Small-molecule Kinase Inhibitors Research Articles

Abstract IA020: Deciphering the mechanisms of neutrophil immune response in bone metastatic prostate cancer

Abstract Metastatic disease of prostate cancer (PCa) is currently incurable. Bone is the most common tissue site for prostate cancer metastasis and progression in bone is largely dictated by tumor-stromal interactions in the bone microenvironment. We showed previously that bone marrow neutrophils inhibit bone metastatic PCa growth, however metastatic PCa becomes resistant to neutrophil response and progresses within the bone compartment. It is unclear how these findings translate to a clinical setting. Further, the mechanisms associated with tumor resistance to neutrophil immune response remain unknown and presents a new avenue for therapeutic intervention. Thus, the goals of this study were: 1) to define the impact of metastatic PCa on neutrophil function throughout prostate cancer progression and 2) to determine the potential of neutrophils as predictive biomarkers of PCa disease progression. To do this, peripheral blood polymorphonuclear neutrophils (PMNs) were collected from 4 patient cohorts (localized PCa, metastatic hormone-sensitive PCa (mHSPC), metastatic castration-resistant (mCRPC) and healthy men) and PMN molecular and functional properties assessed, including: cancer cell cytotoxicity and cell surface markers. There was a significant decrease with disease progression in PMN expression of CD10, a marker for differentiation, while in tandem, there was an increase in pro-inflammatory marker, CD88 (complement receptor) in mCRPC men compared to mHSPC. PMNs from all disease stages were equally cytotoxic to human PCa cell lines (LNCaP, C42B, and PC3). However, second generation androgen deprivation therapy (ADT) suppressed patient PMN cytotoxicity against mCRPC cells (C42B, PC3). This suppression was due to androgen receptor (AR)-mediated regulation of the pleotropic and immune suppressant cytokine, transforming growth factor beta receptor I (TβRI). PMNs from mCRPC patients who received 2nd generation ADT, expressed significantly more TβRI than patients who only received 1st line ADT. Further, we found that PMNs in CRPC patient bone metastases express more TβRI than PMNs in matched liver metastases, which express little to no TβRI. Treatment of mouse bone marrow neutrophils with enzalutamide, increased neutrophil TβRI expression in a dose dependent manner and blocked cytotoxicity against PCa cells. Exogenous testosterone treatment in vivo, pharmacologic inhibition (using Repsox, a small molecule kinase inhibitor of TβRI) or genetic deletion (using conditional TβRI knockout mice) rescued enzalutamide-mediated neutrophil suppression and restored neutrophil cytotoxicity. These data collectively suggest that AR inhibition suppresses anti-tumor neutrophil responses via TβRI however, this study highlights the ability to leverage standard-care ADT to generate neutrophil anti-tumor responses against bone metastatic PCa. Citation Format: Leah M Cook. Deciphering the mechanisms of neutrophil immune response in bone metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr IA020.

Advances in Small-Molecule C-KIT/PDGFRα Inhibitors for the Treatment of Gastrointestinal Stromal Tumors

AbstractStem cell factor receptor (C-KIT) or platelet-derived growth factor receptor α (PDGFRα) gene mutations have been identified as oncogenic drivers for most gastrointestinal stromal tumors (GISTs). Thus, small-molecule inhibitors of C-KIT or PDGFRα have emerged as effective treatments for GISTs. Although the currently approved first- to fourth-line drugs are initially effective against GISTs, the inevitable development of drug resistance remains an unmet challenge. To address secondary mutations leading to drug resistance, several novel selective C-KIT/PDGFRα small-molecule inhibitors have been developed and clinically studied. This review summarizes the pathogenesis, treatment, and drug resistance mechanisms of GISTs and briefly describes current challenges and future efforts for GIST treatment using small-molecule kinase inhibitors.

De novo Drug Design and Repurposing to suppress Liver Cancer via VEGF-R1 Mechanism: Comprehensive Molecular Docking, MolecularDynamics Simulations and ADME Estimation

