Selectivity Profile Research Articles

Discovery and SAR of JTE-151: A Novel RORγ Inhibitor for Clinical Development.

A number of RORγ inhibitors have been reported over the past decade. There were also several examples advancing to human clinical trials, however, none of them has reached the market yet, suggesting that there could be common obstacles for their future development. As was expected from the general homology of nuclear receptor ligands, insufficient selectivity as well as poor physicochemical properties were identified as potential risks for a RORγ program. Based on such considerations, we conducted a SAR investigation by prioritizing drug-like properties to mitigate such potential drawbacks. After an intensive SAR exploration with strong emphasis on "drug-likeness" indices, an orally available RORγ inhibitor, JTE-151, was finally generated and was advanced to a human clinical trial. The compound was confirmed to possess highly selective profiles along with good metabolic stability, and most beneficially, no serious adverse events (SAE) and good PK profiles were observed in the human clinical trial.

Structural and functional perspectives on interactions between synthetic cathinones and monoamine transporters.

Synthetic cathinone derivatives comprise a family of psychoactive compounds structurally related to amphetamine. Over the last decade, clandestine chemists have synthesized a consistent stream of innovative cathinone derivatives to outpace governmental regulatory restrictions. Many of these unregulated substances are produced and distributed as designer drugs. Two of the principal chemical scaffolds exploited to expand the synthetic cathinone family are methcathinone and α-pyrrolidinopentiophenone (or α-pyrrolidinovalerophenone, α-PVP). These compounds' main physiological targets are monoamine transporters, where they promote addiction by potentiating dopaminergic neurotransmission. This chapter describes techniques used to study the pharmacodynamic properties of cathinones at monoamine transporters in vitro. Biochemical techniques described include uptake inhibition and release assays in rat brain synaptosomes and in mammalian expression systems. Electrophysiological techniques include current measurements using the voltage clamp technique. We describe a Ca2+ mobilization assay wherein voltage-gated Ca2+ channels function as reporters to study the action of synthetic cathinones at monoamine transporters. We discuss results from systematic structure-activity relationship studies on simple and complex cathinones at monoamine transporters with an emphasis on identifying structural moieties that modulate potency and selectivity at these transporters. Moreover, different profiles of selectivity at monoamine transporters directly predict compounds associated with behavioral and subjective effects within animals and humans. In conclusion, clarification of the structural aspects of compounds which modulate potency and selectivity at monoamine transporters is critical to identify and predict potential addictive drugs. This knowledge may allow prompt allocation of resources toward drugs that represent the greatest threats after drugs are identified by forensic laboratories.

Selective Profiles among Single or Double Stranded DNA or RNA Viruses Detect their Double or Single Stranded Condition

Selective Profiles among Single or Double Stranded DNA or RNA Viruses Detect their Double or Single Stranded Condition

Identification and engineering of potent cyclic peptides with selective or promiscuous binding through biochemical profiling and bioinformatic data analysis.

As our understanding of biological systems grows, so does the need to selectively target individual or multiple members of specific protein families in order to probe their function. Many targets of current biological and pharmaceutical interest are part of a large family of closely related proteins and achieving ligand selectivity often remains either an elusive or time-consuming endeavour. Cyclic peptides (CPs) occupy a key niche in ligand space, able to achieve high affinity and selectivity while retaining synthetic accessibility. De novo cyclic peptide ligands can be rapidly generated against a given target using mRNA display. In this study we harness mRNA display technology and the wealth of next generation sequencing (NGS) data generated to explore both experimental approaches and bioinformatic, statistical data analysis of peptide enrichment in cross-screen selections to rapidly generate high affinity CPs with differing intra-family protein selectivity profiles against fibroblast growth factor receptor (FGF-R) family proteins. Using these methods, CPs with distinct selectivity profiles can be generated which can serve as valuable tool compounds to decipher biological questions.

Efficacy of Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) Supplementation on Psychological Stress and Selective Fitness Profile Parameters in Female College Students in West Bengal, India.

