Multiple Mutants Research Articles

Abstract B002: Development of bifunctional CRBN-SOS1 degraders for treatment of mutant KRAS cancers

Abstract Although oncogenic Kirsten rat sarcoma virus (KRAS) was once deemed undruggable, recent advances have revealed promising anti-tumor effects with pan-KRAS and KRAS-mutant specific inhibitors in both preclinical and clinical settings. Despite the success of these inhibitors, the diversity of tumors as well as tumor sensitivity results in acquired drug resistance suggesting that combinational therapeutic approaches would be necessary to avoid this problem. An attractive combination strategy would be to couple KRAS inhibition with a blockade of its guanine exchange factor, Son of Sevenless (SOS1). This would keep KRAS in its inactive state as well as mitigate upstream MAPK pathway reactivation notoriously associated with inhibition in this pathway. Utilizing our PRODEGY platform, we developed bifunctional SOS1 degraders for the treatment of patients with KRAS mutant cancers. These degraders have shown rapid and potent CRBN-mediated degradation which exhibit DC50s <10nM in a H358 SOS1-HiBiT cell line. Our high-throughput HiBiT data translated into effective SOS1 degradation in KRAS mutant cells harboring G12C, G12D, G12V, G12S, or G13D mutations. SOS1 degradation was validated to be dependent on the proteasome and CRBN as cotreatment with proteasomal inhibitors (MG132 or MLN4924) or treatment in a CRBN KO cells abolished degradation. Treatment of our SOS1 degraders resulted in the suppression of multiple signaling markers downstream of SOS1. Given its role in RAS activation, we observed a reduction in active KRAS, HRAS and MRAS protein upon treatment of SOS1 degraders in KRAS G12C MIA PaCa-2 cells. Furthermore, pERK and pS6 levels were decreased in a more potent or comparable manner compared to the SOS1 inhibitor, BI3406, in multiple KRAS mutant cell lines. Out competing SOS1 degraders with other CRBN binders highlighted that inhibition of pERK and pS6 was due to CRBN-mediated degradation. To determine functional activity of our SOS1 degraders, we developed and optimized 3D proliferation assays in multiple KRAS mutant (G12C, G12V, G12S, G13D) cell lines which yielded IC50s ranging from 0.5 – 70nM. Similarly, 100- to 300-fold IC50 value shifts were observed in SOS1 KO and CRBN KO H358 cell lines validating that the anti-proliferative activity of SOS1 degraders is specific to its targets. Treatment of SOS1 degraders in KRAS-mutant xenograft models resulted in >90% degradation of SOS1 in tumors and subsequently led to significant tumor growth inhibition as a single agent. Combination treatment of the KRAS G12C inhibitor AMG510 and SOS1 degraders produced strong synergistic antiproliferative effects in H358 and MIA PaCa-2 cell lines. Additionally, synergy was observed with SOS1 degraders and the EGFR inhibitor, Erlotinib, as well as the MEK inhibitor, Trametinib, in KRAS G13D mutant LoVo cells. The ability of our SOS1 degraders to have potency alone as well as in combination with multiple types of different RTK/RAS/MAPK pathway inhibitor highlights the value of SOS1 degraders as a potential therapeutic option for a range of KRAS mutant cancers. Citation Format: Kyle Begovich, Angela Schoolmeesters, Navin Rajapakse, Maneesh Kumar, Elena Martinez-Terroba, Arvind Shakya, Brandon Whitefield, Nataraj Pagadala, Aparajita Chourasia, Leah Fung. Development of bifunctional CRBN-SOS1 degraders for treatment of mutant KRAS cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B002.

Abstract B018: The development of macrocyclic peptide Ras inhibitors

Abstract While there has been tremendous progress developing small molecule covalent K-Ras G12C inhibitors that bind Ras-off (GDP) and inhibit nucleotide exchange, there remains a need for additional K-Ras inhibitors with different Ras isoform/mutant selectivity profiles and Ras-on (GTP) mechanisms of inhibition. Of particular interest are compounds with a pan-K-Ras profile that inhibit multiple oncogenic mutants and wild type K-Ras, but achieve a therapeutic window by sparing H and N-Ras. In addition, addressing cancers driven by K-Ras oncogenic mutants beyond G12C, K-Ras amplification, or upstream dysregulation where K-Ras is maintained in the GTP-bound state will require “Ras-on” compounds that prevent effector binding. We were interested in the potential of peptide macrocycles that bind the Switch II pocket of K-Ras, in a different conformation than the small molecules, to inhibit Ras-on by allosterically blocking Raf and other effector binding. KRpep-2d is a 19-residue disulfide bridged peptide macrocycle discovered by phage display against K-Ras G12D bound to GDP. Due to its high molecular weight (2561 Da), polarity and metabolic instability, KRpep2d is inactive in in vitro cell assays. And while biochemical experiments have shown that it binds the Ras-off GDP-bound conformation of K-Ras with high affinity to inhibit nucleotide exchange, it has low affinity for K-Ras GTP and does not allosterically inhibit Raf binding. Using parallel macrocyclic peptide chemistry, we made 180 analogs of KRpep-2d to explore truncation of the exocyclic residues and replacement of the labile disulfide bond. UNP-6752, the best compound from this exploratory library, had K-Ras affinities similar to KRpep2d with a 50-fold K-Ras GDP/GTP preference, but was only 11 residues and cyclized with a non-reducible linker. Critically this compound lacked the eight arginine residues of KrPep-2d, which both preclude passive-cell permeability and induce mast cell degranulation. A second iteration of 350 compounds designed to explore single or multiple amino acid substitutions in the core of UNP-6752 led to compound UNP-7572 with sub-micromolar affinity for K-Ras GTP and an IC50 in a K-Ras-Raf TR-FRET assay of ~800 nM. Working from the UNP-7572 we are tuning selectivity for K-Ras and our progress further improving allosteric Ras-Raf inhibition, cell permeability and activity will be presented. Citation Format: Kristopher Josephson, Anthony Silvestri, Jennifer L. Fribourgh, Walter M. Bray, Brian David, Cameron C. Hanna, Sai Kumar Chakka, Alex Hyatt, Monique Porcella, Dio Dieppa-Matos, Cameron R. Pye, Joshua Schwochert. The development of macrocyclic peptide Ras inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B018.

