the functional consequences of SMYD2 inhibition. Taken together, these results have broad implications for inhibitor design against KMTs and clearly demonstrate the potential for developing novel therapies against these enzymes.

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZeneca's collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.

The histamine type 3 receptor (H3) is a G protein-coupled receptor implicated in several disorders of the central nervous system. Herein, we describe the radiolabeling and preclinical evaluation of a candidate radioligand for the H3 receptor, 4-(1S,2S)-2-(4-cyclobutylpiperazine-1-carbonyl)cyclopropyl]-N-methyl-benzamide (5), and its comparison with one of the frontrunner radioligands for H3 imaging, namely, GSK189254 (1). Compounds 1 and 5 were radiolabeled with tritium and carbon-11 for in vitro and in vivo imaging experiments. The in vitro binding of [(3)H]1 and [(3)H]5 was examined by (i) saturation binding to rat and nonhuman primate brain tissue homogenate and (ii) in vitro autoradiography on tissue sections from rat, guinea pig, and human brain. The in vivo binding of [(11)C]1 and [(11)C]5 was examined by PET imaging in mice and nonhuman primates. Bmax values obtained from Scatchard analysis of [(3)H]1 and [(3)H]5 binding were in good agreement. Autoradiography with [(3)H]5 on rat, guinea pig, and human brain slices showed specific binding in regions known to be enhanced in H3 receptors, a high degree of colocalization with [(3)H]1, and virtually negligible nonspecific binding in tissue. PET measurements in mice and nonhuman primates demonstrated that [(11)C]5 binds specifically and reversibly to H3 receptors in vivo with low nonspecific binding in brain tissue. Whereas [(11)C]1 showed similar binding characteristics in vivo, the binding kinetics appeared faster for [(11)C]5 than for [(11)C]1.[(11)C]5 has suitable properties for quantification of H3 receptors in nonhuman primate brain and has the potential to offer improved binding kinetics in man compared to [(11)C]1.

A new photoredox-catalyzed C N coupling reaction of sulfoximines with aryl halides has been developed for a general N-arylation of sulfoximines. The reactions proceed in the presence of visible light with high levels of chemoselectivity and a wide range of functionality is tolerated. There is a rapidly increasing interest in sulfoximines as pharmacophores in drug discovery and this new method offers potential in terms of high functional group tolerance and late-stage functionalization of compounds.

Further structure activity relationship studies on a previously reported 8-azabicyclo[3.2.1]octan-3-yloxy-benzamide series of potent and selective kappa opioid receptor antagonists is discussed. Modification of the pendant N-substitution to include a cyclohexylurea moiety produced analogs with greater in vitro opioid and hERG selectivity such as 12 (kappa IC50=172 nM, mu:kappa ratio=93, delta:kappa ratio=174, hERG IC50=33 microM). Changes to the linker conformation and identity as well as to the benzamide ring moiety were also investigated.