Aims: The aim is to halt the progression of liver cancer [Hepatocellular carcinoma] by suppressing the VEGF-R1 receptor using Myricetin and its de novo-designed analogues. Background: VEGF/VEGFR autocrine signalling promotes the growth, progression, and metastasis of Hepatocellular carcinoma, making the development of molecularly targeted therapies highly feasible. Invasive and metastatic behaviours in various cancers, including hepatocellular carcinoma [HCC], are closely monitored through the use of VEGF signalling pathway inhibitors. Specifically in HCC, VEGFR-1 facilitates the invasive capabilities of cancer cells primarily by triggering the epithelial-mesenchymal transition [EMT] process. VEGFR-1 significantly influences the activity of proteolytic enzymes that are critical for the invasive behaviour of HCC cells. Notably, a novel mechanism has been discovered where VEGFR-1 activation leads to the upregulation of MMP-9, thereby enhancing the invasiveness of HCC cells. The scientists, in their study, have elaborated on the various antiangiogenic agents developed for the treatment of HCC. They have highlighted clinical trials that explore the efficacy of these treatments, which include the application of monoclonal antibodies and small-molecule kinase inhibitors designed to target specific pathways involved in tumour angiogenesis and growth. Objective: Creating a pharmaceutical chemistry table regarding ‘’Structure-Activity Relationship of New Compounds on anticancer’’. To do so, Myricetin and its de novo designed structured variants were used in molecular docking, molecular dynamics, cluster analyses, and 1H NMR estimation to specifically understand and enhance the mechanism of suppressing the VEGF-R1 receptor. Method: Proper ligand [Myricetin and its analogues] and receptor [VEGF-R1] preparations, and optimizations were done using the density functional theory [DFT]/B3LYP function along with the 6-31G[d,p] basis set principle in the latest software programs such as Gaussian 09, Gauss View 6.0 and Avogadro. Then using PyRx and Autodock Vina 1.1.2., many molecular docking trials were achieved with 100 posed simulations in each run. An extensive cluster analysis was performed to identify the most optimal docking poses with the highest accumulation and most favourable binding interactions, ensuring the accuracy of the study. The docking configurations that exhibited the most precise and advantageous binding energies were chosen as initial structured data for subsequent Molecular Dynamics [MD] simulations for each drug candidate. To verify the molecular docking results, MD runs were achieved in our supercomputers and the trajectory analyses were made. The data confirmed what was found in molecular docking results, verifying the high efficiency of the druggable molecules’ inhibition towards VEGF-R1. Result: Amine-derivatized Myricetin has a significantly high docking score [-10.56 kcal/mol] and great inhibition constant compared to pristine Myricetin [-4.77 kcal/mol] itself while Fluorinederivatized Myricetin [-6.45 kcal/mol] has an affinity towards VEGF-R1 between the first two molecules. Thus, the structure-activity relationship concerning pharmaceutical chemistry aspects of all the molecules studied, yielded us a great insight into what Myricetin’s organic structure possesses towards inhibiting the progression of Liver Cancer. Also, ADME studies showed that both Amine and Fluorined-derivatized Myricetin molecules are good drug candidates. Conclusion: This study highlighted the significant potential of Myricetin as an anti-cancer drug when modified with specific functional groups. Through comprehensive in silico computational analyses, our research group enhanced Myricetin's inhibitory capabilities by derivatizing its Hydroxyl group with Amine and Fluorine, resulting in improved docking scores and inhibition constants. The findings from molecular docking and molecular dynamics simulations provide a promising foundation for future in vitro and in vivo investigations of this molecule as a potential drug in cancer research.

Inhibition of ATM or ATR in combination with hypo-fractionated radiotherapy leads to a different immunophenotype on transcript and protein level in HNSCC.

The treatment of head and neck tumors remains a challenge due to their reduced radiosensitivity. Small molecule kinase inhibitors (smKI) that inhibit the DNA damage response, may increase the radiosensitivity of tumor cells. However, little is known about how the immunophenotype of the tumor cells is modulated thereby. Therefore, we investigated whether the combination of ATM or ATR inhibitors with hypo-fractionated radiotherapy (RT) has a different impact on the expression of immune checkpoint markers (extrinsic), the release of cytokines or the transcriptome (intrinsic) of head and neck squamous cell carcinoma (HNSCC) cells. The toxic and immunogenic effects of the smKI AZD0156 (ATMi) and VE-822 (ATRi) in combination with a hypo-fractionated scheme of 2x5Gy RT on HPV-negative (HSC4, Cal-33) and HPV-positive (UM-SCC-47, UD-SCC-2) HNSCC cell lines were analyzed as follows: cell death (necrosis, apoptosis; detected by AnxV/PI), expression of immunostimulatory (ICOS-L, OX40-L, TNFSFR9, CD70) and immunosuppressive (PD-L1, PD-L2, HVEM) checkpoint marker using flow cytometry; the release of cytokines using multiplex ELISA and the gene expression of Cal-33 on mRNA level 48h post-RT. Cell death was mainly induced by the combination of RT with both inhibitors, but stronger with ATRi. Further, the immune phenotype of cancer cells, not dying from combination therapy itself, is altered predominantly by RT+ATRi in an immune-stimulatory manner by the up-regulation of ICOS-L. However, the analysis of secreted cytokines after treatment of HNSCC cell lines revealed an ambivalent influence of both inhibitors, as we observed the intensified secretion of IL-6 and IL-8 after RT+ATRi. These findings were confirmed by RNAseq analysis and further the stronger immune-suppressive character of RT+ATMi was enlightened. We detected the down-regulation of a central protein of cytoplasmatic sensing pathways of nucleic acids, RIG-1, and found one immune-suppressive target, EDIL3, strongly up-regulated by RT+ATMi. Independent of a restrictive toxicity, the combination of RT + either ATMi or ATRi leads to comprehensive and immune-modulating alterations in HNSCC. This includes pro-inflammatory signaling induced by RT + ATRi but also anti-inflammatory signals. These findings were confirmed by RNAseq analysis, which further highlighted the immune-suppressive nature of RT + ATMi.

