ATP-competitive Kinase Inhibitors Research Articles

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 (>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<0.01). Combination of capmatinib and radiation significantly delayed growth compared to vehicle control, capmatinib alone or radiation alone (p<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.

Therapy for patients with chronic phase-chronic myeloid leukemia previously treated with ⩾2 tyrosine kinase inhibitors: a systematic literature review.

ATP-competitive tyrosine kinase inhibitors (TKIs) are the current standard of care for patients with chronic phase-chronic myeloid leukemia (CP-CML) in the first-line and second-line (2 L) setting. Treatment after 2 L is not clearly established. The objective of this study was to summarize the available evidence to compare the efficacy and safety of interventions in the treatment of CP-CML patients who had received ⩾2 prior TKIs. A systematic literature review was performed. A systematic literature review (SLR) of studies published until May 2021, reporting clinical outcomes in adult patients with CP-CML who had received ⩾ 2 prior TKIs was performed. Studies were identified through the database searches via Ovid platform (Embase, MEDLINE Epub Ahead of Print, In-Process and Other Non-Indexed Citations, and Cochrane Central Register of Controlled Trials), the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP), bibliographic search of relevant reviews, and proceedings from the previous 3 years of the key conferences in the field of oncology. Our search identified 38 relevant studies. Among the identified studies of the current third-line treatments, the major molecular response (MMR) rate for ponatinib was 19.0-66.7%, 23.3-25.5% for asciminib, 19.2% for omacetaxine, and 13.2% for bosutinib at 6 months. The complete cytogenetic response (CCyR) rate was 21.4-64.8% for ponatinib, 38.7-40.8% for asciminib, 18-24.2% for bosutinib, and 16.1% for omacetaxine at 6 months. The findings from current SLR demonstrated the lack of data for patients with CML treated with ⩾2 TKIs. TKIs such as asciminib, ponatinib, and bosutinib are valid options for those patients. Further research is needed to identify the best treatment option for patients with CML receiving later lines of therapy.

A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling

mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.

CML-466 Asciminib Provides Durable Molecular Responses in Patients With Chronic Myeloid Leukemia in Chronic Phase (CML-CP) With the T315I Mutation: Updated Efficacy and Safety Data From a Phase I Trial

In CML-CP, the BCR::ABL1 T315I mutation confers resistance to previously approved ATP-competitive tyrosine kinase inhibitors (TKIs), except ponatinib and olverembatinib. In a previous analysis of the phase I, dose-escalation trial X2101, asciminib-a BCR::ABL1 inhibitor that binds to the ABL myristoyl pocket-demonstrated efficacy and a favorable safety profile in heavily pretreated patients with T315I-mutated CML. We report updated efficacy and safety data in patients with CML-CP with the T315I mutation (data cutoff: January 6, 2021). Provide updated safety and efficacy data for patients with T315I-mutated CML-CP after added exposure. Patients with T315I-mutated CML-CP and treated with ≥1 prior TKI were enrolled and received asciminib 200mg twice daily (BID). 48 patients were included; 25 patients (52.1%) received ≥3 prior TKIs. At data cutoff, treatment was ongoing in 27 patients (56.3%). 45 of 48 patients were evaluable (BCR::ABL1IS >0.1% at baseline) for major molecular response (MMR); 3 were excluded for BCR::ABL1 atypical transcripts. Among evaluable patients, 19 (42.2%) achieved MMR by week 24 and 22 (48.9%) by week 96. Evaluable patients included 26 ponatinib-pretreated and 19 ponatinib-naive patients; 34.6% and 68.4%, respectively, achieved MMR by week 96. The probability of maintaining MMR for ≥96 weeks was 84% (95% CI, 68.1%-100.0%). 23 of 37 patients (62.2%) with BCR::ABL1IS >1% at baseline achieved BCR::ABL1IS ≤1% by week 96. The safety/tolerability profile of asciminib remained favorable after ≈9 months of added exposure (median duration of exposure, 2.08 years; range, 0.04-4.13 years). The most common (≥10%) grade ≥3 adverse events (AEs) were lipase increase (18.8%, all asymptomatic elevations) and thrombocytopenia (14.6%). Arterial occlusive events occurred in 4 patients (8.3%); none led to dose adjustment/interruption/discontinuation. AEs leading to discontinuation occurred in 5 patients (10.4%). Only 2 study deaths, both due to COVID-19, occurred in this patient population. After a median exposure of >2 years, asciminib monotherapy 200mg BID exhibited a sustained, favorable safety profile and clinical efficacy in patients with T315I-mutated CML-CP-a population with high unmet medical need. This updated analysis confirms asciminib as a treatment option for patients with T315I-mutated CML-CP, including those previously treated with ponatinib.

Advances in RIPK1 kinase inhibitors.

Programmed necrosis is a new modulated cell death mode with necrotizing morphological characteristics. Receptor interacting protein 1 (RIPK1) is a critical mediator of the programmed necrosis pathway that is involved in stroke, myocardial infarction, fatal systemic inflammatory response syndrome, Alzheimer’s disease, and malignancy. At present, the reported inhibitors are divided into four categories. The first category is the type I ATP-competitive kinase inhibitors that targets the area occupied by the ATP adenylate ring; The second category is type Ⅱ ATP competitive kinase inhibitors targeting the DLG-out conformation of RIPK1; The third category is type Ⅲ kinase inhibitors that compete for binding to allosteric sites near ATP pockets; The last category is others. This paper reviews the structure, biological function, and recent research progress of receptor interaction protein-1 kinase inhibitors.