Janus kinases are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling, with constitutive activation of JAK/STAT pathways associated with a wide variety of malignancies. Elevated JAK/STAT signalling leading to increased activation of STAT3 is reported in a wide variety of cancers, including breast, liver, prostate, colorectal, head and neck, oesophageal, pancreatic, bladder, and non-small cell lung, and is implicated in the pathogenesis of diffuse large B-cell lymphoma and nasopharyngeal carcinomas. Overall, up to 70% of human tumours are linked to persistent elevated STAT3 activity which can be associated with poorer prognosis in many of these settings. In addition, elevated pSTAT3 is observed in response to chemotherapy treatment, and also in response to treatment with inhibitors of oncogenic signalling pathways such as EGFR, MAPK and AKT, and is associated with resistance or poorer response to agents targeting these pathways. In many of these cases, JAK1 is believed to be a primary driver of STAT3 phosphorylation and signalling, suggesting inhibition of JAKs as a therapeutic approach to treat these potential resistance mechanisms. The mixed JAK1/2 kinase inhibitor ruxolitinib is approved for the treatment of myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera and has been tested in a variety of tumor settings. Since JAK2 is essential for the signal transduction downstream of erythropoietin, thrombopoietin and related receptors that control erythrocyte and megakaryocyte expansion, dosing of inhibitors that target JAK2 can be limited by toxicities such as thrombocytopenia and anaemia. Starting from a non-kinome selective screening hit, structure-based design was used to optimise a series of aminopyrimidines that led to JAK1-selective candidate drug AZD4205. This compound demonstrates ATP competitive binding with IC50’s in a high ATP concentration enzyme assay against JAK1 of 73 nM (Ki = 2.8 nM), with high selectivity against JAK2 and JAK3 with IC50’s of 13,233 nM and >30,000 nM respectively. In addition it showed potent inhibition of p-STAT3 in a cell based assay of JAK1 activity with an IC50 of 128 nM and excellent selectivity across the kinome. In summary, AZD4205 is a highly potent JAK1-selective kinase inhibitor with excellent preclinical pharmacokinetics with potential for further clinical development. The optimization from screening hit to first disclosure of this candidate drug will be presented. Citation Format: Jason G. Kettle, Qibin Su, Neil Grimster, Sameer Kawatkar, Scott Throner, Richard Woessner, Huawei Chen, Geraldine Bebernitz, Kristen Bell, Erica Anderson, Linette Ruston, Jon Winter-Holt, Paul Lyne, Melissa Vasbinder, Claudio Chuaqui. Discovery of the JAK1 selective kinase inhibitor AZD4205 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 979. doi:10.1158/1538-7445.AM2017-979

A new series of potent and selective histamine-3 receptor (H3R) antagonists was identified on the basis of an azaspiro[2.5]octane carboxamide scaffold. Many scaffold modifications were largely tolerated, resulting in nanomolar-potent compounds in the H3R functional assay. Exemplar compound 6s demonstrated a selective profile against a panel of 144 secondary pharmacological receptors, with activity at only σ2 (62% at 10 μM). Compound 6s demonstrated free-plasma exposures above the IC50 (∼50×) with a brain-to-plasma ratio of ∼3 following intravenous dosing in mice. At three doses tested in the mouse novel object recognition model (1, 3, and 10 mg/kg s.c.), 6s demonstrated a statistically significant response compared with the control group. This series represents a new scaffold of H3 receptor antagonists that demonstrates in vivo exposure and efficacy in an animal model of cognition.

Herein, we describe the discovery of inhibitors of norepinephrine (NET) and dopamine (DAT) transporters with reduced activity relative to serotonin transporters (SERT). Two compounds, 8b and 21a, along with nomifensine were tested in a rodent receptor occupancy study and demonstrated dose-dependent displacement of radiolabeled NET and DAT ligands. These compounds were efficacious in a rat forced swim assay (model of depression) and also had activity in rat spontaneous locomotion assay.

Novel in vitro mGlu(5) positive allosteric modulators with good potency, solubility, and low lipophilicity are described. Compounds were identified which did not rely on the phenylacetylene and carbonyl functionalities previously observed to be required for in vitro activity. Investigation of the allosteric binding requirements of a series of dihydroquinolinone analogs led to phenylacetylene azachromanone 4 (EC(50) 11.5 nM). Because of risks associated with potential metabolic and toxicological liabilities of the phenylacetylene, this moiety was successfully replaced with a phenoxymethyl group (27; EC(50) 156.3 nM). Derivation of a second-generation of mGlu(5) PAMs lacking a ketone carbonyl resulted in azaindoline (33), azabenzimidazole (36), and N-methyl 8-azaoxazine (39) phenylacetylenes. By scoping nitrogen substituents and phenylacetylene replacements in 39, we identified phenoxymethyl 8-azaoxazine 47 (EC(50) 50.1 nM) as a potent and soluble mGlu(5) PAM devoid of both undesirable phenylacetylene and carbonyl functionalities.

Initial high throughput screening efforts identified highly potent and selective kappa opioid receptor antagonist 3 (κ IC50 = 77 nM; μ:κ and δ:κ IC50 ratios >400) which lacked CNS exposure in vivo. Modification of this scaffold resulted in development of a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides showing potent and selectivity κ antagonism as well as good brain exposure. Analog 6c (κ IC50 = 20 nM; μ:κ = 36, δ:κ = 415) was also shown to reverse κ-agonist induced rat diuresis in vivo.