Psychological disparities impact physical activity and fitness in sedentary female college students by affecting cardiovascular efficiency. Ganoderma lucidum, vitality-enhancing herb alleviates health and rejuvenates the mind-body to improve endurance fitness. A double-blinded, randomized, placebo-controlled parallel design study was conducted to determine whether supplementation of G. lucidum in daily dosages of 500 mg (GL500mg group) and 1000 mg (GL1000mg group) improves psychophysiological health capabilities during the different phases of the experimental trial. Analysis for pre-experimental trial (day 0), experimental trial (day 15), and post-experimental trial (after day 30) on anthropometric, psychological, physiological, and physical fitness parameters were executed. Seventy-eight participants (n = 78, age 20.64 ± 3.21 years) were assigned randomly and equally divided (n = 26) to one of the three treatment groups for intragroup and intergroup comparisons. Significant differences in the post-experimental GL1000mg group for heart rate (HR), maximal oxygen consumption (VO2max), physical work capacity (PWC170), and right-hand grip strength (P < 0.05) compared with the placebo group were observed. GL1000mg-supplemented group also significantly improved (P < 0.05) HR, VO2max and PWC170 (P < 0.001) after pre- to post-trials. Experimental trial between placebo and GL1000mg group and post-experimental trial between the GL500mg and GL1000mg group showed significant changes in VO2max(P < 0.001) and PWC170 (P < 0.05). Anxiety, depression, vitality and positive well-being scores significantly improved, leading to improved psychological well-being after GL1000mg supplementation. GL1000mg supplementation for 30 days might act as a longevity-promoting tonic for endurance and strength performance by ameliorating stress to improve the overall psychophysiological health, vitality and quality of life for better cardiovascular efficacy.

Nature-derived Alkaloids as a Promising Bioactive Compound in Drug Discovery to Meet Global Leishmania Needs

Background: Natural products have been important resources for potential drug development. Among the many classes of natural products, alkaloids have the most therapeutic applications. Treatment of leishmaniasis by chemical drugs remains a challenge because of toxic side effects, limited efficacy, and drug resistance. This review focuses to embrace all researches on leishmanicidal alkaloids over a specific range of time, with special consideration the molecular mechanism of action, and structure-related activity. Methods: All publications (in English) from Web of Science, PubMed, Science Direct, Scopus, and Google Scholar from 2000-2021 using a variety of keywords such as natural alkaloids, herbal alkaloids, marine alkaloids along with leishmaniasis were included in the present study. In this summary, the focus is mainly on natural alkaloids of plant, mineral, marine origin, etc., which have empirically demonstrated the antileishmanial effect. Results: Fourteen categories of alkaloids with anti-leishmaniasis activity were extracted from the articles. The highest number of alkaloids belonged to isoquinoline, indole, and quinoline alkaloids (21.78%, 20.67%, and 16.48% respectively). This review indicated that the mentioned alkaloids are able to inhibit the proliferation of parasites, the respiratory chain and protein synthesis, arrest the cell cycle, disrupt the mitochondrial membrane integrity, inhibit leishmanial topoisomerase, induce mitochondrial dysfunction, and changes in the parasite morphology. Discussion: The present study highlighted antileishmanial alkaloids that are active against different species of Leishmania in vitro and some of them are also active in visceral and cutaneous leishmaniasis models. However, more clinical studies are needed to clarify the anti-leishmanial activity of alkaloids against leishmania in detail. Conclusion: Among the reported compounds, two main classes of alkaloids including isoquinoline and indole alkaloids cover a wider range of anti-parasitic compounds, and structure-activity relationships (SAR) studies of these molecular skeletons may be good lead compounds and afford the structural clues to develop novel medicines with more selective therapeutic profiles.

Enhancing the antimycobacterial efficacy of pyridine-4-carbohydrazide: linkage to additional antimicrobial agents via oxocarboxylic acids

Linking pyridine-4-carbohydrazide to other antimicrobial agents via oxocarboxylic acids significantly enhances efficacy against mycobacteria with promising selectivity and safety profiles.

Chlorinated Enyne Fatty Acid Amides from a Marine Cyanobacterium: Discovery of Taveuniamides L-M and Pharmacological Characterization of Taveuniamide F as a GPCR Antagonist with CNR1 Selectivity.

NMR and MS/MS-based metabolomics of a cyanobacterial extract from Piti Bomb Holes, Guam, indicated the presence of unique enyne-containing halogenated fatty acid amides. We isolated three new compounds of this class, taveuniamides L-N (1-3), along with the previously reported taveuniamide F (4), which was the most abundant analog. The planar structures of the new compounds were established using 1D and 2D NMR as well as mass spectrometry. We established the configuration of this chemical class to be R at C-8 via Mosher's analysis of 4 after reduction of the carboxamide group. Our biological investigations with 4 revealed that the compound binds to the cannabinoid receptor CNR1, acting as an antagonist/inverse agonist in the canonical G-protein signaling pathways. In selectivity profiling against 168 GPCR targets using the β-arrestin functional assay, we found that 4 antagonizes GPR119, NPSR1b, CCR9, CHRM4, GPR120, HTR2A, and GPR103, in addition to CNR1. Interestingly, 4 showed a 6.8-fold selectivity for CNR1 over CNR2. The binding mode of 4 to CNR1 was investigated using docking and molecular dynamics simulations with both natural and unnatural stereoisomers, revealing important CNR1 residues for the interaction and also providing a possible reasoning for the observed CNR1/CNR2 selectivity.