Targeting erbB Pathways in Breast Cancer: Dual Kinase Inhibition for Brain Metastasis and Prevention of p185HER2/Neu Tumor Development.

Breast cancer predominantly affects women and poses challenges in the treatment of both local and advanced diseases. In a previous study, we reported the effectiveness of ER121, a structurally resolved small compound specifically designed to target human cancers expressing or overexpressing mutant EGFR and HER2. The objective of this study is to assess the efficacy and toxicity of ER121 in metastatic and triple negative breast cancer (TNBC, HER2+) cells and tumor models. The Herceptin-resistant breast cancer cell line JIMT-1 was used in an in vivo tumor model, and MMTV-erbB2 (Fo5) transgenic mice models were used to evaluate the efficacy and safety of ER121 as neoadjuvant. ER121 treatment focusing on experimental brain metastasis in TNBC, HER2+ model, was quantified by total flux employing the In Vivo Imaging System (IVIS). We also compared the brain tissue from the treated and the controls groups. Additionally, ER121 was evaluated in JIMT-1, a Herceptin-resistant breast cancer cell line, both in vitro and in vivo tumor model. We also administered ER121 orally in neoadjuvant model with the MMTV-erbB2 (Fo5) transgenic mice, the survival rates were compared with the control group. Tumor-free survival of multiple treated groups were analyzed by Kaplan-Meier analysis employing the log-rank test with the Bonferroni correction using R Statistical Software. In this study, we present findings indicating that ER121 treatment significantly attenuated breast tumor growth using a TNBC, HER2+ model, focusing on experimental brain metastasis, as quantified by total flux employing IVIS. These observations were further corroborated by analysis of brain tissue from the treatment group compared to controls. Data is presented as Mean ± S.D. statistical significance was calculated using Student t test (*p < 0.05). Additionally, ER121 significantly inhibited JIMT-1, a Herceptin-resistant breast cancer cell line was used in vivo xenograft model. Additionally, we used a neoadjuvant model with the MMTV-erbB2 (Fo5) transgenics and the tumor-free survival rates exhibited a remarkable difference between the control and treated groups when ER121 was administered orally. We found statistically significant p values of 0.048 employing log-rank test with Bonferroni Correction for comparing ER121high, ER121 Low, Herceptin and PBS groups. All analyses were performed using R Statistical Software. ER121 is a non-toxic small-molecule erbB kinase inhibitor and holds promise as an oral and systemic therapeutic agent for treating progressive erbB-driven tumors in therapeutic settings. Moreover, ER121 shows potential as a preventive therapy in neoadjuvant settings for erbB2-associated tumors and when administered systemically can dramatically limit erbB2 brain metastases in animal models.

Oncolytic Viruses: From Basics to Challenges to Innovation

The landscape of cancer therapeutics has undergone a transformative shift from broad approaches, such as radiation and chemotherapy, to more precise strategies, encompassing small molecule kinase inhibitors and monoclonal antibodies targeting immune checkpoint molecules. Oncolytic viruses (OVs) have recently emerged as a promising and viable option for cancer immunotherapy, particularly for “cold” tumors entrenched in an immunologically-suppressive tumor microenvironment. Genetic attenuation refines the characteristics of OVs, replacing specific genes to enhance conditional viral replication within tumor cells. Despite their potential, the therapeutic use of OVs faces challenges such as pre-mature elimination by the immune system of the host alongside the dense stromal barriers. In this review article, the strategic encapsulation of genetically-engineered OVs within intra-lesional/-tumoral carriers/ vehicles such as mesenchymal stem cells are explored. To further enhance vector delivery strategies and precise targeting, protecting/shielding OVs from host immune responses, via utilizing release-controlled nanoparticles and employing nanofilaments to optimize vector propagation, for example, are discussed. Such innovative approaches not only mitigate challenges related to pre-mature viral clearance and stromal barriers yet also facilitates a localized, controlled, and sustained release of OVs, thereby optimizing their therapeutic bio-safety and -impact. Ongoing clinical investigations are exploring the synergies between OVs and carriers/delivery systems, combined with various anti-cancer therapeutics as radiotherapy, chemotherapy, immune checkpoint inhibitors, and monoclonal antibodies. These studies hold promise for revolutionizing cancer treatment, ensuring both safety and efficacy, aiming to advance bio-pharmaceutical delivery systems from bench-top to bed-side for enhanced therapeutic results, improved patient survival, and enhanced quality of life or QoL.