Abstract PR10: IK-595, a MEK-RAF complex inhibitor, obviates CRAF mediated resistance resulting in superior RAS/MAPK pathway inhibition and anti-tumor activity in RAS/RAF altered cancers

Abstract Alterations in the RAS/RAF/MEK/ERK pathway are the most common drivers of oncogenesis. Although MEK is a clinically validated cancer target and several MEK inhibitors have been approved by the FDA, their clinical utility is limited to BRAFV600 mutant cancers and NF1 mutant neurofibromas. The limitations of these approved MEK inhibitors are inherently related to a narrow therapeutic window and their inability to completely inhibit MAPK signaling. Specifically, RAS mutant cancer cells have a stronger dependency on CRAF for MAPK signal transduction. The MEK inhibition and the subsequent reduction of phosphorylated ERK (pERK) by the approved MEK inhibitors triggers the relief of the ERK-dependent negative feedback control on CRAF, leading to CRAF mediated MEK reactivation and pERK rebound. In addition, CRAF has kinase independent functions that contribute to its anti-tumor apoptotic activities. We developed IK-595, a potent inhibitor of the MEK-RAF complex, to overcome the limitations of available MEK inhibitors. IK-595 stabilizes the MEK-BRAF and MEK-CRAF protein complexes both biochemically and in cells. By trapping MEK in an inactive complex with RAF, it blocks RAF-dependent MEK phosphorylation, limiting CRAF mediated MEK reactivation that hinders the efficacy of approved MEK inhibitors in RAS mutant tumors. Importantly, IK-595 demonstrates persistent and further inhibition of ERK phosphorylation than trametinib and VS-6766. This translates to potent inhibition of cell proliferation across a variety of MAPK pathway altered cancer cell lines, including cells with KRAS and NRAS mutations and RAF alterations. Moreover, IK-595 synergizes with KRASG12C, pan-RAF, and mTOR inhibitors in vitro, leading to enhanced anti-proliferation in cancer cells. In addition, IK-595 inhibits MEK and ERK phosphorylation and ERK target gene DUSP6 expression in vivo, driving robust anti-tumor efficacy in multiple KRAS and NRAS mutant, and CRAF amplified, mouse tumor models. Lastly, the pharmacokinetic properties of IK-595 provide a broader therapeutic window compared to available MEK inhibitors. Together, IK-595 is a novel MEK-RAF complex inhibitor that prolongs pathway inhibition, minimizing the potential for resistance, while providing an optimal therapeutic window for patients. Citation Format: Eric Haines, Michael Burke, Rachel Catterall, Victor De Jesus, Bin Li, Joseph D. Manna, George Punkosdy, Oksana Zavidij, Sarah R. Wessel, Grace Werosta, Jill Cavanaugh, Sheila Newhouse, Aravind Basavapathruni, Lan Xu, Sergio Santillana, X. Michelle Zhang, Sabine K. Ruppel. IK-595, a MEK-RAF complex inhibitor, obviates CRAF mediated resistance resulting in superior RAS/MAPK pathway inhibition and anti-tumor activity in RAS/RAF altered cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR10.

