ATP-competitive Kinase Inhibitors Research Articles

Dual-Action Kinase Inhibitors Influence p38α MAP Kinase Dephosphorylation.

Reversible protein phosphorylation directs essential cellular processes including cell division, cell growth, cell death, inflammation, and differentiation. Because protein phosphorylation drives diverse diseases, kinases and phosphatases have been targets for drug discovery, with some achieving remarkable clinical success. Most protein kinases are activated by phosphorylation of their activation loops, which shifts the conformational equilibrium of the kinase towards the active state. To turn off the kinase, protein phosphatases dephosphorylate these sites, but how the conformation of the dynamic activation loop contributes to dephosphorylation was not known. To answer this, we modulated the activation loop conformational equilibrium of human p38α ΜΑP kinase with existing kinase inhibitors that bind and stabilize specific inactive activation loop conformations. From this, we discovered three inhibitors that increase the rate of dephosphorylation of the activation loop phospho-threonine by the PPM serine/threonine phosphatase WIP1. Hence, these compounds are "dual-action" inhibitors that simultaneously block the active site and stimulate p38α dephosphorylation. Our X-ray crystal structures of phosphorylated p38α bound to the dual-action inhibitors reveal a shared flipped conformation of the activation loop with a fully accessible phospho-threonine. In contrast, our X-ray crystal structure of phosphorylated apo human p38α reveals a different activation loop conformation with an inaccessible phospho-threonine, thereby explaining the increased rate of dephosphorylation upon inhibitor binding. These findings reveal a conformational preference of phosphatases for their targets and suggest a new approach to achieving improved potency and specificity for therapeutic kinase inhibitors.

Structural Study of Selectivity Mechanisms for JNK3 and p38α with Indazole Scaffold Probing Compounds.

Selectivity is a primary focus in medicinal chemistry for ATP-competitive kinase inhibitors due to the highly conserved ATP binding pockets in the kinome. A decade of medicinal chemistry efforts has been carried out to develop selective inhibitors for JNKs, resulting in the identification of numerous promising scaffolds that even exhibit isoform selectivity. Thiophene-indazole is one of the scaffolds explored for isoform selectivity. Some iterations of this scaffold have also shown selectivity for p38α. In this study, we utilized four compounds derived from thiophene-indazole to investigate the mechanisms of selectivity for JNK3 and p38α. We determined crystal structures of the inhibitors bound to either JNK3 or p38α and subjected them to molecular dynamics (MD) simulations to understand the binding mechanism and critical interactions that govern affinity and selectivity for these two important kinases. The findings from this study provides valuable information for improving current lead inhibitors and developing a new generation of JNK3 isoform inhibitors.

Identification of kinase inhibitors as potential host-directed therapies for intracellular bacteria

The emergence of antimicrobial resistance has created an urgent need for alternative treatments against bacterial pathogens. Here, we investigated kinase inhibitors as potential host-directed therapies (HDTs) against intracellular bacteria, specifically Salmonella Typhimurium (Stm) and Mycobacterium tuberculosis (Mtb). We screened 827 ATP-competitive kinase inhibitors with known target profiles from two Published Kinase Inhibitor Sets (PKIS1 and PKIS2) using intracellular infection models for Stm and Mtb, based on human cell lines and primary macrophages. Additionally, the in vivo safety and efficacy of the compounds were assessed using zebrafish embryo infection models. Our screen identified 11 hit compounds for Stm and 17 hit compounds for Mtb that were effective against intracellular bacteria and non-toxic for host cells. Further experiments were conducted to prioritize Stm hit compounds that were able to clear the intracellular infection in primary human macrophages. From these, two structurally related Stm hit compounds, GSK1379738A and GSK1379760A, exhibited significant activity against Stm in infected zebrafish embryos. In addition, we identified compounds that were active against intracellular Mtb, including morpholino-imidazo/triazolo-pyrimidinones that target PIK3CB, as well as 2-aminobenzimidazoles targeting ABL1. Overall, this study provided insights into kinase targets acting at the host–pathogen interface and identified several kinase inhibitors as potential HDTs.

Enzyme Is the Name-Adapter Is the Game.

Signaling proteins in eukaryotes usually comprise a catalytic domain coupled to one or several interaction domains, such as SH2 and SH3 domains. An additional class of proteins critically involved in cellular communication are adapter or scaffold proteins, which fulfill their purely non-enzymatic functions by organizing protein-protein interactions. Intriguingly, certain signaling enzymes, e.g., kinases and phosphatases, have been demonstrated to promote particular cellular functions by means of their interaction domains only. In this review, we will refer to such a function as "the adapter function of an enzyme". Though many stories can be told, we will concentrate on several proteins executing critical adapter functions in cells of the immune system, such as Bruton´s tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K), and SH2-containing inositol phosphatase 1 (SHIP1), as well as in cancer cells, such as proteins of the rat sarcoma/extracellular signal-regulated kinase (RAS/ERK) mitogen-activated protein kinase (MAPK) pathway. We will also discuss how these adaptor functions of enzymes determine or even undermine the efficacy of targeted therapy compounds, such as ATP-competitive kinase inhibitors. Thereby, we are highlighting the need to develop pharmacological approaches, such as proteolysis-targeting chimeras (PROTACs), that eliminate the entire protein, and thus both enzymatic and adapter functions of the signaling protein. We also review how genetic knock-out and knock-in approaches can be leveraged to identify adaptor functions of signaling proteins.