ATM Kinase Small Molecule Inhibitors Prevent Radiation-Induced Apoptosis of Mouse Neurons In Vivo

ATM kinase is becoming an important therapeutic target for tumor radiosensitization. Radiation is known to cause neuro-inflammation and neurodegeneration; however, the effects of small molecule ATM inhibitors (ATMi’s) and radiation on normal tissue, including healthy brain, are largely unexplored. Therefore, we examined the mouse CNS after ATMi radiosensitization with a focus on the fate of neurons. We used several approaches to assess the effects on the DNA damage response (DDR) and apoptosis of neurons using immunostaining. In vivo, a significant decrease in viable neurons and increase in degenerating neurons and apoptosis was observed in mice treated with radiation alone. On the other hand, an ATMi alone had little to no effect on neuron viability and did not induce apoptosis. Importantly, the ATMi’s did not further increase radiation toxicity. In fact, multiplex immunostaining showed that a clinical candidate ATMi (AZD1390) protected mouse neurons from apoptosis by 90% at 4 h after radiation. We speculate that the lack of toxicity to neurons is due to a normal ATM–p53 response that, if blocked transiently with an ATMi, is protective. Altogether, in line with previous work using ATM knockout mice, we provide evidence that ATM kinase inhibition using small molecules does not add to neuronal radiation toxicity, and might, in fact, protect them from radiation-induced apoptosis at least in the short term.

Inhibition of WNK Kinases in NK Cells Disrupts Cellular Osmoregulation and Control of Tumor Metastasis

Introduction: The serine/threonine with-no-lysine (WNK) kinase family function in blood pressure control, electrolyte homeostasis, and cellular osmoregulation. These kinases and their downstream effectors are considered promising therapeutic targets in hypertension and stroke. However, the role of WNK kinases in immune cells remains poorly understood. Methods: Using the small-molecule WNK kinase inhibitors WNK463 and WNK-IN-11, we investigated how WNK kinase inhibition affects natural killer (NK) cell physiology. Results: WNK kinase inhibition with WNK463 or WNK-IN-11 significantly decreased IL-2-activated NK cell volume, motility, and cytolytic activity. Treatment of NK cells with these inhibitors induced autophagy by activating AMPK and inhibiting mTOR signaling. Moreover, WNK kinase inhibition increased phosphorylation of Akt and c-Myc by misaligning activity of activating kinases and inhibitory phosphatases. Treatment of tumor-bearing mice with WNK463 impaired tumor metastasis control by adoptively transferred NK cells. Conclusion: The catalytic activity of WNK kinases has a critical role of multiple aspects of NK cell physiology and their pharmacologic inhibition negatively impacts NK cell function.

Slowing Down the "Magic Bullet": Encapsulation of Imatinib in Fe-MOF for Cardiotoxicity Reduction and Improvement in Anticancer Activity.

Imatinib, a small molecule kinase inhibitor, is used as a cancer growth blocker. However, one of its most serious side effects is congestive cardiac failure. Reducing drug toxicity may be achieved through the use of drug delivery systems. Biocompatible metal-organic framework (MOF) materials, namely FeMIL-100 and FeMIL-101-NH2, were employed as potential imatinib carriers. They efficiently delivered the drug as an anticancer agent while minimizing cardiotoxicity. Notably, the release of imatinib from FeMIL-100 was rapid in acidic conditions and slower in pH-neutral environments, allowing targeted delivery to cancer cells. The carrier's pH-dependent stability governed the drug release mechanism. Two release models-Korsmeyer-Peppas and Weibull-were fitted to the experimental data and discussed in terms of drug release from a rigid microporous matrix. Cytotoxicity tests were conducted on two cell lines: HL60 (a model cell line for acute myeloid leukemia) and H9c2 (a cell line for cardiomyocytes). Overall, the metal-organic framework (MOF) carriers mitigated imatinib's adverse effects without compromising its effectiveness.