Discovery of imidazo[1,2-b]pyridazine macrocyclic derivatives as novel ALK inhibitors capable of combating multiple resistant mutants

Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI) often loses effectiveness against non-small cell lung malignancies (NSCLCs) with ALK gene rearrangements (ALK+). 19 novel imidazo[1,2-b]pyridazine macrocyclic derivatives were designed, synthesized, and tested for their biological activities in an effort to develop ALK inhibitors that would overcome second-generation ALK-TKIs, particularly the G1202R mutation and the lorlatinib-resistant L1196M/G1202R double mutations. Of all the target substances, O-10 had the most effective enzymatic inhibitory activity, with IC50 values for ALKWT, ALKG1202R, and ALKL1196M/G1202R of 2.6, 6.4, and 23 nM, respectively. O-10, on the other hand, reduced the growth of ALK-positive Karpas299, BaF3-EML4-ALKG1202R, and BaF3-EML4-ALKL1196M/G1202R cells with IC50 values of 38, 52, and 64 nM, respectively. This was equally effective to the reference drug Repotrectinib (IC50 = 40, 164, and 208 nM). The kinase selectivity profile, liver microsome stability test and in vivo pharmacokinetic properties in SD rats of compound O-10 were further evaluated. O-10 was regarded as an effective ALK inhibitor for the treatment of mutations overall.

Identifying Key Residues in Lysine Decarboxylase for Soluble Expression Using Consensus Design Soluble Mutant Screening (ConsenSing)

Although recent advances in deep learning approaches for protein engineering have enabled quick prediction of hot spot residues improving protein solubility, the predictions do not always correspond to an actual increase in solubility under experimental conditions. Therefore, developing methods that rapidly confirm the linkage between computational predictions and empirical results is essential to the success of improving protein solubility of target proteins. Here, we present a simple hybrid approach to computationally predict hot spots possibly improving protein solubility by sequence-based analysis and empirically explore valuable mutants using split GFP as a reporter system. Our approach, Consensus design Soluble Mutant Screening (ConsenSing), utilizes consensus sequence prediction to find hot spots for improvement of protein solubility and constructs a mutant library using Darwin assembly to cover all possible mutations in one pot but still keeps the library as compact as possible. This approach allowed us to identify multiple mutants of Escherichia coli lysine decarboxylase, LdcC, with substantial increases in soluble expression. Further investigation led us to pinpoint a single critical residue for the soluble expression of LdcC and unveiled its mechanism for such improvement. Our approach demonstrated that following a protein's natural evolutionary path provides insights to improve protein solubility and/or increase protein expression by a single residue mutation, which can significantly change the profile of protein solubility.

Abstract LB328: Discovery & characterization of a next-generation FGFR4 inhibitor overcoming resistant mutations

Abstract Introduction: Aberrant activation of FGF19-FGFR4 signaling pathway plays an essential role in the tumorigenesis of Hepatocellular carcinoma (HCC) and FGFR4 inhibitors have shown preliminary efficacy in recent clinical trials for patients with FGF19 overexpression. However, the observed responses only lasted a few months before tumors relapse. Acquired FGFR4 resistant mutations were found in ~30% of FGFR4 inhibitor responsive patients. Similar FGFR4 mutations haven also been found de novo in about 7-10% of Rhabdomyosarcoma (RMS) and ER-treated invasive lobular carcinoma patients. First generation FGFR4 inhibitors have minimal activity against these de novo or acquired resistant mutations. Therefore, next-generation of FGFR4 inhibitors are needed to overcome these resistant FGFR4 mutations to provide better treatment options for patients. Using advanced computation-aided structural analysis and medicinal chemistry design, we have discovered a next-generation small molecule FGFR4 inhibitor, ABSK012, and demonstrated its strong activities against de novo and acquired resistant FGFR4 mutations while retaining inhibition for wild-type FGFR4. Method: Inhibitory activity of ABSK012 against FGFR4 and FGFR4 mutants was evaluated by MSA assay and its inhibition on FGFR4-dependent cell proliferation was evaluated by Celltiter-Glo assay in wile type FGFR4-dependent cancer cell lines or mutant FGFR4-dependent Ba/f3 cell lines. Its selectivity against other FGFR family member and kinases was analyzed by cellular and KinomeScan profiling. Efficacy studies were conducted in HCC xenograft models and mutant FGFR4-dependent xenograft models including a RMS PDX model harboring FGFR4 V550L mutation. Results: ABSK012 demonstrated strong potency over multiple FGFR4 mutants that are insensitive to a first generation FGFR4 inhibitor BLU-554. It also inhibited wild-type FGFR4 with IC50<5 nM in biochemical assay and exhibited great selectivity against other kinases. In multiple mutant and wild-type FGFR4-dependent cell lines, ABSK012 demonstrated significantly improved anti-proliferation activity. In preclinical in vivo studies in HCC models, oral administration of ABSK012 strongly inhibited the tumor growth at doses without obvious toxicities. More importantly, in a RMS PDX xenograft harboring FGFR4 V550L mutation, ABSK012 also showed significant anti-tumor activity. Other ADME and safety profiling demonstrated excellent drug-like properties of ABSK012. Conclusion: ABSK012, presented here by Abbisko Therapeutics, is a highly potent, selective, and next-generation small molecule FGFR4 inhibitor overcoming FGFR4 mutations resistant to first-generation inhibitors. Its superior preclinical profile supports its fast-track development into clinic. Citation Format: Haiyan Ying, Nannan Zhang, Haibing Deng, Fei Yang, Wenqun Xin, Bin Shen, Hongping Yu, Zhui Chen, Yao-chang Xu. Discovery & characterization of a next-generation FGFR4 inhibitor overcoming resistant mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB328.