Efficacy of GCN2 inhibition by a novel small molecule AP030 in acute leukemia.

6532 Background: GCN2 is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR) that is activated in response to amino acid scarcity. Active GCN2 phosphorylates translation initiation factor eIF2α resulting in the attenuation of global protein synthesis. ISR signaling primarily promotes cell survival but may trigger cell death, dependent on the cellular context. In hematological tumors GCN2 promotes tumor cell survival under conditions of nutrient scarcity. Several GCN2 inhibitors are in clinical development for treatment of both solid and hematological tumors. Here, we describe the preclinical results of a novel, selective ATP-competitive inhibitor of GCN2. Methods: GCN2 inhibition by AP030 was determined using a biochemical Lanthascreen assay and a cell based HTRF assay measuring eIF2a phosphorylation following stimulation with Borrelidin. Kinase selectivity was determined using KinomeScan, and bespoke biochemical and cell-based assays. Interaction of AP030 with GCN2 was determined using X-ray crystallography. AP030 activity in disease-relevant tumor cell lines was determined using cell viability, caspase activation, protein expression and qPCR gene expression endpoints. Inhibition of hematological tumor growth and induction of cell death was investigated in patient samples, and ALL and AML in vivo animal models. Results: Potent inhibition of GCN2 in the Lanthascreen assay was demonstrated with AP030 (Ki of 4.4nM). Following stimulation with Borrelidin, AP030 inhibited eIF2a phosphorylation with an IC50 of 50.8nM and inhibited downstream targets CHAC1 and DDIT3 measured by qPCR. X-ray crystallography confirmed that AP030 binds to the ATP-binding site of GCN2. The KinomeScan confirmed that AP030 was highly selective against the human kinome. AP030 led to partial or complete reduction of AML cell line viability as a single agent and acted synergistically with asparaginase in ALL cell lines. Caspase 3/7 induction was observed in the AML and ALL cell lines evaluated and AP030 increased the proportion of cells undergoing apoptosis in AML patient primary samples. In the CCRF-CEM ALL systemic in vivo tumor model AP030 inhibited tumor growth in combination with asparaginase (TGI 79.33%). Treatment with 0.5-5mg/kg (q.d.) of AP030 resulted in dose-dependent tumor growth inhibition and regression of the MOLM-16 AML in vivo model (TGI 97.37% 5mg/kg). RNA sequencing of residual tumor cells revealed disruption of amino acid and protein metabolism, consistent with GCN2 inhibition. Conclusions: Targeting hematological tumor reliance on the GCN2 arm of the ISR during nutrient scarcity is a novel approach to preventing tumor cell survival. AP030 a novel, potent, selective, ATP-competitive kinase inhibitor has been shown to lead to impressive efficacy in acute leukemia. Based on these preclinical results, a phase 1/2 study has been initiated in AML.

A phase 1, open-label, dose escalation and dose expansion study to evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of PF-07799544 (ARRY-134) as a single agent and in combination with PF-07799933 BRAF dimer inhibitor, in participants 16 years and older with advanced solid tumors.

TPS3180 Background: The MAPK pathway plays a role in several key signaling and phosphorylation events that contribute to tumorigenesis. Inhibition of MEK, along with RAF kinases, has proven to be a successful transformative strategy for melanoma and other MAPK pathway-altered tumors. However, the duration of clinical benefit is limited by a narrow therapeutic index, de novo and acquired resistance and poor brain penetration. PF-07799544 is a next-generation, fully brain penetrant MEK inhibitor. PF-07799933 is an oral selective ATP-competitive small-molecule RAF kinase inhibitor that suppresses BRAF signaling in BRAF V600-mutant and non-V600-BRAF mutant tumors. It displays significantly less paradoxical activation than approved BRAFi and is not “pan RAF” as it spares non-BRAF mutated-containing RAF dimers. We describe here the design of the Phase 1 study of PF-07799544 as Monotherapy or in Combination with a next generation BRAF dimer inhibitor PF-07799933 in participants with BRAF mutated (BRAFm) advanced solid tumors. Methods: The purpose of this first-in-human study is to investigate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and potential clinical benefits of PF-07799544 administered as a single agent in participants with advanced solid tumors (Phase 1a) and in combination with PF-07799933 in participants with BRAF Class 1, 2 and 3 mutated solid tumors with or without brain involvement (Phase 1b). Phase 1a will enroll participants with advanced solid tumors who have progressed on standard of care. The primary objective is to determine monotherapy MTD/RDE of PF-07799544. Once PK measurements indicate the potential for significant WT MEK mutation target coverage, participants with untreated and symptomatic brain metastases (bm) will be allowed to enroll. Phase 1b is comprised of multiple sub-studies, all of which evaluate PF-07799544 in combination with PF-07799933 in participants with BRAFm solid tumors who have progressed on standard of care therapies, including Class 1 BRAFi where indicated. Sub-study B: BRAFm melanoma (V600 and non-V600) will determine the MTDc/RDEc of the combination (Part 1 primary objective), followed by dose expansion cohorts (Part 2) (~80 participants): Cohort 1: BRAF V600 melanoma (asymptomatic and untreated bm permitted), Cohort 2: BRAF V600 melanoma (symptomatic bm), Cohort 3 BRAFm Class 2/3 melanoma (asymptomatic and untreated bm permitted); Sub-study C: BRAFm Class 2 or 3 advanced solid tumor Cohort 1 (asymptomatic and untreated bm permitted and Cohort 2 (symptomatic bm) (~20 each cohort). The main objectives of the dose expansion cohorts in all substudies, will be to evaluate anti-tumor activity, safety, PK and PD. Clinical trial information: NCT05538130 .