Machine Learning Prediction of On/Off Target-driven Clinical Adverse Events.

Currently, 90% of clinical drug development fails, where 30% of these failures are due to clinical toxicity. The current extensive animal toxicity studies are not predictive of clinical adverse events (AEs) at clinical doses, while current computation models only consider very few factors with limited success in clinical toxicity prediction. We aimed to address these issues by developing a machine learning (ML) model to directly predict clinical AEs. Using a dataset with 759 FDA-approved drugs with known AEs, we first adapted the ConPLex ML model to predict IC values of these FDA-approved drugs against their on-target and off-target binding among 477 protein targets. Subsequently, we constructed a new ML model to predict clinical AEs using IC values of 759 drugs' primary on-target and off-target effects along with tissue-specific protein expression profiles. The adapted ConPLex model predicted drug-target interactions for both on- and off-target effects, as shown by co-localization of the 6 small molecule kinase inhibitors with their respective kinases. The coupled ML models demonstrated good predictive capability of clinical AEs, with accuracy over 75%. Our approach provides a new insight into the mechanistic understanding of in vivo drug toxicity in relationship with drug on-/off-target interactions. The coupled ML models, once validated with larger datasets, may offer advantages to directly predict clinical AEs using in vitro/ex vivo and preclinical data, which will help to reduce drug development failure due to clinical toxicity.

Characterization and Animal Skin Irritations Investigation of Vemurafenib Microemulsion-Based Hydrogel Using Oily Ionic Liquid

Cutaneous melanoma accounts for a yearly mortality rate of 55,500 persons. The use of oral small-molecule kinase inhibitors, specifically targeting BRAFv600, has been licensed as the principal treatment strategy for managing both locally progressed and metastatic presentations of the condition. Approximately 30% of people on vemurafenib, a BRAFv600 inhibitor, have side effects when are taken orally. Objective: This research was attempted to develop a vemurafenib microemulsion by substituting conventional oil phases with ionic liquids (ILs). The microemulsions were created by dissolving vemurafenib in a combination of ionic liquid (1-Butyl-3-methylimidazolium hexafluorophosphate and 1-Octyl-3-methylimidazolium hexafluorophosphate) and surfactant (Triton x-100). Multiple tests were conducted to measure physical stability, pH determination, content homogeneity examination, and in vitro medicine release analysis. Four formulations of Vemurafenib microemulsions successfully met all criteria in the microemulsion characterisation and assessment tests. The droplet sizes in these microemulsions fell inside the range of microemulsions, which is less than 200 nm. They were then used to create microemulsion-based hydrogels, employing Carbamer 340 as a gelling agent by conducting a simple mixing method. Hydrogels formed from microemulsions containing 1-Octyl-3-methylimidazolium hexafluorophosphate demonstrated the ability to form clear hydrogels with desirable consistency. Regarding Ex-vivo permeability study and skin deposition the permeability profiles of GOT3 formula exhibits a permeability measurement of 33569± 344 mP.s at 6 rpm, whereas GOT4 demonstrates a permeability measurement of 54723± 380 mP.s at 6 rpm. During the skin irritation test, there was no apparent erythema and edema were observed when compared to the negative group. This may indicate that the designed microemulsion-based gel formulation demonstrates good biocompatibility with skin tissue. Topical delivery of vemurafenib is a promising route of drug administration to melanoma skin. Ionic liquids (ILs) have permeation-enhancing properties with either hydrophilic or lipophilic characteristics‎‎‎‎‎.

Synthetic Approaches and Clinical Application of Representative Small-Molecule Inhibitors of Cyclin-Dependent Kinase for Cancer Therapy.

The regulation of the cancer cell cycle heavily relies on cyclin-dependent kinases (CDKs). Targeting CDKs has been identified as a promising approach for effective cancer therapy. In recent years, there has been significant attention paid towards developing small-molecule CDK inhibitors in the field of drug discovery. Notably, five such inhibitors have already received regulatory approval for the treatment of different cancers, including breast tumors, lung malignancies, and hematological malignancies. This review provides an overview of the synthetic routes used to produce 17 representative small-molecule CDK inhibitors that have obtained regulatory approval or are currently being evaluated through clinical trials. It also discusses their clinical applications for treating CDK-related diseases and explores the challenges and limitations associated with their use in a clinical setting, which will stimulate the further development of novel CDK inhibitors. By integrating therapeutic applications, synthetic methodologies, and mechanisms of action observed in various clinical trials involving these CDK inhibitors, this review facilitates a comprehensive understanding of the versatile roles and therapeutic potential offered by interventions targeting CDKs.

