Abstract The small membrane-bound GTPase KRAS functions as an on/off switch for multiple important cellular signaling pathways, including RAF/MEK/ERK and PI3K/AKT. As one of the most prevalent cancer drivers, KRAS alterations occur in approximately 17% of all solid tumors, including pancreatic, colorectal, lung adenocarcinoma, and esophagogastric cancers. Despite recent advances, KRAS G12C-selective inhibitors face clinical challenges in achieving durable therapeutic efficacy due to acquired drug resistance, while the mutational diversity of KRAS-driven cancers restricts their utility across patient populations, representing a significant unmet medical need. On the other hand, targeting all RAS proteins, including HRAS and NRAS, might raise potential toxicity risk, as the RAS family is essential in normal cells. Here, we report an oral pan-KRAS (ON/OFF) inhibitor ISM7713 that selectively targets all major oncogenic KRAS variants in both inactive and active conformations across multiple solid tumors while sparing HRAS and NRAS. Surface plasmon resonance revealed that ISM7713 binds to GDP-bound KRAS (inactive, or “off”) with picomolar affinity and to GTP-bound KRAS (active, or “on”) with sub-nanomolar affinity. Nucleotide exchange assays and RAS-cRAF binding assays further supported dual inhibitory function, together identifying sub-nanomolar IC50 values against KRAS variants (G12D, G12V, G12C, and WT). In cellular assays, ISM7713 suppressed cell growth with single-digit nanomolar IC50 values across cancer cell lines harboring major KRAS mutations or KRAS amplification, including GP2D (KRAS G12D), SW620 (KRAS G12V), NCI-H358 (KRAS G12C), and MKN1 (KRAS WT amplification). Treatment in vivo at 10-30 mg/kg (BID) induced tumor regression in multiple CDX models driven by different KRAS alterations (KRAS G12D/V/C mutations or KRAS amplification), along with dose-dependent pharmacodynamic responses, including changes in ERK phosphorylation and DUSP6 transcription. ISM7713 demonstrated reasonable solubility and permeability. In vivo, it exhibited good plasma clearance and bioavailability across mouse, rat, dog, and monkey. Additionally, mini-Ames and off-target safety panel profiling indicated no potential risk. Collectively, these findings highlight the potential of this novel molecule as a potent pan-KRAS (ON/OFF) inhibitor for the treatment of solid tumors with KRAS alterations. Citation Format: Sujing Shi, Defeng Shen, Jianping Wu, Tingting Liu, Jinxin Liu, Qingshuo Meng, Zuoxiao Shi, Suguna Rachakonda, David Gennert, Luoheng Qin, Xin Cai, Man Zhang, Feng Ren, Alex Zhavoronkov. ISM7713, a novel oral pan-KRAS (ON/OFF) inhibitor, shows robust anti-tumor activity in solid tumors with KRAS alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 425.

Introduction and Objective: Diabetic foot ulcers (DFUs) are chronic, slow-healing wounds characterized by unresolved inflammation affecting a significant proportion of patients with diabetes. Dysregulated NLRP3 inflammasome activation in diabetic wounds contributes to chronic inflammation, impeding tissue repair and promoting ulcer chronicity. NLRP3 expression is significantly upregulated in patients with DFUs compared to healthy controls. Methods: THP-1 derived macrophages and primary cells were used to evaluate compound activity in vitro. Selectivity was assessed by testing against other inflammasomes and a 44-panel safety assay. Hyperglycemia was induced in mice with STZ and a full-thickness wound created on the dorsal skin. Efficacy was assessed by wound closure rate. Results: Using our AI-based drug discovery platforms, we developed ISM5059, a peripherally restricted NLRP3 inhibitor with nanomolar inhibition potency of IL-1β (IC50 10.2 nM) and IL-18 (IC50 9.1 nM) release in THP-1-derived macrophages. ISM5059 showed similar activity in primary human monocytes and mouse bone marrow-derived macrophages. ISM5059 displays high selectivity, with no inhibitory activity on AIM2, NLRC4, NLRP1 inflammasomes, and a clean safety profile in a 44-panel screen. In vivo efficacy evaluated in an STZ-induced diabetic foot ulcer mouse model found ISM5059 accelerated wound healing in a dose-dependent fashion; at 6 mg/kg, its effect was comparable to the positive control, dapagliflozin. Combined treatment with dapagliflozin further accelerated wound closure, achieving healing rates similar to those observed in non-diabetic, healthy mice. ISM5059 exhibited favorable drug-like properties, including optimal in vitro ADMET profiles, strong in vivo exposure and clearance, and excellent oral bioavailability across multiple preclinical species. Conclusion: These findings establish ISM5059 as an effective and selective peripheral NLRP3 inhibitor with robust efficacy and a promising safety margin for potential treatment of diabetic foot ulcers. Disclosure Z. Cao: Employee; Current; Insilico Medicine. M. Zhang: Employee; Current; Insilico Medicine. X. Cai: Employee; Current; Insilico Medicine. C. Satler: Employee; Current; Insilico Medicine. D. Gennert: Employee; Current; Insilico Medicine. J. Wu: Employee; Current; Insilico Medicine. L. Qin: Employee; Current; Insilico Medicine. F. Ren: Employee; Current; Insilico Medicine. A. Zhavoronkov: Employee; Current; Insilico Medicine.