Structure-activity relationship study of 1,6-naphthyridinone derivatives as selective type II AXL inhibitors with potent antitumor efficacy

The role of AXL in various oncogenic processes has made it an attractive target for cancer therapy. Currently, kinase selectivity profiles, especially circumventing MET inhibition, remain a scientific issue of great interest in the discovery of selective type II AXL inhibitors. Starting from a dual MET/AXL-targeted lead structure from our previous work, we optimized a 1,6-naphthyridinone series using molecular modeling-assisted compound design to improve AXL potency and selectivity over MET, resulting in the potent and selective type II AXL-targeted compound 25c. This showed excellent AXL inhibitory activity (IC50 = 1.1 nM) and 343-fold selectivity over the highly homologous kinase MET in biochemical assays. Moreover, compound 25c significantly inhibited AXL-driven cell proliferation, dose-dependently suppressed 4T1 cell migration and invasion, and induced apoptosis. Compound 25c also showed noticeable antitumor efficacy in a BaF3/TEL-AXL xenograft model at well-tolerated doses. Overall, this study presented a potent and selective type II AXL-targeted lead compound for further drug discovery.

Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.

Targeting Alpha7 Nicotinic Acetylcholine Receptors in Lung Cancer: Insights, Challenges, and Therapeutic Strategies.

Alpha7 nicotinic acetylcholine receptor (α7 nAChR) is an ion-gated calcium channel that plays a significant role in various aspects of cancer pathogenesis, particularly in lung cancer. Preclinical studies have elucidated the molecular mechanism underlying α7 nAChR-associated lung cancer proliferation, chemotherapy resistance, and metastasis. Understanding and targeting this mechanism are crucial for developing therapeutic interventions aimed at disrupting α7 nAChR-mediated cancer progression and improving treatment outcomes. Drug research and discovery have determined natural compounds and synthesized chemical antagonists that specifically target α7 nAChR. However, approved α7 nAChR antagonists for clinical use are lacking, primarily due to challenges related to achieving the desired selectivity, efficacy, and safety profiles required for effective therapeutic intervention. This comprehensive review provided insights into the molecular mechanisms associated with α7 nAChR and its role in cancer progression, particularly in lung cancer. Furthermore, it presents an update on recent evidence about α7 nAChR antagonists and addresses the challenges encountered in drug research and discovery in this field.

Design, synthesis, and in silico insights into dual-inhibition of CDK-6/Aurora A kinase by 2-phenylbenzimidazole-based small molecules

A novel series of 2-phenylbenzimidazole scaffold was designed as dual tyrosine kinase inhibitors based on targeted chemotherapy. All compounds were assessed for their antiproliferative activity against 60 NCI-cancer cell lines, where four compounds 14, 15, 17, and 18 showed superb growth inhibition values, and meanwhile proved safe towards WI-38 normal cells (IC50 range of 39.14–86.66 µM) compared to Doxorubicin (IC50= 6.72 µM). Hence, they were further in vitro evaluated for dual inhibition of Aurora A and CDK-6 enzymes after kinase selectivity profiling, using an extensive docking study to filter off-target enzymes. The four compounds displayed dual enzymatic inhibition, with compounds 14 and 18 being even more potent regarding CDK-6 with IC50 values of 0.197 µM and 0.172 µM, respectively, than Staurosporine (IC50=0.319 µM), and highly potent towards Aurora A, with IC50 values of 74 nM and 62 nM, respectively, compared to 47 nM for Danusertib. Compounds 14, 15, 17, and 18 triggered cell cycle arrest at the G1 phase and induced total apoptosis of HCT-15 cells by 30.96 % - 45.63 %. In Silico studies including molecular docking, physicochemical properties, compliance with Lipinski's rule, and radar plot technique were applied and were found to be in good agreement with the biological results.