Abstract LB222: An mRNA-based cancer vaccine multi-targeting KRAS mutations inhibits tumor growth by increasing immune response in KRAS mutant LL/2 mouse model

Abstract Kirsten rat sarcoma viral oncogene (KRAS) mutations are present in 26% of non-small cell lung cancer (NSCLC) patients (The Cancer Genome Atlas data). Studies have reported the presence of spatial or intra-tumoral heterogeneity of KRAS mutations. Though resistance to mutant KRAS inhibitors targeting a specific form of major KRAS mutations (e.g, G12C or G12D) is multifaceted, intra-tumoral heterogeneity of KRAS mutation is one of the crucial factors for the intrinsic resistance to the inhibitor. Multi-targeting of different KRAS mutations can prevent resistance due to the heterogeneity of KRAS mutations within a tumor. To achieve this, we incorporated an mRNA-based cancer vaccine containing transcripts for multiple KRAS mutant antigens. Our KRAS cancer vaccine (CV) candidate showed significantly attenuated tumor growth by 37% in the syngeneic KRAS G12C-expressing LL/2 tumor-bearing mice. CV treatment group showed significantly decreased tumor size by 45.4% after biopsy. Moreover, the mice treated with KRAS CV showed spleen enlarged by 42.6% indicating increased immune responses. To confirm, the expansion of T cells in the cancer vaccine-treated group, we collected tumor tissues and analyzed proportions of CD4+ and CD8+ T lymphocytes in the tumor. We found a 14.5-fold increase in infiltration of CD8+ T cells and a 0.5-fold increase in CD8+ CD44+ memory/effector T cells in the tumors from mice treated with KRAS CV. Whereas CD4+ Foxp3+ Treg cells were decreased by 3.9-fold in the tumor. HLA-A*02 is the most common MHC class I allele. To investigate the changes in MHC expression, we conducted FACS analysis using HLA-A*02 antibodies after ex vivo treatment of KRAS CV on the human peripheral blood mononuclear cells (hPBMCs). KRAS CV treatment facilitated higher proportions of HLA-A*02+ monocytes (CD14+) and B cells (CD19+). Though LL/2 tumor model has been considered an immunosuppressive model, our results suggest that the KRAS CV can significantly enhance immune responses, thereby suppressing tumor growth. Citation Format: Seung-Hyun Shin, Youngjin Han, Chang Gyu Lim, Yong Ho Heo, Seongju Jeong, Yu-Yon Kim, In Young Choi. An mRNA-based cancer vaccine multi-targeting KRAS mutations inhibits tumor growth by increasing immune response in KRAS mutant LL/2 mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB222.

Molecular epidemiology of potential candidate markers for chloroquine resistance in imported Plasmodium vivax malaria cases in Iran

BackgroundThe spread of Plasmodium vivax strains resistant to chloroquine (CQ) has posed a challenge to control strategies aimed at eliminating malaria. Molecular analysis of candidate resistance markers is very important for monitoring the P. vivax resistance to CQ in different endemic regions. In the present study, the multidrug resistance 1 (pvmdr1) gene, a possible marker for CQ resistance in P. vivax, was evaluated by molecular methods.MethodsA simple PCR–RFLP method was developed for mutation analysis in pvmdr1 gene. A number of 120 blood spots were obtained from patients with P. vivax mono-infection in 2021. All of the samples were collected from Pakistani patients who travelled to Iran.ResultsNone of the samples had any mutation at codon 976 of pvmdr1, while the 1076 mutation was detected in 96.2% of the examined isolates. Only two pvmdr1 haplotypes were identified, including the single mutant (Y976/1076L) as the most prevalent haplotype (with 96.2% frequency) and the wild type (Y976/F1076; with 3.8% frequency).ConclusionsIn this study, the major CQ resistance-mediating mutation and multiple mutant haplotypes of the pvmdr1 gene was not detected. However, continuous monitoring of drug resistance markers and close supervision of the efficacy of CQ is essential to detect the potential emergence of CQ-resistant P. vivax isolates in Iran. This data is important for performing future epidemiological surveillance to monitor CQ resistance in this endemic area and the bordering regions.

Abstract 1578: Novel Cereblon mediated bifunctional degraders of SOS1 for treatment of pan-KRAS mutant tumors