Cyclin-dependent kinases 12 and 13 (CDK12/13) safeguard genomic integrity by preferentially regulating gene expression in the DNA damage response (DDR). The CDK12/13-mediated upregulation of DDR genes and pathways significantly contributes to both tumorigenesis and the development of resistance to antitumor therapies. Thus, the functional inhibition of CDK12/13 offers an attractive strategy to combat carcinogenesis, particularly for refractory and treatment-resistant cancers. Here, we report the discovery of compound 12b as a novel, potent, orally available covalent CDK12/13 dual inhibitor with a promising safety profile and robust in vivo antitumor properties.

Abstract Ubiquitin-specific protease 1 (USP1), a well-characterized member of the deubiquitinating enzymes family, cleaves ubiquitin from various target proteins. USP-1 expression is dysregulated in many cancers where it acts as an oncogenic driver with roles in various DNA damage repair processes including translesion synthesis and the Fanconi anemia pathway. Previous data show that USP1 plays a critical role in protecting the replication fork in BRCA1-deficient cells and that its loss results in reduced cell survival and replication fork degradation, suggesting that USP1 inhibitors may be particularly useful in BRCA-deficient tumors. Though PARP inhibitors (PARPi) have shown clinical benefit in BRCA-mutated (BRCAm) populations, some patients either do not respond to therapy or develop resistance. USP1 inhibitors may have the potential to address this unmet clinical need. We evaluated ISM3091, a novel and selective USP1 inhibitor, against tumor cell lines and in vivo models with BRCAm and other homologous recombination repair deficiency (HRD), as well as in homologous recombination (HR)-proficient models.ISM3091 demonstrated potent anti-proliferation activity (IC50, 20 nM) in the BRCA1m triple negative breast cancer (TNBC) cell line, MDA-MB-436, with a ~1500-fold selectivity for BRCAm vs BRCA WT. Synthetic lethality between USP1 and HRD was due to persistent monoubiquitinated PCNA. In vitro data revealed that the combination of ISM3091 and olaparib, a PARPi, had synergistic activity in cell lines with HRD. Multiple in vivo CDX and PDX models confirmed the dose-dependent, single-agent inhibitory activity of ISM3091, with Tumor Growth Inhibition (TGI) of 66% at 30 mg/kg BID (TNBC) and 60% at 50 mg/kg BID (ovarian). The combination of ISM3091 and olaparib yielded a more robust and durable anti-tumor response, even at low doses of ISM3091, with TGI of 91% (TNBC; ISM3091, 3 mg/kg BID + olaparib, 50 mg/kg QD) and 110% (ovarian; ISM3091, 50 mg/kg BID + olaparib, 50 mg/kg QD). Notably, ISM3091 displayed strong single-agent activity (TGI 72% at 50 mg/kg BID) in an ovarian PDX model with acquired resistance to olaparib (BRCA WT), indicating that it has the potential to treat tumors with HRD beyond BRCAm, and also to overcome PARPi resistance. In vivo evaluation against the HR-proficient lung adenocarcinoma cell line, NCIH1792, showed potent efficacy (TGI 86% at 30 mg/kg BID) suggesting a broader ISM3091 potential. ISM3091also displayed very favorable ADME properties and PK profiles, and GLP toxicology studies indicated that it was well tolerated without significant gastrointestinal toxicity or hematological toxicity. These data support the future clinical development of ISM3091 as a potential best-in-class USP1 inhibitor not only for PARPi-resistant/responsive HRD-mutant cancers, both, as a single agent as well as in combination with PARPi, but also for subsets of HR-proficient cancers. Citation Format: Yangguang Li, Jianping Wu, Jinxin Liu, Luoheng Qin, Xin Cai, Junwen Qiao, Ling Wang, Sujata Rao, Feng Ren, Alex Zhavoronkov. ISM3091, a novel selective USP1 inhibitor as a targeted anticancer therapy [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 502.