Abstract C160: HMPL-653, a highly potent and selective CSF-1R inhibitor, targeting both tumor cells and tumor microenvironment

Abstract Background: Colony stimulating factor 1 receptor (CSF-1R) and its ligand CSF-1 (CSF-1R/CSF-1) signaling regulates the function and survival of tumor-associated macrophages (TAM), which are involved in tumor progression and suppression of anti-tumor immunity. Moreover, activation of CSF-1R/CSF-1 axis including CSF-1 fusions or CSF-1R activating mutations has also been implicated in the pathogenesis of certain tumors such as tenosynovial giant cell tumor and histiocytic neoplasms. HMPL-653 is a highly potent and selective CSF-1R small molecule inhibitor, discovered and being currently developed in phase I clinical trial (NCT05277454) by HUTCHMED. Methods: The inhibition on CSF-1R kinase was determined using Z-LYTE™ kinase assay. The selectivity of HMPL-653 was assessed against a panel of 413 kinases by Eurofins. Cellular target inhibition on phosphorylation of CSF-1R (p-CSF-1R) was detected by ELISA. In vitro cell viability was measured by CCK-8 assay. Multiple CSF-1/CSF-1R driven tumor models in Nu/Nu nude mice were applied to determine the anti-tumor activity of HMPL-653 as a single agent. To demonstrate the immunoregulatory effect of HMPL-653, a murine tumor model, B16F10, was established in C57BL/6J mice, and the combination regimen, HMPL-653 plus PCP (poly (I:C)+CpG+anti-PD-1) was evaluated. Results: HMPL-653 strongly inhibited CSF-1R kinase with IC50 of 5 nM, showing &amp;gt;7-fold potency than that of the approved CSF-1R inhibitor pexidartinib (IC50=39 nM). And the selectivity profiling in 413 kinase panels revealed that HMPL-653 was highly potent only against CSF-1R, indicating a lower off-target risk than paxidatinib reported. HMPL-653 potently inhibited CSF-1 stimulated p-CSF-1R in THP-1 cells with IC50 of 5 nM. In CSF-1/CSF-1R-dependent cell lines, M-NFS-60 (mouse myelogenous leukemia) and three cell lines stably expressed BCR-CSF-1R or CSF-1R mutations (Ba/F3-BCR-CSF-1R, Ba/F3-CSF-1RW450-E456del, and Ba/F3-CSF-1RY546-K551del), HMPL-653 significantly down regulated p-CSF-1R and its downstream signaling p-ERK and p-AKT. As a consequence, HMPL-653 robustly inhibited the cell viability of above cell models (IC50s: 3~40 nM). Besides the direct anti-tumor effect, HMPL-653 also demonstrated strong inhibition on macrophage survival and M2 macrophage polarization, indicating a potential effect on targeting tumor microenvironment. In vivo, oral administration of HMPL-653 induced remarkable and dose-dependent anti-tumor efficacies in M-NFS-60 ascites model and subcutaneous tumor models harboring above mentioned CSF-1R alterations. And the PK/PD analysis revealed that the anti-tumor effect correlated well with p-CSF-1R inhibition. Moreover, HMPL-653 significantly improved the anti-tumor activity of PCP regimen in B16F10 model by reducing TAMs including M2 macrophages and increasing the ratio of M1/M2 macrophages. Conclusion: HMPL-653 is a highly potent and selective CSF-1R small molecule inhibitor which targets both CSF-1/CSF-1R-dependent tumors and tumor associated macrophages, warranting further clinical evaluation. Citation Format: Jia Hu, Liang Ge, Hui Zhang, An Jiang, Juntao Yu, Weifang Xue, Jian Wang, Yang Sai, Na Yang, Weiguo Qing, Yongxin Ren, Michael Shi, Weiguo Su. HMPL-653, a highly potent and selective CSF-1R inhibitor, targeting both tumor cells and tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C160.

Abstract B116: Preclinical characterization of orally bioavailable, highly potent pan-KRAS inhibitors with selectivity over HRAS and NRAS