Asciminib in Newly Diagnosed Chronic Myeloid Leukemia

BackgroundPatients with newly diagnosed chronic myeloid leukemia (CML) need long-term therapy with high efficacy and safety. Asciminib, a BCR::ABL1 inhibitor specifically targeting the ABL myristoyl pocket, may offer better efficacy and safety and fewer side effects than currently available frontline ATP-competitive tyrosine kinase inhibitors (TKIs).MethodsIn a phase 3 trial, patients with newly diagnosed CML were randomly assigned in a 1:1 ratio to receive either asciminib (80 mg once daily) or an investigator-selected TKI, with randomization stratified by European Treatment and Outcome Study long-term survival score category (low, intermediate, or high risk) and by TKI selected by investigators before randomization (including imatinib and second-generation TKIs). The primary end points were major molecular response (defined as BCR::ABL1 transcript levels ≤0.1% on the International Scale [IS]) at week 48, for comparisons between asciminib and investigator-selected TKIs and between asciminib and investigator-selected TKIs in the prerandomization-selected imatinib stratum.ResultsA total of 201 patients were assigned to receive asciminib and 204 to receive investigator-selected TKIs. The median follow-up was 16.3 months in the asciminib group and 15.7 months in the investigator-selected TKI group. A major molecular response at week 48 occurred in 67.7% of patients in the asciminib group, as compared with 49.0% in the investigator-selected TKI group (difference, 18.9 percentage points; 95% confidence interval [CI], 9.6 to 28.2; adjusted two-sided P<0.001]), and in 69.3% of patients in the asciminib group as compared with 40.2% in the imatinib group within the imatinib stratum (difference, 29.6 percentage points; 95% CI, 16.9 to 42.2; adjusted two-sided P<0.001). The percentage of patients with a major molecular response at week 48 was 66.0% with asciminib and 57.8% with TKIs in the second-generation TKI stratum (difference, 8.2 percentage points; 95% CI, −5.1 to 21.5). Adverse events of grade 3 or higher and events leading to discontinuation of the trial regimen were less frequent with asciminib (38.0% and 4.5%, respectively) than with imatinib (44.4% and 11.1%) and second-generation TKIs (54.9% and 9.8%).ConclusionsIn this trial comparing asciminib with investigator-selected TKIs and imatinib, asciminib showed superior efficacy and a favorable safety profile in patients with newly diagnosed chronic-phase CML. Direct comparison between asciminib and second-generation TKIs was not a primary objective. (Funded by Novartis; ASC4FIRST ClinicalTrials.gov number, NCT04971226).

Identification of kinase modulators as host-directed therapeutics against intracellular methicillin-resistant Staphylococcus aureus.

The increasing prevalence of antimicrobial-resistant Staphylococcus aureus strains, especially methicillin-resistant S. aureus (MRSA), poses a threat to successful antibiotic treatment. Unsuccessful attempts to develop a vaccine and rising resistance to last-resort antibiotics urge the need for alternative treatments. Host-directed therapy (HDT) targeting critical intracellular stages of S. aureus emerges as a promising alternative, potentially acting synergistically with antibiotics and reducing the risk of de novo drug resistance. We assessed 201 ATP-competitive kinase inhibitors from Published Kinase Inhibitor Sets (PKIS1 and PKIS2) against intracellular MRSA. Seventeen hit compounds were identified, of which the two most effective and well-tolerated hit compounds (i.e., GW633459A and GW296115X) were selected for further analysis. The compounds did not affect planktonic bacterial cultures, while they were active in a range of human cell lines of cervical, skin, lung, breast and monocyte origin, confirming their host-directed mechanisms. GW633459A, structurally related to lapatinib, exhibited an HDT effect on intracellular MRSA independently of its known human epidermal growth factor receptor (EGFR)/(HER) kinase family targets. GW296115X activated adenosine monophosphate-activated protein kinase (AMPK), thereby enhancing bacterial degradation via autophagy. Finally, GW296115X not only reduced MRSA growth in human cells but also improved the survival rates of MRSA-infected zebrafish embryos, highlighting its potential as HDT.