Cyclin-dependent kinase 12 (CDK12) belongs to the cyclin-dependent kinase (CDK) family of serine/threonine protein kinases. The CDK12/cyclin K complex regulates the elongation step of RNA transcription by phosphorylating Ser2 on the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II, and selectively affects the expression of genes, such as BRCA1 and BRCA2, involved in DNA Damage Response (DDR). CDK12 mutations as well as overexpression have been reported in different types of malignancies, making it an attractive cancer target. In this study, a series of covalent CDK12 inhibitors were designed and profiled in biochemical and cellular assays. ISM6466A was identified as a lead compound, demonstrating good in vitro efficacy, ADME properties, safety pharmacology profile (e.g., CYP, hERG), and a reasonable in vivo PK profile. Biochemically, ISM6466A exhibited reasonable selectivity over other CDKs, and inhibited cell proliferation in a panel of tumor cell lines with IC50 values in the nanomolar range (e.g., IC50 of ~100nM in the TNBC and AML cell lines, SUM149PT and MV4-11, respectively). Cell-based assays showed that ISM6466A has a selective inhibitory action for the phosphorylation of Ser2 on the CTD, but not for that of Ser5 or Ser7. Notably, ISM6466A demonstrated excellent in vivo anti-tumor efficacy in the SUM149PT breast cancer CDX model (Tumor Growth Inhibition, TGI, of 114% at 60 mg/kg QD) and in the MV4-11 AML CDX model (TGI of 95% at 30 mg/kg QD), without body weight loss. Data from an endpoint PKPD study in the MV4-11 CDX model revealed that ISM6466A inhibited phosphorylation of Ser2 on the CTD, and also inhibited DDR genes such as BRCA1 and ATR. Results from a 28-day rat DRF study revealed that the compound did not show evident signs of toxicity at the tested doses, suggesting a reasonable safety window for ISM6466A.In summary, the novel, covalent, small-molecule CDK12 inhibitor, ISM6466A, demonstrated excellent preclinical efficacy along with favorable drug-like properties. Our results underscore the therapeutic potential of ISM6466A for the treatment of cancer. Citation Format: Lihua Min, Hongfu Lu, Yihong Zhang, Jianping Wu, Xin Cai, Min Zheng, Hui Cui, Junwen Qiao, Xiao Ding, Sujata Rao, Feng Ren, Alex Zhavoronkov. Discovery and evaluation of ISM6466A, a novel covalent CDK12 inhibitor for the treatment of cancer. [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 4989.

Cyclin-dependent kinase 8 (CDK8), as a kinase subunit of the Mediator complex, is involved in the regulation of RNA polymerase II-mediated transcription, thereby modulating multiple signaling pathways and multiple transcription factors involved in oncogenic control. CDK8 deregulation has been implicated in human diseases, particularly in acute myeloid leukemia (AML) and advanced solid tumors, where it has been reported as a putative oncogene. Here, we report the successful optimization of an azaindole series of CDK8 inhibitors that were identified and further progressed through a structure-based generative chemistry approach. In several optimization cycles, we improved in vitro microsomal stability, kinase selectivity, and in vivo pharmacokinetic profile cross-species, leading to the discovery of compound 23, which demonstrated robust tumor growth inhibition in multiple in vivo efficacy models after oral administration.

STUDY DESIGN:An experimental animal study of treatment of spinal cord injury (SCI).OBJECTIVE:This report aims to evaluate the in vivo effects of butylphthalide NBP on SCI biology and to explore its potential mechanism.SUMMARY OF BACKGROUND DATA:SCI causes great damage to humans. The inflammatory and reconstructive processes after SCI is regulated by activation of astroglial and microglial cells. Activated microglia/macrophages can be divided into M2 (anti-inflammatory) and M1 (pro-inflammatory) phenotypes. Butylphthalide (3-n-butylphthalide or NBP) treatment can significantly alleviate ischemic brain damage, and further study has confirmed that central neuroprotective effects can be realized by converting M1 polarized microglia/macrophages to the M2 phenotype. Thus far, it remains unknown whether NBP can modulate the transition of macrophages/microglia between the M1 and M2 phenotypes.METHODS:We randomly divided male mice into three groups (sham group, SCI group, SCI+ NBP group). Molecular and histological tests were performed to detect the macrophage/microglia polarization as well as the potential mechanism of NBP in vivo and in vitro.RESULT:It was found that NBP treatment significantly attenuated the motor dysfunction and neuronal apoptosis induced by SCI. Treatment with NBP could also reduce pro-inflammatory cytokine release after SCI and could facilitate macrophage/microglia M2 polarization and inhibit M1 polarization after SCI. To verify the findings in animal experiments, we examined the effect of NBP on BV2 cell polarization, the results showed that NBP treatment could enhance M2 polarization and inhibit M1 polarization, and that M2 polarization occurred in a p38-dependent manner.CONCLUSION:NBP plays an important role in the anti-inflammatory response in SCI via the facilitation of macrophage/microglia M2 polarization as well as the inhibition of macrophage/microglia M1 polarization. The M2 polarization of macrophages/microglia occurs via activation of p38 pathway.LEVEL OF EVIDENCE:3.