Abstract KRAS is altered in ~16% of all cancers and is an oncogenic driver in non-small cell lung, pancreatic, colorectal, and other cancers. Next generation KRAS inhibitors designed to target multiple oncogenic KRAS mutations, while simultaneously sparing wild-type (WT) HRAS and NRAS inhibition, are expected to offer expanded activity and favorable safety. We have discovered a series of highly potent and selective pan-KRAS inhibitors with activity against KRAS G12C, G12D, and G12V mutants, that also display high selectivity over WT HRAS and NRAS, thus providing an expanded therapeutic index. Here, we describe the preclinical profile of these pan-KRAS inhibitors. Compound potency and selectivity were measured using surface plasmon resonance (SPR) assays and cell-based assays measuring inhibition of p-ERK and 3D cell growth of KRAS-mutant tumor cell lines. These pan-KRAS inhibitors have IC50 values ranging from 3-14 nM for KRAS G12C, G12D, G12V, and WT KRAS in phospho-ERK cell-based assays and selectivity of &amp;gt;200-fold over NRAS WT and &amp;gt;100-fold over HRAS WT. These pan-KRAS inhibitors show a clean safety profile in a 133 off-target panel screen. These pan-KRAS inhibitors demonstrate favorable in vitro ADME properties and oral bioavailability in preclinical species. Tumor growth inhibition and PK/PD studies were performed in mice. In vivo, the pan-KRAS inhibitors administered orally demonstrated dose-dependent target inhibition in KRAS-mutant xenograft models. These data demonstrate that our pan-KRAS inhibitors potently and selectively inhibit KRAS G12D, G12C, and G12V mutations and WT KRAS, while sparing HRAS, NRAS, and other off-targets. We hypothesize that this potency and selectivity profile, along with high oral bioavailability, will provide efficacy and tolerability for patients with KRAS-mutant-driven cancers. An IND submission is planned in 2024. Citation Format: Lourdes Prieto Vallejo, Chandrasekar Iyer, Noelle Goggin, Binghui Li, Peiyi Yang, Huimin Bian, Jessica Podoll, Stefan Grotegut, Manuj Tandon, Bryan D Anderson, Andrew Capen, Min Xiao, Tao Wang, Trent R Stewart, Sean Aronow, Desta Bume, Isabel Rojo Garcia, Chong Si, Andrew Cooke, Robert Bondi, Lakshmi Kelamangalath, Ross Wallace, Gabrielle Kolakowski, Lauire LeBrun, James R Henry, Tim Kercher. Preclinical characterization of orally bioavailable, highly potent pan-KRAS inhibitors with selectivity over HRAS and NRAS [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B116.

Abstract C127: Discovery of AUR-112, a novel MALT1 protease inhibitor for the treatement of B cell lymphomas

Abstract MALT1 is a key regulator of antigen receptor signaling in B and T cells. MALT1 partners with BCL10 and CARMA1 to form the CBM complex in which protease activity of MALT1 cleaves the negative regulators (A20, CYLD, RelB) leading to activation of canonical NF-kB pathway. Constitutive activation of NF-kB signaling is a key driver in B-cell lymphomas including diffuse large B-cell lymphomas (DLBCL). Therapeutic targeting of MALT1 protease activity can therefore be a useful approach for the treatment of B-cell lymphomas with dysregulated NF-kB signaling. We sought to discover and develop MALT1 inhibitors with the “best-in-class” profile with an emphasis on lowering plasma protein binding (PPB) to achieve improved pharmacological outcome. After extensive SAR efforts utilizing various computational methods, AUR-112 has been identified as a potential development candidate. The allosteric inhibitor AUR-112 exhibited potent and selective inhibition of MALT1 protease activity in both biochemical and cellular assays. Inhibition of MALT1 correlated with potent antiproliferative activity in an ABC-DLBCL cell line. The mechanistic activity of AUR-112 is supported by the inhibition of IL-10 in a cell line and IL-2 in human whole blood. Consistent with our objective, AUR112 showed low plasma protein binding across multiple species, which translated to greater potency in whole blood compared to published MALT1 inhibitors, positioning it as a best-in-class contender. AUR-112 has excellent oral bioavailability across species and exhibited desirable selectivity profile in kinase, protease and CEREP safety screen panels. Oral administration of AUR-112 led to dose dependent anti-tumor activity including durable tumor regression in cell line and patient derived xenograft models. Treatment with AUR-112 led to potent pharmacodynamic modulation in xenograft models as measured by significant and sustained reduction in serum IL-10 levels that correlated with the compelling efficacy. AUR-112 also demonstrated dose dependent inhibition of T-reg cells in vitro and in vivo supporting the immune modulatory role of MALT1 inhibition. AUR-112 is identified as a development candidate with potentially best-in-class physico-chemical properties resulting in a high free fraction for treatment of ABC-DLBCL patients. IND enabling studies are currently ongoing to support the clinical development of AUR-112. Citation Format: Wesley Roy Balasubramanian, Sammeta Srinivasa Raju, Vasantha K.Y, Archana Bhumi Reddy, Janith Mary Maben, Sandeep Sadashiv Patil, Raghavendra N.R, Kiran Aithal B, Charamanna K.B, Dinesh Chikkanna, Kavitha Nellore, Samiulla D.S, Girish Daginakatte, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Discovery of AUR-112, a novel MALT1 protease inhibitor for the treatement of B cell lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C127.