Abstract KRAS, a GTPase that regulates cellular growth, is the most frequently mutated oncogene with implications in non-small cell lung, pancreatic and colorectal cancers. Hyperactivation of KRAS in mutant alleles favors KRAS in its active GTP-bound state over its inactive GDP-bound state. Once deemed undruggable, recent advances have revealed promising anti-tumor effects with pan-KRAS and KRAS-mutant specific inhibitors in both preclinical and clinical settings. Despite the success of these inhibitors, the diversity of tumors as well as tumor sensitivity results in acquired drug resistance suggesting that combinational therapeutic approaches would be necessary to avoid this problem. An attractive dual approach would be to couple KRAS inhibition with a blockade of its guanine exchange factor (GEF), SOS1, to keep KRAS in its inactive state as well as prevent upstream pathway reactivation. Utilizing our PRODEGY platform, we developed bifunctional SOS1 degraders for the treatment of patients with KRAS mutant cancers. These degraders have shown rapid and potent Cereblon (CRBN)-mediated degradation which exhibit DC50s <10nM in a H358 SOS1-HiBiT cell line. Our high- throughput HiBiT data translated into effective SOS1 degradation in KRAS mutant cells harboring G12C, G12D, G12V, G12S, or G13D mutations. SOS1 degradation was validated to be dependent on the proteasome and CRBN as cotreatment with proteasomal inhibitors (MG132 or MLN4924) or treatment in a CRBN KO cells abolished degradation. Additionally, treatment of SOS1 degraders resulted in suppression of downstream signaling markers, pERK and pS6, in both 3D immunoblots and high-throughput assays with pERK IC50s <10nM. Chemical competitor assays also revealed that inhibition of pERK and pS6 was dependent on CRBN-mediated competition. To determine functional activity of our SOS1 degraders, we developed and optimized 3D proliferation assays in multiple KRAS mutant (G12C, G12D, G12V, G12S, G13D) cell lines which yielded IC50s ranging from 0.5 - 70nM. Similarly, 100- to 300-fold IC50 value shifts were observed in SOS1 KO and CRBN KO H358 cell lines validating that the anti-proliferative activity of SOS1 degraders is specific to its targets. Treatment of SOS1 degraders in KRAS-mutant xenograft models resulted in >75% degradation of SOS1 in tumors and subsequently led to significant tumor growth inhibition as a single agent. SOS1 degradation also provided synergistic anti-proliferation effects with other RAS/MAPK/AKT inhibition in in vitro studies. For example, combination treatment of AMG510 and SOS1 degraders resulted in strong synergistic effects in KRAS G12C H358 cells. Together, our data SOS1 degradation could be a valuable potential treatment option for a range of KRAS mutant cancers. Citation Format: Kyle Begovich, Angela Schoolmeesters, Navin Rajapakse, Maneesh Kumar, Arvind Shakya, Brandon Whitefield, Elena Martinez-Terroba, Nataraj Pagadala, Shenlin Huang, Aparajita H. Chourasia, Leah Fung. Novel Cereblon mediated bifunctional degraders of SOS1 for treatment of pan-KRAS mutant tumors [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 1578.

Abstract 1660: Preclinical investigation of orally bioavailable, potent KRASMulti inhibitor JAB-23425

Abstract Background: Mutant KRAS is an important driver of tumor and has remained an undruggable target for decades. Despite the success of mutant-specific KRASG12C inhibitors, there is limited therapeutics for most KRAS-mutated patients (e.g., KRASG12D, KRASG12V and KRASG13D). Developing KRASMulti inhibitor targeting a broad range of KRAS-driven cancers is another promising strategy. Methods: The Induced Allosteric Drug Discovery Platform (IADDP) integrating medicinal chemistry, biophysical studies and computational techniques was applied for the development of JAB-23425. Surface Plasmon Resonance (SPR) was applied to determine JAB-23425 binding affinity on KRAS. Guanine nucleotide exchange assays and effector interaction assays were applied to determine the functional inhibition of GDP- and GTP-bound KRAS in biochemical level. ERK phosphorylation assays and CellTiter-Glo assays in both KRAS-dependent and KRAS-independent cells were performed to evaluate the inhibitory activities of downstream signaling of KRAS and cell viability. In vivo PK-PD study was conducted to evaluate the relationship between drug concentration and the level of tumor ERK phosphorylation. Antitumor activity of JAB-23425 was evaluated in multiple mouse models of KRAS mutant cancer. Results: Affinity measurement by SPR indicated strong binding of JAB-23425 to KRASG12D, KRASG12V, KRASG13D, KRASG12A, KRASG12R and KRASQ61H mutants as well as wild-type KRAS in both GDP- and GTP-bound states, with most KD in the sub-nanomolar range. Biochemical assays consistently demonstrated high potency of JAB-23425 on multiple KRAS mutants, and good selectivity over HRAS and NRAS. At the cellular level, JAB-23425 significantly reduced downstream ERK phosphorylation (most IC50 around 1 nM) and inhibited cell viability (most IC50 lower than 10 nM) on KRAS-dependent tumor cells harboring various KRAS mutations and amplification, with good selectivity against normal cells and KRAS-independent tumor cells (e.g., NRASmut and BRAFV600E). Above in vitro data showed that JAB-23425 is a potent and selective KRASMulti inhibitor and can spare HRAS and NRAS inhibition. In vivo PK-PD study showed that JAB-23425 exposure in plasma and tumor has good correlation with inhibition of tumor ERK phosphorylation. Furthermore, JAB-23425 monotherapy by oral administration showed potent antitumor activity and good tolerability in multiple KRASG12D, KRASG12V and KRASG13D mouse models. Conclusions: Our preclinical results identified JAB-23425 as a highly potent, orally bioavailable KRASMulti inhibitor, that can target multiple KRAS-driven cancers. Citation Format: Peng Wang, Yanping Wang, Xin Sun, Dan Liu, Xiaoyu Liu, Wei Zhang, Xueting He, Rui Zhou, Cunbo Ma, Amin Li, Yiwei Lin, Wei Long. Preclinical investigation of orally bioavailable, potent KRASMulti inhibitor JAB-23425 [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 1660.