Abstract 6489: Paradoxical activation of kinases occurs directly with ATP-competitive kinase inhibitors and is observable biochemically at physiologically relevant drug concentrations

Abstract We sought to screen all FDA approved, cancer-indicated, kinase targeting drugs using the gold standard radiometric biochemical activity assay (HotSpot) against the largest collection of kinases in the world (&amp;gt;100 drugs against 735 total kinases including 395 wild type and 340 mutants). One of these drugs, mitapivat, is an activator of Pyruvate Kinase. Here, we show mitapivat-driven biochemical inhibition of several, but not all, mutants of c-KIT. We also observed the equal and opposite phenomenon whereby many ATP-competitive kinase inhibitors demonstrate biochemical activation of certain kinases, several of which seem particularly susceptible to the effect. Previous work by other investigators (at Genentech and the Rosen lab, MSKCC) demonstrated that PLX4720 (precursor to Vemurafenib) paradoxically activated wild type BRAF, but not BRAF mutants, by driving dimerization of the kinase in drug concentrations insufficient to inhibit both members of the dimerized pair. Our data suggests this phenomenon may be more widespread than previously appreciated and fully observable in vitro. Such paradoxical activation could be driving both toxicities and inefficacy against cancers expressing some of these activatable kinases. This information could suggest novel opportunities for combination therapy and improve outcomes by contraindicating certain medication from use based on tumor expression profile. Citation Format: Christian M. Loch, Mia Eason, Shuguang Liang, Alison Goupil, Anthony Acinapura, Jamie Purcell, Maia Domiguez. Paradoxical activation of kinases occurs directly with ATP-competitive kinase inhibitors and is observable biochemically at physiologically relevant drug concentrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6489.

Abstract 5957: Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer

Abstract Introduction Transcription regulators, such as cyclin-dependent kinase 9 (CDK9), have been implicated in the super-enhancer driven transcriptional control of oncogenes in multiple cancers including pancreatic cancer. CDK9 has been shown to be employed by cancer cells for the constant production of short-lived oncoproteins such as c-Myc to maintain their survival. Due to its critical role in regulating transcription in cancer cells, CDK9 has emerged as a potential therapeutic target. Here, we report the antitumor activity of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, in preclinical models for pancreatic cancer. Experimental Procedures Fragment-based MolecuLern driven AI workflow methods were used to design a series of ATP-competitive CDK9 specific kinase inhibitors. BLX-3030 was selected as one of the most potent leads with an IC50 of 1.1 nM in a cell-free CDK9 kinase assay. A Sulforhodamine B (SRB) assay was used to evaluate the cell growth inhibitory activity of BLX-3030 in a panel of 10 pancreatic ductal adenocarcinoma (PDAC) and 2 pancreatic neuroendocrine tumor (pNET) cell lines. Western blotting was used to determine the effects of BLX-3030 treatment on c-Myc expression in cancer cells. The combination effects between BLX-3030 and standard of care regimen, gemcitabine (GEM) and nab-paclitaxel (NP), was assessed using the SRB assay. Finally, the in vivo antitumor activity of BLX-3030 alone or in combination with GEM and NP was evaluated in a mouse cell line xenograft model for pancreatic cancer. Results BLX-3030 exhibited potent cell growth inhibitory activity in the panel of pancreatic cancer cell lines with IC50 values ranging from 133.5 nM to 508.1 nM. The potency of BLX-3030 in the cell lines appears to correlate with the expression level of c-Myc in the cell lines. BLX-3030 showed similar activities in PDAC and pNET cell lines with mean IC50 values of 244.5 nM and 194.6 nM, respectively. However, treatment with BLX-3030 for 48 hours did not seem to significantly reduce c-Myc expression in PSN-1 PDAC cells. When combined with GEM and NP, BLX-3030 demonstrated mostly additive effects in PSN-1 cells. Preliminary data from the in vivo study show that BLX-3030 improves the antitumor activity of GEM and NP. Conclusion In summary, BLX-3030 potently inhibited the growth of both PDAC and pNET cells in vitro. The growth inhibitory activity of BLX-3030 in PDAC cell lines correlates with their c-Myc expression levels. BLX-3030 showed an additive effect when combined with standard of care drugs (GEM and NP) in PDAC cell lines. Overall, the novel CDK9 inhibitor, BLX-3030, demonstrated potent activity in pancreatic cancer cell line models and presents a promising preclinical lead for pancreatic cancer. (This work was supported in part by Biolexis Therapeutics and the Seena Magowitz Foundation). Citation Format: Yesenia Barrera-Millan, Zhaoliang Li, Hariprasad Vankayalapati, David J. Bearss, Daniel D. Von Hoff, Haiyong Han. Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5957.

Integrated Stress Response Potentiates Ponatinib-Induced Cardiotoxicity.