Abstract A037: Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in non-small cell lung cancer

Abstract Molecular therapies targeting EGFR-mutant non-small cell lung cancer (NSCLC) have dramatically improved prognosis for patients with the classical activating mutations, L858R and exon 19 deletion. However, tumors harboring EGFR exon 20 insertions have been historically more difficult to target. Agents such as amivantamab and mobocertinib have recently been granted accelerated approval for treatment of lung cancer patients with exon 20 insertions, but agents with an improved selectivity profile versus wild-type EGFR are still needed. BAY 2927088, currently in phase I clinical trials, is a novel reversible inhibitor that targets EGFR exon 20 insertion mutations with decreased activity towards wild-type EGFR. Although most patients with advanced EGFR-mutant NSCLC eventually progress due to acquired resistance to EGFR inhibitors, the mechanisms of resistance to BAY 2927088 are currently unknown. Using two different methods of in vitro resistance generation by inhibitor treatment of sensitive cell lines, we identified mechanisms of intrinsic and acquired resistance to BAY 2927088 and other EGFR inhibitors. Incubation with a high concentration of inhibitor selected for drug-tolerant persister cells that underwent a transcriptional state transition resembling EMT (epithelial-mesenchymal transition). Conversely, gradual escalation of inhibitor concentrations resulted in acquisition of oncogenic NRAS mutations. In a parallel approach, we performed a deep mutational scanning assay of the EGFR D770_N771insSVD kinase domain, replacing each amino acid with every other possible amino acid, identifying both known and novel mutations that cause resistance to BAY 2927088 and other EGFR inhibitors. As expected, the C797S mutation was enriched as a mechanism of acquired resistance only to irreversible EGFR exon 20 insertion inhibitors. Surprisingly, the T790M gatekeeper mutation was found to cause resistance to all EGFR exon 20 insertion inhibitors tested, including irreversible inhibitors. Several novel resistance mutations clustering predominantly in exons 20 and 21 were also identified. Citation Format: Gizem Karsli Uzunbas, Quinn McVeigh, Akansha Gupta, Stephanie Hoyt, Kristen Doucette, Hasmik Keshishian, Steven Carr, Xiaoping Yang, David Root, Andrew D Cherniack, Franziska Siegel, Stephan Siegel, Matthew Meyerson, Heidi Greulich. Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A037.

Abstract B160: A phase 1 open-label, dose-escalation study of central nervous system–penetrant cyclin-dependent kinase (CDK)4/6 inhibitor PRT3645 in patients with select advanced or metastatic solid tumors

Abstract Introduction: CDK4/6 mediate cell cycle progression and are important for cancer initiation, growth, and survival. CDK4/6 inhibitors are approved for patients with advanced HR+/HER2– breast cancer but are ineffective against primary or metastatic brain tumors. PRT3645 is an investigational, highly selective, and potent CDK4/6 inhibitor, designed to achieve high tissue and brain penetration. This phase 1 dose-escalation study (NCT05538572) aims to evaluate safety, preliminary efficacy, and pharmacokinetic/pharmacodynamic profile of PRT3645. Methods: Patient eligibility criteria include: age ≥18 years; Eastern Cooperative Oncology Group/Karnofsky performance status ≤1/≥80; adequate organ function; histologically confirmed advanced, recurrent, or metastatic solid tumor malignancy: breast cancer (HR+/HER2– or HR+/HER2+), glioblastoma (isocitrate dehydrogenase [IDH] wild type), CDKN2A/B homozygous deleted IDH–mutant astrocytoma, non-small cell lung cancer (KRAS mutant), or select solid tumors with CDK pathway alterations; and progression on, ineligibility for, or refusal of standard therapy. Patients with treated and untreated brain metastases (lesion &amp;lt;1.5 cm) could enroll if they had no neurological symptoms and did not require immediate local treatment. PRT3645 was taken orally once daily for 28 days (1 cycle) with dose escalation employing a Bayesian optimal interval design to evaluate the safety, tolerability, incidence of dose-limiting toxicities, and pharmacokinetic/pharmacodynamic profile. Results: At data cutoff (May 18, 2023), 8 patients received 20 and 40 mg once daily (first 2 dose levels of dose escalation). No dose-limiting toxicities were observed and the maximum tolerated dose was not reached. Median age was 62 (range, 53-76) years; 50% of patients were male; in these early dose cohorts 75% of patients discontinued therapy (all due to progressive disease; median duration of therapy: 8 [range, 4-16] weeks); 5 patients had glioblastoma, 2 had breast cancer, and 1 had sarcoma. All-cause, any-grade adverse events (AEs) reported in ≥1 patients were anemia and dizziness. One grade 1 AE (fatigue) was considered related to PRT3645. No patients had grade ≥3 AEs, serious AEs, diarrhea, QTcF prolongation, or hepatotoxicity. No patients had dose reductions, interruptions, withdrawals, or deaths due to AEs. Between pre-dose and 2 weeks post first dose, a reduction in phosphorylated Rb (&amp;gt;70% in 3/4 patients) and Ki67 (&amp;gt;60% in 2/4 patients) expression was seen at 40 mg; both markers showed a correlation between histochemical score and drug exposure. Conclusions: Treatment with the next-generation CDK4/6 inhibitor PRT3645 was associated with a substantial decrease in phosphorylated Rb and Ki67 expression, indicating a high level of target engagement at a low dose. PRT3645 exhibited tolerable dose escalation in the initial 2 cohorts of patients with no significant gastrointestinal or hematologic events reported to date, leveraging its enhanced selectivity profile. Dose escalation is ongoing. Citation Format: Amita Patnaik, Mohamad A. Salkeni, Manish R. Patel, Atrayee Basu-Mallick, Dale Shepard, Christine Lihou, William Sun, Jennifer Xavier, Sri Sahasranaman, Neelesh Sharma, Erika Hamilton. A phase 1 open-label, dose-escalation study of central nervous system–penetrant cyclin-dependent kinase (CDK)4/6 inhibitor PRT3645 in patients with select advanced or metastatic solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B160.