KLSD: a kinase database focused on ligand similarity and diversity.

Due to the similarity and diversity among kinases, small molecule kinase inhibitors (SMKIs) often display multi-target effects or selectivity, which have a strong correlation with the efficacy and safety of these inhibitors. However, due to the limited number of well-known popular databases and their restricted data mining capabilities, along with the significant scarcity of databases focusing on the pharmacological similarity and diversity of SMIKIs, researchers find it challenging to quickly access relevant information. The KLIFS database is representative of specialized application databases in the field, focusing on kinase structure and co-crystallised kinase-ligand interactions, whereas the KLSD database in this paper emphasizes the analysis of SMKIs among all reported kinase targets. To solve the current problem of the lack of professional application databases in kinase research and to provide centralized, standardized, reliable and efficient data resources for kinase researchers, this paper proposes a research program based on the ChEMBL database. It focuses on kinase ligands activities comparisons. This scheme extracts kinase data and standardizes and normalizes them, then performs kinase target difference analysis to achieve kinase activity threshold judgement. It then constructs a specialized and personalized kinase database platform, adopts the front-end and back-end separation technology of SpringBoot architecture, constructs an extensible WEB application, handles the storage, retrieval and analysis of the data, ultimately realizing data visualization and interaction. This study aims to develop a kinase database platform to collect, organize, and provide standardized data related to kinases. By offering essential resources and tools, it supports kinase research and drug development, thereby advancing scientific research and innovation in kinase-related fields. It is freely accessible at: http://ai.njucm.edu.cn:8080.

N-phenyl pyrazoline derivative inhibits cell aggressiveness and enhances paclitaxel sensitivity of triple negative breast cancer cells

Protein kinase dysregulation induces cancer cell aggressiveness leading to rapid tumor progression and poor prognosis in TNBC patients. Many small-molecule kinase inhibitors have been tested in clinical trials to treat TNBC patients. In the previous study, we found that N-phenylpyrazoline small molecule acts as a protein kinase inhibitor in cervical cancer cells. However, there remains unknown about N-phenyl pyrazoline potency as a kinase inhibitor and its anti-cancer activity in TNBC cells. In this study, we investigated the activity of N-phenyl pyrazoline against TNBC cells via tyrosine kinase inhibition. Based on the MTT assay, the IC50 values for the N-phenyl pyrazoline 2, 5, A, B, C, and D against Hs578T were 12.63 µM, 3.95 µM, not available, 18.62 µM, 30.13 µM, and 26.79 µM, respectively. While only P5 exhibited the IC50 against MDA MB 231 (21.55 µM). Further, N-phenyl pyrazoline 5 treatment significantly inhibited the cell proliferation rate of Hs578T and MDA MB 231 cells. The migration assay showed that treatment with the compound N-phenyl pyrazoline 5 with 4 µM concentration significantly reduced cell migration of Hs578T cells. N-phenyl pyrazoline 5 treatment at 1 µM and 2 µM was able to reduce the tumorsphere size of Hs578t cells. A combination treatment of P5 and paclitaxel showed a synergistic effect with a combination index score > 1 in both TNBC cells. Further, the P5 predictively targeted the protein kinases that significantly correlated to breast cancer prognosis. The GSEA analysis result shows that receptor tyrosine kinase, Notch3, Notch4, and Ephrin signaling pathways were targeted by P5. The P5 treatment reduced the EGFR expression level and activation in TNBC cells.

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 .

Efficacy and safety of surufatinib combined with EP regimen and serplulimab in first-line treatment of NEC.