Abstract 5739: Targeting WEE1 to improve the therapy of KRAS G12C mutant non-small cell lung cancer

Abstract Background: While sotorasib is the first clinically approved small-molecule inhibitor of KRAS G12C in non-small-cell lung cancer (NSCLC), efficacy of sotorasib alone is limited, presumably due to adaptive resistance mechanisms in cancer cells. To expand therapeutic potential of sotorasib, combination strategies need to be developed. In this study, we conducted a large-scale screening to identify novel combination candidates with sotorasib. In addition, molecular mechanisms of synergistic effects were investigated in multiple preclinical models. Methods: To identify novel combination partners of sotorasib, we conducted high-throughput screening using a kinase inhibitor library containing 1,400 chemical compounds. Anti-proliferative and apoptotic effects in vitro were assessed by ATP-based cell viability assay and/or clonogenic assay, and caspase 3/7 assay and/or Annexin V assay, respectively. Synergistic effects were evaluated by calculating the BLISS index and visualized by SynergyFinder Plus. Immunoblotting was performed using antibodies against pCDK1 (Tyr15), pERK, pAkt, MCL1, BIM, PUMA, BCL2, or BCL-xL. In vivo efficacy studies were performed using a KRAS G12C mutant H358 xenograft nude mouse model. Results: Through the large-scale unbiased combination screening of -1,400 kinase inhibitors, we identified a WEE1 inhibitor (AZD1775), a G2/M checkpoint abrogator in clinical-stage development, as a promising combination partner of sotorasib in KRAS G12C mutant NSCLC. The synergistic effects in vitro were broadly observed in multiple KRAS G12C mutant NSCLC cell lines, regardless of single-agent sensitivity to sotrasib or co-occurring oncogenic mutation profiles. The combination treatment upregulated pro-apoptotic protein BIM, leading to apoptosis. In vivo efficacy studies in H358-xenografted models also demonstrated remarkable tumor regression and durable response in mice treated with sotorasib and AZD1775 compared to ones with a single agent alone. Conclusion: We identify AZD1775, a WEE1 inhibitor, as a novel combination candidate, which enhanced the anti-tumor activity of sotorasib both in vitro and in vivo preclinical models. These findings can lead to a novel therapeutic strategy for KRAS G12C mutant NSCLC. Citation Format: Gaku Yamamoto, Kosuke Tanaka, Ryo Kamata, Shunta Mori, Jie Liu, Toyohiro Yamauchi, Yuta Sakae, Akihiro Ohashi, Susumu S. Kobayashi. Targeting WEE1 to improve the therapy of KRAS G12C mutant non-small cell lung 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 5739.

Design, synthesis and biological evaluation of novel indolin-2-one derivatives as potent second-generation TRKs inhibitors

Tropomyosin receptor kinases (TRKs) are effective targets for anti-cancer drug discovery. The first-generation type I TRKs inhibitors, larotrectinib and entrectinib, exhibit durable disease control in the clinic. The emergence of acquired resistance mediated by secondary mutations in the TRKs domain significantly reduces the therapeutic efficacy of these two drugs, indicating an unmet clinical need. In this study, we designed a potent and orally bioavailable TRK inhibitor, compound 24b, using a molecular hybridization strategy. Compound 24b exhibited significant inhibitory potency against multiple TRK mutants in both biochemical and cellular assays. Furthermore, compound 24b induced apoptosis of Ba/F3-TRKAG595R and Ba/F3-TRKAG667C cells in a dose-dependent manner. Additionally, compound 24b exhibited moderate kinase selectivity. In vitro stability revealed that compound 24b showed excellent plasma stability (t1/2 > 289.1 min) and moderate liver microsomal stability (t1/2 = 44.3 min). Pharmacokinetic studies have revealed that compound 24b is an orally bioavailable TRK inhibitor with a good oral bioavailability of 116.07%. These results indicate that compound 24b be used as a lead molecule for further modifications to overcome drug-resistant mutants of TRK.

Exploring the metabolic and physiological roles of HQT in S. lycopersicum by gene editing.