Abstract B115: Preclinical characterization of LY3962673, an orally bioavailable, highly potent, and selective KRAS G12D inhibitor

Abstract KRAS G12D mutations are activating oncogenic events that occur in approximately 35%, 13%, and 4% of pancreatic, colorectal, and non-small cell lung cancers, respectively and less commonly in other cancers. Here, we describe LY3962673, a highly potent inhibitor of KRAS G12D that is selective against wild-type (WT) KRAS and orally bioavailable. Compound potency and selectivity were measured using surface plasmon resonance (SPR) and cell-based assays monitoring the inhibition of pERK (Thr202/Tyr204) and cell growth. In SPR assays, LY3962673 had Kd values of 0.058 and 4.7 nM for GDP-bound KRAS G12D and WT KRAS, respectively (81-fold selective). In KRAS G12D-mutant HPAC, LY3962673 inhibited ERK1/2 phosphorylation with an IC50 of 4.2 nM contrasting 231 nM IC50 in WT KRAS MKN45 cells (55-fold selective). Similarly, LY3962673 inhibited cell growth in KRAS G12D and WT KRAS cell lines with IC50 values of 7.7 nM and 1800 nM, respectively (233-fold selective). LY3962673 shows a clean safety profile in a 133 off-target panel screen. LY3962673 demonstrates favorable in vitro ADME properties. LY3962673 shows oral bioavailability across preclinical species when dosed either as freebase or salt form. Tumor growth inhibition and PK/PD studies were performed in mice. In vivo, LY3962673 administered orally demonstrated tumor regressions in KRAS G12D-driven tumor models, without body weight loss. This wide therapeutic index is predicted to allow for maximizing dose intensity and efficacy in patients. These data demonstrate that LY3962673 potently inhibits KRAS G12D while sparing WT KRAS and other off-targets with its selectivity. We hypothesize that LY3962673’s low nanomolar potency, desirable selectivity profile, and oral bioavailability will provide efficacy and tolerability in patients with KRAS G12D-driven cancers. An IND submission is planned in the first half of 2024. Citation Format: Chandrasekar Iyer, Binghui Li, Trent R Stewart, Tao Wang, Andrew Capen, Rachel Cavitt, Bryan D Anderson, Wayne Bocchinfuso, Gaiying Zhao, Michael J Rodriguez, Santiago Carballares, Andrew Cooke, Robert Bondi, Lee Burns, Lakshmi Kelamangalath, Ross Wallace, Gabrielle Kolakowski, James R Henry, Chong Si. Preclinical characterization of LY3962673, an orally bioavailable, highly potent, and selective KRAS G12D inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B115.