Mitochondrial dysfunction is a primary driver of cardiac contractile failure; yet, the cross talk between mitochondrial energetics and signaling regulation remains obscure. Ponatinib, a tyrosine kinase inhibitor used to treat chronic myeloid leukemia, is among the most cardiotoxic tyrosine kinase inhibitors and causes mitochondrial dysfunction. Whether ponatinib-induced mitochondrial dysfunction triggers the integrated stress response (ISR) to induce ponatinib-induced cardiotoxicity remains to be determined. Using human induced pluripotent stem cells-derived cardiomyocytes and a recently developed mouse model of ponatinib-induced cardiotoxicity, we performed proteomic analysis, molecular and biochemical assays to investigate the relationship between ponatinib-induced mitochondrial stress and ISR and their role in promoting ponatinib-induced cardiotoxicity. Proteomic analysis revealed that ponatinib activated the ISR in cardiac cells. We identified GCN2 (general control nonderepressible 2) as the eIF2α (eukaryotic translation initiation factor 2α) kinase responsible for relaying mitochondrial stress signals to trigger the primary ISR effector-ATF4 (activating transcription factor 4), upon ponatinib exposure. Mechanistically, ponatinib treatment exerted inhibitory effects on ATP synthase activity and reduced its expression levels resulting in ATP deficits. Perturbed mitochondrial function resulting in ATP deficits then acts as a trigger of GCN2-mediated ISR activation, effects that were negated by nicotinamide mononucleotide, an NAD+ precursor, supplementation. Genetic inhibition of ATP synthase also activated GCN2. Interestingly, we showed that the decreased abundance of ATP also facilitated direct binding of ponatinib to GCN2, unexpectedly causing its activation most likely because of a conformational change in its structure. Importantly, administering an ISR inhibitor protected human induced pluripotent stem cell-derived cardiomyocytes against ponatinib. Ponatinib-treated mice also exhibited reduced cardiac function, effects that were attenuated upon systemic ISRIB administration. Importantly, ISRIB does not affect the antitumor effects of ponatinib in vitro. Neutralizing ISR hyperactivation could prevent or reverse ponatinib-induced cardiotoxicity. The findings that compromised ATP production potentiates GCN2-mediated ISR activation have broad implications across various cardiac diseases. Our results also highlight an unanticipated role of ponatinib in causing direct activation of a kinase target despite its role as an ATP-competitive kinase inhibitor.

Real-World Effectiveness of Asciminib in Patients with Chronic Myeloid Leukemia (CML) Harboring the T315I Mutation: A Global Chart Review Study of Patients Treated in the Asciminib Managed Access Program (MAP)

Background: In patients (pts) with CML, the BCR::ABL1 T315I gatekeeper mutation confers treatment resistance to all approved ATP-competitive tyrosine kinase inhibitors (TKIs) except ponatinib and olverembatinib; thus pts harboring this mutation have limited treatment options. Asciminib, a first-in-class TKI that works by specifically targeting the ABL myristoyl pocket (STAMP), has a distinct mechanism of action and no known overlapping resistance profile with ATP-competitive TKIs. In clinical trials, asciminib has demonstrated efficacy and acceptable safety in adult pts with Ph+ CML in chronic phase and the T315I mutation. The asciminib MAP (NCT04360005) enables access to asciminib treatment for pts with CML who are resistant or intolerant to available treatments or for whom available treatments are contraindicated. This global chart review study aimed to better understand the effectiveness and safety of asciminib in pts with CML and the T315I mutation treated as part of the MAP. Methods: This non-interventional chart review analyzed data from existing routine medical charts of pts enrolled in the MAP who received at least one dose of asciminib. Pts with CML aged ≥18 years old were included in the study if they had confirmed presence of the T315I mutation detected at any point prior to commencement of asciminib and had received their first dose of asciminib between 01 November 2018 and 30 April 2022. Data was abstracted from CML diagnosis date to the first dose of asciminib (index date) and up until 12 months after the start of asciminib. Treatment effectiveness was assessed in terms of major molecular response (MMR) in pts in MMR at index date or not, at and by 3, 6, 9, and 12 months. Other variables included demographic and disease characteristics, treatment patterns, response to prior TKIs and safety. All analyses were descriptive. Results: 31 pts were included across 8 countries (Netherlands, UK, Italy, Hong Kong, Pakistan, Spain, Australia, and US). Median age at CML diagnosis was 47 (range 18‒74) years and most pts were in chronic phase ( Table 1). Pts were heavily pretreated, with the majority having received ≥2 prior TKIs. Nearly all pts (28/31; 90.3%) had received prior ponatinib treatment at any time before the switch to asciminib. Median prior TKI treatment duration was longest for ponatinib (43.4 months, range 0.3‒117.3) vs other TKIs. The most common reason for discontinuation of all previous TKIs at any time before asciminib was lack of response to treatment, with the exception of ponatinib, where the most common reason was due to adverse events (AEs). Ponatinib was the last TKI prior to commencement of asciminib in 27/31 (87.1%) pts. Overall, 22/31 pts (71.0%) experienced AEs that led to a switch to another TKI at any time prior to asciminib, whereas nearly half of pts discontinued the last TKI before starting asciminib due to intolerance (14 pts, 45.2%). A total of 12/31 patients (38.7%) were already in MMR at index date ( Table 2), confirming that intolerance was an important reason for switching to asciminib. Of the pts not in MMR at the start of asciminib, 4/10 (40.0%) and 5/12 (41.7%) evaluated pts achieved MMR by 6 and 12 months, respectively. Of the 12 pts in MMR at the start of asciminib, 6/12 (50.0%) maintained MMR by 6 months. Overall, 10/31 pts (32.3%) discontinued asciminib before completion of the 12-month follow-up (death, 7 [4 serious AEs, SAEs; 2 CML; 1 unknown]; physician decision, 3). SAEs were reported in 9 pts (29.0%) that received asciminib; 8 SAEs led to hospitalization, 4 to death (1 cerebrovascular accident, 1 CML, 1 pneumonia, 1 fungal infection), 2 to asciminib discontinuation, and 2 were considered life-threatening. SAEs reported for 1 pt (hematotoxicity and procedural hemorrhage) were assessed as asciminib-related. Conclusions: Asciminib demonstrated effectiveness in pts with CML and the T315I mutation in routine medical practice, including ponatinib pre-treated pts, a population with limited available treatment options. Effectiveness estimates were consistent with those observed in clinical trials. Although the number of pts was small, this global study reports on one of the largest cohorts of pts with CML and the T315I mutation.