e15123 Background: Neuroendocrine carcinomas (NEC) are more challenging than neuroendocrine tumors (NET) due to poor differentiation, aggressive invasion, and early diagnosis difficulties, often marked by local and distant metastasis at diagnosis, leading to poor outcomes. The ORR for NEC as first-line treatment with EP or EC regimens is approximately 30%, highlighting a substantial treatment gap. Surufatinib, a targeted small molecule kinase inhibitor, acts on vascular endothelial growth factor receptors (VEGFR-1/2/3), fibroblast growth factor receptor (FGFR-1), and colony-stimulating factor-1 receptor (CSF-1R). Its efficacy focuses on anti-angiogenesis and vascular normalization, alongside its role in modulating tumor-associated macrophages (TAMs) and boosting the effectiveness of immunotherapy. Methods: This single-arm, open-label, multicenter clinical study comprised a safety run-in (SR) phase and a dose-expansion (DE) phase. In the SR phase, six patients (pts) were planned to be enrolled to receive treatment with surufatinib (250 mg, QD, PO, Q3W) combined with the EP regimen (etoposide injection: 100mg/m2, d1-3, IV, Q3W; cisplatin: 25mg/m2, d1-3, IV, Q3W) and serplulimab (300mg, d1, IV, Q3W) for a maximum of six cycles, followed by a maintenance phase of surufatinib plus serplulimab until disease progression or other criteria as specified in the protocol. The dose of surufatinib in the DE phase was based on results from the SR phase. The primary endpoints were recommended Phase 2 dose (RP2D) and PFS, while secondary endpoints included ORR, DCR, OS, and safety. Results: As of December 27, 2023, this reports results from the SR phase. Six pts, median age 61.5 years (range 38-70), 66.7% male, all with ECOG performance status 1, were enrolled and completed their first treatment cycle. A majority (83.3%) had gastrointestinal-origin tumors with a median Ki-67% index of 70% (range 30%-90%). During the SR phase, one patient experienced Grade 4 hematologic toxicity (neutropenia and leukopenia) as a dose-limiting toxicity (DLT). Consequently, RP2D of surufatinib was established at 250mg for further research in the DE phase. The most common adverse events were neutropenia (50%), decreased white blood cell count (33.3%), fatigue (33.3%), and diarrhea (33.3%). Among those enrolled in the SR phase, 5 pts underwent at least one assessment of efficacy, yielding an objective response rate (ORR) of 60% and a disease control rate (DCR) of 100%, with the median progression-free survival (mPFS) not yet mature. Conclusions: Surufatinib combined with the EP regimen and serplulimab for NEC as first-line treatment showed notable efficacy and safety. The study established a phase II recommended surufatinib dose of 250mg, alongside immunotherapy and chemotherapy. Clinical trial information: NCT05747729 .

A green and highly sensitive microwell spectrofluorimetric method with high throughput for the determination of pemigatinib based on dual fluorescence enhancement by photoinduced electron transfer blocking and micellization: Application to the analysis of tablets, content uniformity testing, and human plasma.

Pemigatinib (PGT) is a recently FDA-approved small molecule kinase inhibitor used for the treatment of relapsed or refractory myeloid/lymphoid neoplasms in adults. This study introduces the development of a first microwell spectrofluorimetric method (MW-SFM) for quantifying PGT in FDA-approved tablets and plasma samples. The method utilized the enhancement of PGT's weak native fluorescence by blocking photoinduced electron transfer (PET) and micellization with sodium lauryl sulfate (SLS). The MW-SFM was performed in 96-microwell plates, and fluorescence signals were measured using a fluorescence microplate reader with excitation at 290 nm and emission at 350 nm. The method exhibited a linear range of 2-250 ng mL-1, and a limit of quantitation was 6.5ng mL-1. The accuracy and precision of the method were confirmed with recovery rates ranging from 96.5% to 102.8% and relative standard deviations of 1.52% to 3.51%. The MW-SFM successfully analyzed Pemazyre® tablets, assessed content uniformity, and analyzed PGT-spiked human plasma samples. The greenness of the MW-SFM was verified using three different metric tools. In conclusion, the proposed MW-SFM is a valuable tool in supporting quality assessment of dosage forms, conducting pharmacokinetic studies, and monitoring therapeutic outcomes.

Efficacy and safety of surufatinib, toripalimab, nab-paclitaxel in combination with radiotherapy or surgery in the first-line treatment of esophageal squamous cell cancer: A single-centered prospective clinical trial.