Abstract B073: Characterization of selective, allosteric inhibitors of human XRN1

Abstract Background: 5′→3′ exoribonuclease 1 (XRN1) plays an important role in innate immunity by preventing double stranded RNA (dsRNA) accumulation. In addition, XRN1 has emerged as a potential target for drug discovery efforts as tumors with high interferon signaling are sensitive to XRN1 loss using genetic tools. To date, no XRN1-selective inhibitors have been disclosed. This study details characterization of two small-molecule inhibitors of XRN1, 3’-5’ adenosine diphosphate (pAp) and Compound 1, through biochemical, biophysical, and structural analysis. Materials and Methods: Enzymatic and biophysical assays for XRN1 were developed, and characterization of XRN1 inhibitors was performed. To enable structure-based drug design, novel constructs of human XRN1 were designed and characterized for both catalytic activity and compound binding using biochemical and biophysical methods. In addition, cocrystal structures of XRN1 were determined for each inhibitor. Results: pAp was found to be a potent, RNA-competitive inhibitor of XRN1 dsRNA degradation with similar activity for XRN2. Cocrystal structures of XRN1 with pAp confirm the nucleotide analog binds in the active site of the enzyme. Enzymatic and biophysical techniques indicate Compound 1 is also a substrate-competitive inhibitor with single-digit micromolar affinity for XRN1. However, Compound 1 is highly selective for XRN1 and does not bind or inhibit XRN2. High-resolution crystal structures indicate that Compound 1 binds in an allosteric binding site, confirming the mechanism of inhibition and selectivity profile determined by in vitro assays. Conclusions: This work demonstrates XRN1 can be selectively inhibited by small-molecule inhibitors. Human XRN1 structures provide a tool for structure-based drug design efforts against this compelling oncology target.Conflict of Interests: All authors are current or former employees and shareholders of Accent Therapeutics, Inc. Citation Format: Gordon J Lockbaum, E. Allen Sickmier, Brian A Sparling, Kevin Knockenhauer, Nicholas Holt, Julie Liu, Maureen M Lynes, Simina Grigoriu, Kenneth W Duncan, Robert A Copeland, P. Ann Boriack-Sjodin. Characterization of selective, allosteric inhibitors of human XRN1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B073.

Safety Profile in Patients with Chronic Lymphocytic Leukemia Undergoing Treatment with 1st and 2nd Generation Bruton Tyrosine Kinase Inhibitors: Multicentric Real-World Experience

INTRODUCTION Chronic lymphocytic leukemia (CLL) is the most frequent leukemia, being more frequent in older adults. Bruton's tyrosinkinase inhibitors (BTKi) have revolutionized the treatment, displacing immunochemotherapy treatment in all patient profiles. The selectivity profile in the action of these drugs means that the toxicity of first and second generation BTKi is different, resulting in higher rates of atrial fibrillation and arterial hypertension, mainly in patients treated in the second line or later, while maintaining their efficacy. Real-life studies are needed to be able to demonstrate the effects in first-line treatment in a more heterogeneous patient profile, without the strict exclusion and inclusion criteria used in clinical trials. METHODS An observational, retrospective, multicenter, retrospective study was performed. Patients with in treatment with Ibrutinib or Acalabrutinib in first line or later were included. Demographic data, comorbidity, cytogenetic and mutational disease data, adverse effect profile, comprehensive geriatric assessment (if applicable) and treatment response evaluation data are collected. RESULTS A total of 91 patients were included with a median age of 73.22 years SD 11.06. Fifty-three (58.2%) were male. Fifty-three (58.2%) were treated in frontline, 31.9% (29) in second line and 9.9% (9) in subsequent lines. Acalabrutinib was used in 25 (27.5%) patients and Ibrutinib in 66 (72.5%). Patient characteristics are shown in Table 1. Differences were revealed between these two BTKis in terms of line of treatment, with the majority being treated first (P=0.006) and Rai stage, with Rai II predominating (P=0.014). No differences were found between the two BTKis according to the rest of the characteristics. In terms of toxicity, 14 (15.4%) of patients treated with Ibrutinib developed arterial hypertension, compared to no patients in the Acalabrutinib arm (p=0.009). With respect to the development of cardiac toxicity, 19 (20.9%) patients suffered some cardiotoxic event, 3 (3.3%) in patients on acalabrutinb and 16 (17.6%) on ibrutinib, with no significant differences found between the two drugs (p=0.2). No significant differences were found with respect to other toxicities (hematologic, digestive, bleeding and infections). Table 2 No differences were found in terms of progression-free survival or overall survival. CONCLUSIONS The iBTKs have changed the treatment of CLL, although the second-generation iBTKs have a safer cardiovascular profile and are just as effective in our cohort, even in the frontline.