Clickable Photoreactive ATP-Affinity Probe for Global Profiling of ATP-Binding Proteins.

Adenosine triphosphate (ATP) is the major energy carrier in organisms, and there are many cellular proteins that can bind to ATP. Among these proteins, kinases are key regulators in several cell signaling processes, and aberrant kinase signaling contributes to the development of many human diseases, including cancer. Hence, small-molecule kinase inhibitors have been successfully used for the treatment of various diseases. Since the ATP-binding pockets are similar for many kinases, it is very important to evaluate the selectivity of different kinase inhibitors. We report here a clickable ATP photoaffinity probe for the global profiling of ATP-binding proteins. After incubating the protein lysate with the ATP probe followed by ultraviolet (UV) irradiation, ATP-binding proteins were labeled with an alkyne handle for subsequent biotin conjugation through click chemistry. Labeled proteins were enriched with streptavidin beads, digested with trypsin, and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). More than 400 ATP-binding proteins, including approximately 200 kinases, could be identified in a single LC-MS/MS run in the data-dependent acquisition mode. We then applied this method to the analysis of targets of three selected ATP-competitive kinase inhibitors. We were able to successfully identify some of their reported target proteins from label-free quantification results and validated the results using Western blot analyses. Together, we developed a clickable ATP photoaffinity probe for proteome-wide profiling of ATP-binding proteins and demonstrated that this chemoproteomic method is amenable to high-throughput target identification of kinase inhibitors.

MET Inhibitor Capmatinib Radiosensitizes MET Exon 14-Mutated and MET-Amplified Non-Small Cell Lung Cancer

The objective of this study was to investigate the effects of inhibiting the MET receptor with capmatinib, a potent and clinically relevant ATP-competitive tyrosine kinase inhibitor, in combination with radiation in MET exon 14-mutated and MET-amplified non-small cell lung (NSCLC) cancer models. In vitro effects of capmatinib and radiation on cell proliferation, colony formation, MET signaling, apoptosis, and DNA damage repair were evaluated. In vivo tumor responses were assessed in cell line xenograft and patient-derived xenograft models. Immunohistochemistry was used to confirm the in vitro results. In vitro clonogenic survival assays demonstrated radiosensitization with capmatinib in both MET exon 14-mutated and MET-amplified NSCLC cell lines. No radiation-enhancing effect was observed in MET wild-type NSCLC and a human bronchial epithelial cell line. Minimal apoptosis was detected with the combination of capmatinib and radiation. Capmatinib plus radiation compared with radiation alone resulted in inhibition of DNA double-strand break repair, as measured by prolonged expression of γH2AX. In vivo, the combination of capmatinib and radiation significantly delayed tumor growth compared with vehicle control, capmatinib alone, or radiation alone. Immunohistochemistry indicated inhibition of phospho-MET and phospho-S6 and a decrease in Ki67 with inhibition of MET. Inhibition of MET with capmatinib enhances the effect of radiation in both MET exon 14-mutated and MET-amplified NSCLC models.

How Ligands Interact with the Kinase Hinge.

ATP-competitive kinase inhibitors form hydrogen bond interactions with the kinase hinge region at the adenine binding site. Thus, it is crucial to explore hinge-ligand recognition as part of a rational drug design strategy. Here, harnessing known ligand-bound kinase structures and experimental assay resources, we first created a kinase structure-assay database (KSAD) containing 2705 nM ligand-bound kinase complexes. Then, using KSAD, we systematically investigate hinge-ligand binding patterns using interaction fingerprints, thereby delineating 15 different hydrogen-bond interaction modes. We believe these results will be valuable for de novo drug design and/or scaffold hopping of kinase-targeted drugs.