The most abundant phenolic compound in Solanaceous plants is chlorogenic acid (CGA), which possesses protective properties such as antimicrobial and antioxidant activities. These properties are particularly relevant when plants are under adverse conditions, such as pathogen attack, excess light, or extreme temperatures that cause oxidative stress. Additionally, CGA has been shown to absorb UV-B light. In tomato and potato, CGA is mainly produced through the HQT pathway mediated by the enzyme hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase. However, the absence of natural or induced mutants of this gene has made it unclear whether other pathways contribute to CGA production and accumulation. To address this question, we used CRISPR technology to generate multiple knock-out mutant lines in the tomato HQT gene. The resulting slhqt plants did not accumulate CGA or other caffeoylquinic acids (CQAs) in various parts of the plant, indicating that CQA biosynthesis depends almost entirely on the HQT pathway in tomato and, likely, other Solanaceous crops. We also found that the lack of CGA in slhqt plants led to higher levels of hydroxycinnamoyl-glucose and flavonoids compared to wild-type plants. Gene expression analysis revealed that this metabolic reorganization was partly due to flux redirection, but also involved modulation of important transcription factor genes that regulate secondary metabolism and sense environmental conditions. Finally, we investigated the physiological role of CGA in tomato and found that it accumulates in the upper epidermis where it acts as a protector against UV-B irradiation.

The crosstalk between ubiquitination and endocrine therapy.

Endocrine therapy (ET), also known as hormone therapy, refers to the treatment of tumors by regulating and changing the endocrine environment and hormone levels. Its related mechanism is mainly through reducing hormone levels and blocking the binding of hormones to corresponding receptors, thus blocking the signal transduction pathway to stimulate tumor growth. However, with the application of ET, some patients show resistance to ET, which is attributed to abnormal accumulation of hormone receptors (HRs) and the production of multiple mutants of HRs. The targeted degradation of abnormal accumulation protein mediated by ubiquitination is an important approach that regulates the protein level and function of intracellular proteins in eukaryotes. Here, we provide a brief description of the traditional and novel drugs available for ET in this review. Then, we introduce the link between ubiquitination and ET. In the end, we elaborate the clinical application of ET combined with ubiquitination-related molecules. KEY MESSAGES: • A brief description of the traditional and novel drugs available for endocrine therapy (ET). •The link between ubiquitination and ET. •The clinical application of ET combined with ubiquitination-related molecules.

Development of fluorine-substituted NH2-biphenyl-diarylpyrimidines as highly potent non-nucleoside reverse transcriptase inhibitors: Boosting the safety and metabolic stability.

Our recent studies for nonnucleoside reverse transcriptase inhibitors identified a highly potent compound JK-4b against WT HIV-1 (EC50=1.0nmol/L), but the poor metabolic stability in human liver microsomes (t 1/2=14.6min) and insufficient selectivity (SI=2059) with high cytotoxicity (CC50=2.08μmol/L) remained major issues associated with JK-4b. The present efforts were devoted to the introduction of fluorine into the biphenyl ring of JK-4b, leading to the discovery of a novel series of fluorine-substituted NH2-biphenyl-diarylpyrimidines with noticeable inhibitory activity toward WT HIV-1 strain (EC50=1.8-349nmol/L). The best compound 5t in this collection (EC50=1.8nmol/L, CC50=117μmol/L) was 32-fold in selectivity (SI=66,443) compared to JK-4b and showed remarkable potency toward clinically multiple mutant strains, such as L100I, K103N, E138K, and Y181C. The metabolic stability of 5t was also significantly improved (t 1/2=74.52min), approximately 5-fold higher than JK-4b in human liver microsomes (t 1/2=14.6min). Also, 5t possessed good stability in both human and monkey plasma. No significant invitro inhibition effect toward CYP enzyme and hERG was observed. The single-dose acute toxicity test did not induce mice death or obvious pathological damage. These findings pave the way for further development of 5t as a drug candidate.

Machine Learning Guided Design of High-Affinity ACE2 Decoys for SARS-CoV-2 Neutralization.

A potential therapeutic strategy for neutralizing SARS-CoV-2 infection is engineering high-affinity soluble ACE2 decoy proteins to compete for binding to the viral spike (S) protein. Previously, a deep mutational scan of ACE2 was performed and has led to the identification of a triple mutant variant, named sACE22.v.2.4, that exhibits subnanomolar affinity to the receptor-binding domain (RBD) of S. Using a recently developed transfer learning algorithm, TLmutation, we sought to identify other ACE2 variants that may exhibit similar binding affinity with decreased mutational load. Upon training a TLmutation model on the effects of single mutations, we identified multiple ACE2 double mutants that bind SARS-CoV-2 S with tighter affinity as compared to the wild type, most notably L79V;N90D that binds RBD similarly to ACE22.v.2.4. The experimental validation of the double mutants successfully demonstrates the use of machine learning approaches for engineering protein-protein interactions and identifying high-affinity ACE2 peptides for targeting SARS-CoV-2.

Structural insights into the complex between Arf1 and ASAP1 at the membrane surface by NMR.

ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs) are enzymes that need to bind to membranes to catalyze the hydrolysis of GTP bound to the small GTP-binding protein Arf1. Simultaneous binding of the Pleckstrin homology (PH) domain of the ArfGAP ASAP1 to membranes containing the phosphatidylinositol phosphate PIP(4,5)P2 and to Arf1.GTP is key for maximum GTP hydrolysis, but is not fully understood. Recently, we showed that binding of multiple PI(4,5)P2 molecules to the ASAP1 PH domain triggers an allosteric conformational switch (PIP switch) involving regions distant from the membrane interface. Here, we examined the interface between Arf1 and ASAP1 PH at the membrane surface of nanodiscs by Nuclear Magnetic Resonance (NMR) methods. Chemical Shift Perturbations (CSPs) observed upon complex formation and distance restraints obtained from Paramagnetic Relaxation Enhancement (PRE) NMR experiments between multiple MTSL-tagged Arf1 mutants and isotopically labeled ASAP1 PH were combined to build a model of the Arf1:ASAP1 PH complex at the membrane. Interestingly, the PIP switch resides at the interface between Arf1 and ASAP1 PH, shedding light on how PIP binding to ASAP1 PH may affect GTP hydrolysis.

Pharmacochaperones as a novel approach to rescue misfolding rod opsin mutants associated with retinitis pigmentosa.

The correct trafficking of folded, functional rhodopsin (Rho) is critical to sustaining vision. Inherited destabilizing mutations in the rhodopsin (RHO) gene lead to a spectrum of blinding diseases called retinitis pigmentosa (RP) with currently no cure available. Therapeutic approaches to correct these impartments focus on the development of pharmacological chaperones that stabilize rod opsin. The native ligand, 11-cis-retinal, improves protein stability, folding, and trafficking of some mutated Rho variants. However, its chemical instability, toxicity, and poor pharmacokinetics limit its use as a therapeutic. In our laboratory, we focus our attention on finding chaperones with natural-like product scaffolds due to their broad chemical diversity. Furthermore, ∼50% of marketed small molecule drugs are derived from natural products scaffold, and some of them target GPCRs. Thus, we integrated computational, biochemical, biophysical, and in vivo analysis to evaluate non-retinoid ligands with diverse chemical scaffolds as potential chaperones of misfolding Rho variants. Our study led to the discovery of a novel chromenone-containing small molecule with favorable pharmacological properties that through direct binding to the orthosteric pocket enhanced rod opsin stability. This compound improved the membrane trafficking of multiple rod opsin mutants in vitro. Importantly, our studies unraveled the pharmacological potential of this new chromenone rod opsin ligand to slow down the progression of photoreceptor degeneration in the P23H Rho knock-in mice, a model of RP. Together, our studies encourage further search and evaluation of novel non-retinoid compounds that could mitigate the misfolding and mistrafficking of the Rho mutants to prolong the survival of photoreceptors in the retina, and thus delaying vision loss in RP.

Evolution and mammalian adaptation of H3 and H10 subtype avian influenza viruses in wild birds in Yancheng Wetland of China

Wild birds play a critical role in avian influenza virus (AIV) ecology and some outbreaks of avian influenza in human originate from wild birds, suggesting that epidemiological surveillance and interspecies-transmission analysis of AIVs in wild birds are critical. Since 2019, we have performed sampling in Yancheng Wetland along the East Asian-Australasian Flyway. Totally, 2054 fecal swabs were collected and one H3N8, two H3N1, one H10N8, and three H10N1 were isolated. Three H3 gene of AIVs we isolated belonged to Eurasian lineage, but the four H10 gene clustered into North American lineage. What’s more, the H3 and the foreign H10 gene had generated novel reassortants in Yancheng wetland. Receptor binding assay indicated that nearly all strains, except D369/H10N1, presented a dual receptor-binding profile and bound to avian-type receptor preferentially. In animal experiment, all isolates could infect mice without prior adaptation and induce histopathological changes in mice lungs, moreover, all H3 subtype AIVs obviously triggered weight loss of mice. In addition to lung and turbinate, D322/H3N1, D338/H3N8, D211/H10N8 and D266/H10N1 could spread to brain and kidney or liver or spleen, showing a wider range of tissue tropism. Multiple mutants associated with mammalian adaptation were also detected in all isolates according to molecular analysis. These findings revealed that H3 and H10 AIVs circulating in wild birds in Yancheng Wetland underwent complex reassortment and increased mammalian adaptation, which highlighted the necessity to monitor the diverse reassortment of AIVs in wild birds and evaluate the risks of H3 and H10 viruses to human health.

The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems

Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to generate multiple knockout mutants of C. orbiculare efficiently. We first found that CRISPR-Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR-Cas9 vector, more than 90% of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre-loxP marker recycling system for C. orbiculare because a limited availability of desired selective markers hampers sequential gene disruption. In this system, the integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling the reuse of the same selective marker for the next transformation. Using our CRISPR-Cas9 and Cre-loxP systems, we attempted to identify functional sugar transporters involved in fungal virulence. Multiple disruptions of putative quinate transporter genes restricted fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. However, our analyses showed that quinate acquisition was dispensable for infection to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants that we established in this study, we inferred that repetitive rounds of gene disruption using CRISPR-Cas9 and Cre-loxP systems do not cause adverse effects on fungal virulence and growth. Therefore, these systems will be powerful tools to perform a systematic loss-of-function approach for C. orbiculare.