Protein kinase inhibitor ceritinib blocks ectonucleotidase CD39 – a promising target for cancer immunotherapy

BackgroundAn important mechanism, by which cancer cells achieve immune escape, is the release of extracellular adenosine into their microenvironment. Adenosine activates adenosine A2A and A2B receptors on immune cells constituting...

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We set up six light conditions to investigate the changes in the development of Arabidopsis thaliana hypocotyls and roots. Seedlings grown for 96 h under darkness were scored with shorter roots and longer hypocotyls. In shoot-shaded conditions, seedlings were unable to carry out photosynthesis, resulting in insufficient stored nutrients for root development. In the three groups of different light intensities applied to the roots, total light caused stress in the entire seedlings and the length of roots and hypocotyls were shorter than in conditions when roots were growing within light-dark gradients. Importantly, root lengths were higher within light-dark gradients than in total light. Different light treatments did significantly affect root growth and hypocotyl growth. The addition of ATP-competitive mTOR kinase inhibitor (AZD), drastically reduced root, however, this did not occur with hypocotyl length.   Key words: Total light, total dark, gradient light, shoot dark with light blocker, light blocker, shoot dark.

P704: ASCIMINIB PROVIDES DURABLE MOLECULAR RESPONSES IN PATIENTS (PTS) WITH CHRONIC MYELOID LEUKEMIA IN CHRONIC PHASE (CML-CP) WITH THE T315I MUTATION: UPDATED EFFICACY AND SAFETY DATA FROM A PHASE I TRIAL

Background: In pts with CML, the BCR::ABL1 T315I mutation is associated with poor clinical outcomes and confers resistance to previously approved ATP-competitive tyrosine kinase inhibitors (TKIs). Until recently, ponatinib (PON) was the only TKI available for these pts, but its use may be limited by associated cardiovascular events. In the primary analysis of the phase I trial X2101, asciminib—the 1st BCR::ABL1 inhibitor to Specifically Target the ABL Myristoyl Pocket (STAMP)—demonstrated efficacy and a favorable safety profile in heavily pretreated pts with CML with T315I. These results supported the FDA approval of asciminib as a new treatment option for pts with CML-CP with T315I (NCCN 2021). We report updated efficacy and safety data in these pts (data cutoff: January 6, 2021). Aims: Provide updated safety and efficacy data for pts with CML-CP with T315I treated with asciminib monotherapy 200 mg twice daily (BID) after added exposure. Methods: Pts with CML-CP with T315I were enrolled if treated with ≥1 prior TKI and no other effective therapy was available, provided informed consent, and received asciminib 200 mg BID. Results: 48 pts with T315I were included; 2 (4.2%) pts had additional BCR::ABL1 mutations at baseline. Eight (16.7%), 15 (31.3%) and 25 (52.1%) pts received 1, 2, and ≥3 prior TKIs, respectively. At data cutoff, treatment was ongoing in more than half (27 [56.3%]) of pts; the predominant reason for treatment discontinuation was physician’s decision (11 [22.9%]), mainly due to lack of efficacy. Of the 48 pts, 45 were evaluable (BCR::ABL1IS >0.1% at baseline) for major molecular response (MMR); 3 were excluded for BCR::ABL1 atypical transcripts. Among evaluable pts, 19 (42.2%) achieved MMR by wk 24 and 22 (48.9%) by wk 96; 19 were still in MMR at the cutoff date. Evaluable pts included 26 PON-pretreated and 19 PON-naive pts; 34.6% and 68.4%, respectively, achieved MMR by the cutoff date (Table). The probability of pts maintaining MMR for ≥96 wks was 84% (95% CI, 68.1-100.0). Thirteen (28.9%) and 11 (24.4%) pts achieved MR4 and MR4.5, respectively. Twenty (54.1%) and 23 (62.2%) of 37 pts with BCR::ABL1IS >1% at baseline achieved BCR::ABL1IS ≤1% by wk 48 and 96, respectively. The median duration of exposure was 2.08 (range, 0.04-4.13) yrs with more than half (27 [56.3%]) of pts receiving treatment for ≥96 wks; the median daily dose intensity was 398.3 (range, 179-400) mg/day. The safety/tolerability profile of asciminib remained favorable after ≈9 months of added follow-up (Table). The most common (≥5%) grade ≥3 adverse events (AEs) were lipase increase (18.8%, all asymptomatic elevations), thrombocytopenia (14.6%), and vomiting, ALT increase, abdominal pain, hypertension, anemia, neutropenia, and neutrophil count decrease (6.3% each). Arterial occlusive events occurred in 4 (8.3%) pts; none led to dose adjustment/interruption/discontinuation. AEs leading to discontinuation were reported in 2 new pts since the previous data cutoff; both pts discontinued and died due to COVID-19. These were the only study deaths reported in this pt population. Image:Summary/Conclusion: Asciminib monotherapy 200 mg BID exhibited a sustained, favorable safety profile after added exposure with no new safety signals in pts with CML-CP with T315I—a population with high unmet medical need. The clinical efficacy of asciminib is demonstrated by the high proportion of pts achieving durable MMR and BCR::ABL1IS ≤ 1%. The updated analysis confirms asciminib as a treatment option for pts with CML-CP with T315I, including those for whom treatment with PON has failed.