Inhibition Of Isoforms Research Articles

Abstract CT119: A first-in-human phase 1 study of LOXO-435, a potent, highly isoform-selective FGFR3 inhibitor in advanced solid tumors with FGFR3 alterations (trial in progress)

Abstract Background: Alterations in the fibroblast growth factor receptor 3 (FGFR3) have been identified as oncogenic drivers in several solid tumor malignancies, including urothelial carcinoma (UC). Available pan-FGFR inhibitors target all 4 isoforms of FGFR (FGFR1 - 4) and consequently their efficacy can be limited by off-target toxicity (i.e. inhibition of other non-altered FGFR isoforms) including gastrointestinal, oral mucositis, and cutaneous/nail disorders, as well as FGFR1-mediated hyperphosphatemia. Moreover, clinical studies of pan-FGFR inhibitors have reported the development of drug resistance through acquired gatekeeper mutations (e.g. V555M and V565F) at the time of disease progression. LOXO-435 is a potent, highly isoform-selective FGFR3 inhibitor that is designed to preserve activity in the setting of gatekeeper resistance mutations. Preclinically, LOXO-435 has demonstrated potent in vitro and in vivo activity as a selective inhibitor of both wild-type and oncogenically activated FGFR3, including FGFR3 fusions, point mutations, and acquired gatekeeper resistance mutations without evidence of FGFR1-mediated hyperphosphatemia. Methods: LOXO-FG3-22001 is a multicenter, open-label, first-in-human phase 1a/b study of LOXO-435 in patients (pts) with FGFR3-altered advanced solid tumors, including metastatic UC (mUC) (NCT05614739). Eligible pts (≥ 18 years) must have an advanced or metastatic solid tumor with an FGFR3 alteration detected in tumor or ctDNA, ECOG PS of 0-1, and have received all standard therapy or have no known available options to provide benefit. The study will be conducted in 2 phases: phase 1a dose escalation and phase 1b dose expansion. Dose escalation (cohort A) will begin as a single-patient accelerated design, allowing intra-patient dose escalation, followed by the modified toxicity probability interval-2 method. Additional pts will be allowed to backfill to previously cleared dose levels that demonstrate therapeutically relevant exposures or direct evidence of clinical activity. Dose escalation will assess safety, PK/PD, and antitumor activity of LOXO-435 as monotherapy to determine the recommended phase 2 dose (RP2D). Dose expansion will include 4 cohorts of pts with prespecified activating FGFR3 alterations. Cohort B will enroll pts with mUC and include 2 cohorts to evaluate LOXO-435 as monotherapy (B1, B2) and 1 cohort to evaluate LOXO-435 in combination with pembrolizumab (B3). Cohort C will enroll pts with non-UC advanced solid tumors and will evaluate LOXO-435 as monotherapy (C1). Pts in cohort B1 must have progressed on or were intolerant to a prior FGFR inhibitor, and pts in cohorts B2, B3, and C1 must be FGFR inhibitor treatment naive. Dose expansion will evaluate the safety, PK/PD, and antitumor activity (per RECIST v1.1) of LOXO-435 at the RP2D. Citation Format: Gopa Iyer, Arlene Siefker-Radtke, Matthew Milowsky, Neal Shore, Xin Gao, Melissa A. Reimers, Noah Hahn, Rasha Cosman, Nobuaki Matsubara, Andrea Necchi, Debbie Robbrecht, Armelle Vinceneux, Enrique Grande, Jae-Lyun Lee, Tian Zhang, Tormod Guren, Ulrich M. Lauer, Michal Sarfaty, Bernhard Eigl, Syed Hussain, Xiang Zhao, Arjun V. Balar, Kaitlyn Francese, Ryan Widau, Benjamin Garmezy. A first-in-human phase 1 study of LOXO-435, a potent, highly isoform-selective FGFR3 inhibitor in advanced solid tumors with FGFR3 alterations (trial in progress) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT119.