e16047 Background: Chemotherapy(CT) plus immune checkpoint inhibitors (ICIs) has become the current standard of care in the first-line treatment of metastatic esophageal squamous cell cancer(ESCC). In locally advanced setting, radiochemotherapy with or without resection remains the standard of care. Surufatinib, a novel small-molecule kinase inhibitor targeting VEGFR1-3, FGFR and CSF-1R, in combination with PD-1 inhibitors have demonstrated synergistic anti-tumor activities in previous studies. This single-centered prospective clinical trial aims to evaluate the efficacy and safety of surufatinib(SUR), toripalimab(TOR), nab-paclitaxel(NAB) in combination with radiotherapy or surgery in the first-line treatment of locally advanced(LA) or metastatic(R/M) ESCC. Methods: Eligible patients(pts) with confirmed LA or R/M, ECOG PS 0-2 and no prior systematic treatment were enrolled in the trial. Pts would receive 260mg/m2 NAB and TOR 240mg on day 1 and SUR 250mg once daily in each 21-day cycle as induction therapy for two cycles, followed by surgery or another two cycles of concurrent chemoradiotherapy(CCRT) with NAB, SUR and radiotherapy, in which involved-field irradiation were conducted at the dose of 50-63 Gy/25-30F, 1.8-2 Gy per fraction, 5 fractions per week. As maintenance therapy, daily SUR and TOR every three week were administered until disease progression or unacceptable toxicities. Progression-free survival (PFS) was the primary endpoint. Results: As of Jan 7, 2024, 16 pts were enrolled, of whom 15 were evaluable. 14(87.5%) pts were male. The median age was 69.5 (range: 45-79). Tumor located in upper(18.8%), middle(25%) and lower(56.2%) thoracic. 1(6.2%) is Stage II disease, 12(75%) Stage III and 3(18.8%) Stage IV. 8 (50%) pts were with PD-L1 CPS greater than 1, 5 (31.25%) pts greater than 5, and 3 (18.75%) pts greater than 10. PD-L1 CPS of 6(37.50%) pts was unknown. With a median follow-up time of 5 (range: 1-12) months, despite the immatureness of PFS, the 12-month PFS rate was 77.14% (95% CI: 48.89%-100%). The objective response rate was 93.3% (14/15), and disease control rate was 100% (15/15). Five of the included pts met the resection criteria during treatment and underwent subsequent R0 resection. The most common adverse events (≥20%) of all grades were hypoesthesia (62.5%), nausea and vomiting (50%), and white blood cell decrease (43.75%). Conclusions: The combination ofsurufatinib, toripalimab, nab-paclitaxel with radiotherapy or surgery has demonstrated promising efficacy and a well-tolerated safety profile as a potential first-line treatment for LA or R/M ESCC. This ongoing trial merits further investigation and holds significant implications for clinical practice.

Targeting CK2 eliminates senescent cells and prolongs lifespan in Zmpste24-deficient mice

Senescent cell clearance is emerging as a promising strategy for treating age-related diseases. Senolytics are small molecules that promote the clearance of senescent cells; however, senolytics are uncommon and their underlying mechanisms remain largely unknown. Here, we investigated whether genomic instability is a potential target for senolytic. We screened small-molecule kinase inhibitors involved in the DNA damage response (DDR) in Zmpste24−/− mouse embryonic fibroblasts, a progeroid model characterized with impaired DDR and DNA repair. 4,5,6,7-tetrabromo-2-azabenzamidazole (TBB), which specifically inhibits casein kinase 2 (CK2), was selected and discovered to preferentially trigger apoptosis in Zmpste24−/− cells. Mechanistically, inhibition of CK2 abolished the phosphorylation of heterochromatin protein 1α (HP1α), which retarded the dynamic HP1α dissociation from repressive histone mark H3K9me3 and its relocalization with γH2AX to DNA damage sites, suggesting that disrupting heterochromatin remodeling in the initiation of DDR accelerates apoptosis in senescent cells. Furthermore, feeding Zmpste24-deficient mice with TBB alleviated progeroid features and extended their lifespan. Our study identified TBB as a new class senolytic compound that can reduce age-related symptoms and prolong lifespan in progeroid mice.

Structure-Based De Novo Design for the Discovery of Miniprotein Inhibitors Targeting Oncogenic Mutant BRAF.

Being a component of the Ras/Raf/MEK/ERK signaling pathway crucial for cellular responses, the VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has emerged as a promising target for anticancer drug discovery due to oncogenic mutations that lead to pathway hyperactivation. Despite the discovery of several small-molecule BRAF kinase inhibitors targeting oncogenic mutants, their clinical utility has been limited by challenges such as off-target effects and suboptimal pharmacological properties. This study focuses on identifying miniprotein inhibitors for the oncogenic V600E mutant BRAF, leveraging their potential as versatile drug candidates. Using a structure-based de novo design approach based on binding affinity to V600E mutant BRAF and hydration energy, 39 candidate miniprotein inhibitors comprising three helices and 69 amino acids were generated from the substructure of the endogenous ligand protein (14-3-3). Through in vitro binding and kinase inhibition assays, two miniproteins (63 and 76) were discovered as novel inhibitors of V600E mutant BRAF with low-micromolar activity, with miniprotein 76 demonstrating a specific impediment to MEK1 phosphorylation in mammalian cells. These findings highlight miniprotein 76 as a potential lead compound for developing new cancer therapeutics, and the structural features contributing to its biochemical potency against V600E mutant BRAF are discussed in detail.