A phase 1, open-label, dose escalation and dose expansion study to evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of PF-07799933 (ARRY-440) as a single agent and in combination therapy in participants 16 years and older with advanced solid tumors with BRAF alterations.

TPS3164 Background: Approved BRAF inhibitors (BRAFi) are inactive against RAF dimers. Accordingly, the acquisition of BRAF dimers ( e.g., through drug-acquired splice variants or BRAF amplification) may lead to therapy resistance. Non-V600 (class II and III) BRAF alterations that occur de novo in diverse tumor types also signal as dimers and therefore have no approved targeted therapeutic options. Additional liabilities of approved BRAFi include toxicity from paradoxical activation of wild-type (WT) RAF and limited brain penetration, a common primary and metastatic site for BRAF-altered cancers. In contrast to approved agents, investigational BRAFi inhibit WT and mutant RAF proteins (A, B, and C-RAF, monomers, dimers), and therefore may be limited by on-target toxicity from “pan-RAF” inhibition. PF-07799933 is an oral selective ATP-competitive small-molecule RAF kinase inhibitor that suppresses BRAF signaling in BRAF V600-mutant and non-V600- BRAF mutant tumors. It displays significantly less paradoxical activation than approved BRAFi and is not “pan RAF” as it spares non-BRAF-containing RAF dimers. We describe here the Phase 1 study of PF-07799933 as monotherapy or in combination with binimetinib or cetuximab in participants with BRAF-altered advanced solid tumors. Methods: The purpose of this first-in-human study is to investigate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and potential clinical benefits of PF-07799933 administered as a single agent and in combination in participants with BRAF Class I, II and III mutated solid tumors with or without brain involvement. Part 1 (monotherapy dose escalation) and Part 2 (combination dose escalation) will enroll participants with BRAF V600 mutation who have progressed on a approved BRAFi or with de novo Class II or III alterations who have progressed on standard of care therapy. The primary objective of Part 1 is to determine monotherapy MTD/RDE of PF-07799933. Participants in Part 1 will be allowed to add on rational combination with either binimetinib or cetuximab at the time of disease progression. Once PK measurements indicate the potential for significant BRAF inhibition in the brain, participants with untreated and symptomatic brain metastases will be allowed to enroll. The primary objective of Part 2 is combination MTDc/RDEc of PF-07799933 with binimetinib or cetuximab. The primary objective of Part 3 is efficacy in defined cohorts (total ~120 participants): Cohort 1-2: BRAF V600 mutant melanoma; Cohort 3: BRAF Class II altered melanoma; Cohorts 4-5: BRAF V600 and Class II altered CRC; Cohort 6: other BRAF V600, Class II/III altered solid tumor not qualifying for Cohorts 1-5. Clinical trial information: NCT05355701 .

Doubly Constrained C-terminal of Roc (COR) Domain-Derived Peptides Inhibit Leucine-Rich Repeat Kinase 2 (LRRK2) Dimerization.

Missense mutations along the leucine-rich repeat kinase 2 (LRRK2) protein are a major contributor to Parkinson's Disease (PD), the second most commonly occurring neurodegenerative disorder worldwide. We recently reported the development of allosteric constrained peptide inhibitors that target and downregulate LRRK2 activity through disruption of LRRK2 dimerization. In this study, we designed doubly constrained peptides with the objective of inhibiting C-terminal of Roc (COR)-COR mediated dimerization at the LRRK2 dimer interface. We show that the doubly constrained peptides are cell-permeant, bind wild-type and pathogenic LRRK2, inhibit LRRK2 dimerization and kinase activity, and inhibit LRRK2-mediated neuronal apoptosis, and in contrast to ATP-competitive LRRK2 kinase inhibitors, they do not induce the mislocalization of LRRK2 to skein-like structures in cells. This work highlights the significance of COR-mediated dimerization in LRRK2 activity while also highlighting the use of doubly constrained peptides to stabilize discrete secondary structural folds within a peptide sequence.

Abstract 2412: MET inhibition enhances the effect of radiation in MET dysregulated non-small cell lung cancer models

Abstract Objective: Alterations in the MET receptor are seen in up to 10% of patients with non-small cell lung cancer (NSCLC). A mutation in exon 14, which results in deletion of the intracellular juxtamembrane domain of the receptor, leading to enhanced signaling, is the most common MET mutation in NSCLC. MET is involved in multiple pathways associated with apoptosis and radiation response. We investigated the effects of inhibiting MET with capmatinib, a potent and clinically relevant ATP-competitive kinase inhibitor, in combination with radiation in MET-dependentNSCLC models. Methods: MET exon 14 mutated and MET amplified NSCLC cell lines treated with radiation or capmatinib were assessed with proliferation, clonogenic survival, apoptosis, and DNA damage signaling and repair assays. MET signaling proteins were evaluated by Western blotting. In vivo growth responses to the MET inhibitor capmatinib with and without radiation were evaluated in patient derived xenograft (PDX) models established from both MET amplified and MET exon 14 skipping mutation containing patient tumors. Radiation was delivered in 10 daily fractions of 2 Gy and capmatinib was administered by oral gavage daily 1 hour prior to radiation at a dose 20 mg/kg for capmatinib. Immunohistochemistry (IHC) was performed to evaluate MET signaling and proliferation. Results: Radiation increased the expression and capmatinib inhibited phospho-AKT, phospho-s6,phospho-ERK, and phospho-STAT3 in our MET-driven NSCLC cell lines. Combination of capmatinib and radiation in clonogenic assays increased sensitivity to radiation in MET dysregulated cell lines. No radiation-enhancing effect was observed in MET wild-type and human bronchial epithelial cell lines. Campatinib alone significantly inhibited tumor growth in both ourMET exon 14 mutated and MET amplified xenograft models (p&amp;lt;0.01). Combination of capmatinib and radiation significantly delayed growth compared to vehicle control, capmatinib alone or radiation alone (p&amp;lt;0.01). IHC demonstrated inhibition of phospho-MET and phospho-S6 and a decrease in Ki67 with inhibition of MET. A significant decrease Ki67 proliferation was observed in the combination arm compared to radiation along or drug alone. Conclusion:Inhibition of MET enhanced the effect of radiation in multiple MET-altered NSCLC PDX models.Additional studies are currently underway evaluating the mechanisms of radiation sensitization with a focus on MET-induced apoptosis. Citation Format: Shrey K. Ramesh, Saahil N. Javeri, Kwangok P. Nickel, Ahmet Cifci, Rachel L. Minne, Randall J. Kimple, Andrew M. Baschnagel. MET inhibition enhances the effect of radiation in MET dysregulated non-small cell lung cancer models [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 2412.

Oxazolo[5,4-f]quinoxaline-type selective inhibitors of glycogen synthase kinase-3α (GSK-3α): Development and impact on temozolomide treatment of glioblastoma cells

The 2-(3-pyridyl)oxazolo[5,4-f]quinoxalines CD-07 and FL-291 are ATP-competitive GSK-3 kinase inhibitors. Here, we investigated the impact of FL-291 on neuroblastoma cell viability and showed that treatment at 10 μM (i.e. ∼500 times the IC50 against the GSK-3 isoforms) has no significant effect on the viability of NSC-34 motoneuron-like cells. A study performed on primary neurons (non-cancer cells) led to similar results. The structures co-crystallized with GSK-3β revealed similar binding modes for FL-291 and CD-07, with their hinge-oriented planar tricyclic system. Both GSK isoforms show the same orientations for the amino acids at the binding pocket except for Phe130 (α) and Phe67 (β), leading to a larger pocket on the opposite side of the hinge region for the α isoform. Calculations of the thermodynamic properties of the binding pockets highlighted the required features of potential ligands; these should have a hydrophobic core (which could be larger in the case of GSK-3β) surrounded by polar areas (a little more polar in the case of GSK-3α). A library of 27 analogs of FL-291 and CD-07 was thus designed and synthesized by taking advantage of this hypothesis. While the introduction of substituents at different positions of the pyridine ring, the replacement of the pyridine by other heterocyclic moieties, or the replacement of the quinoxaline ring by a quinoline moiety did not lead to any improvement, the replacement of the N-(thio)morpholino of FL-291/CD-07 by a slightly more polar N-thiazolidino led to a significant result. Indeed, the new inhibitor MH-124 showed clear selectivity for the α isoform, with IC50 values of 17 nM and 239 nM on GSK-3α and GSK-3β, respectively. Finally, the efficacy of MH-124 was evaluated on two glioblastoma cell lines. Although MH-124 alone did not have a significant impact on cell survival, its addition to temozolomide (TMZ) significantly reduced the TMZ IC50 values on the cells tested. The use of the Bliss model allowed a synergy to be evidenced at certain concentrations.

Approved Small-Molecule ATP-Competitive Kinases Drugs Containing Indole/Azaindole/Oxindole Scaffolds: R&D and Binding Patterns Profiling.

Kinases are among the most important families of biomolecules and play an essential role in the regulation of cell proliferation, apoptosis, metabolism, and other critical physiological processes. The dysregulation and gene mutation of kinases are linked to the occurrence and development of various human diseases, especially cancer. As a result, a growing number of small-molecule drugs based on kinase targets are being successfully developed and approved for the treatment of many diseases. The indole/azaindole/oxindole moieties are important key pharmacophores of many bioactive compounds and are generally used as excellent scaffolds for drug discovery in medicinal chemistry. To date, 30 ATP-competitive kinase inhibitors bearing the indole/azaindole/oxindole scaffold have been approved for the treatment of diseases. Herein, we summarize their research and development (R&D) process and describe their binding models to the ATP-binding sites of the target kinases. Moreover, we discuss the significant role of the indole/azaindole/oxindole skeletons in the interaction of their parent drug and target kinases, providing new medicinal chemistry inspiration and ideas for the subsequent development and optimization of kinase inhibitors.