Role of the iNOS isoform in the cardiovascular dysfunctions of male rats with 6-OHDA-induced Parkinsonism

IntroductionAvailable studies have shown the involvement of nitric oxide (NO) in the processes that lead to neurodegeneration in Parkinson's disease (PD). Also, the use of inhibitors of the inducible isoform of NO-synthase (iNOS) promotes neuroprotection and attenuates dopamine (DA) loss in experimental models of Parkinsonism. In addition, NO also appears to be involved in cardiovascular changes in 6-hydroxydopamine (6-OHDA)-induced Parkinsonism. The current study aimed to evaluate the effects of iNOS inhibition on cardiovascular and autonomic function in animals that were subjected to Parkinsonism by the administration of 6-OHDA. Materials and methodsThe animals underwent stereotaxic surgery for bilateral microinfusion of the neurotoxin 6-OHDA (6 mg/mL in 0.2% ascorbic acid in sterile saline solution) or vehicle solution for the Sham group. From the day of stereotaxis until the day of femoral artery catheterization, the animals were treated with the iNOS inhibitor, S-methylisothiourea (SMT; 10 mg/kg; i. p.) or saline solution (0.9%; i. p.) for 7 days. The animals were divided into four groups: Sham-Saline, Sham-SMT, 6-OHDA-Saline, and 6-OHDA-SMT. Subsequent analyses were performed on these four groups. After 6 days, they underwent catheterization of the femoral artery, and 24 h later, mean arterial pressure (MAP) and heart rate (HR) were recorded. Another group of animals (the 6-OHDA and Sham groups) was assessed for aortic vascular reactivity after 7 days of bilateral infusion of 6-OHDA or vehicle, in which cumulative concentration-effect curves (CCEC) were made for phenylephrine (Phenyl), acetylcholine and sodium nitroprusside (NPS). Also, CCEC in the presence of Nw-nitro-arginine-methyl-ester (l-NAME) (10-5 M), SMT (10-6 M), and indomethacin (10-5 M) blockers were made. ResultsThe effectiveness of the 6-OHDA lesion was confirmed with the reduction of DA in 6-OHDA animals. However, treatment with SMT could not reverse the loss of DA. Concerning the baseline parameters, SBP and MAP values were lower in 6-OHDA animals compared to their Sham control, with no effect of treatment with SMT. In the analysis of SBP variability, a decrease in variance, the VLFabs component, and the LFabs component were observed in the 6-OHDA groups when compared to their controls, regardless of treatment with SMT. It was also observed that intravenous injections of SMT resulted in an increase in BP and a decrease in HR. However, the response was not different between the Sham and 6-OHDA groups. In vascular function, there was a hyporeactivity to Phenyl in the 6-OHDA group, and when investigating the mechanisms of this hyporeactivity, it was seen that the Rmax to Phenyl increased with incubation with SMT, indicating that iNOS could be involved in the vascular hyporeactivity of animals with Parkinsonism. ConclusionThus, the set of results presented in this study suggests that part of the cardiovascular dysfunction in animals subjected to 6-OHDA Parkinsonism may be peripheral and involve the participation of endothelial iNOS.

Abstract 4444: Targeting PI3K isoforms to improve the effectiveness of T cell mediated immunotherapy

Abstract Hyperactivation of the PI3K pathway has been reported to correlate with resistance to immune checkpoint blockade therapy (ICB) in melanoma, highlighting the therapeutic potential of combining PI3K inhibition (PI3Ki) with ICB. To maximize the clinical benefit of PI3Ki-based immune oncology (IO) combination, we characterized the role of PI3K isoforms in tumor and T cells and determined the immunological impacts of PI3Ki alone or in combination with ICB. Inhibitions of PI3K were achieved by either genetic knockdown (KD) or the bioactive compound in PTEN-present (B16/MC38), PTEN-absent (BP/D4M) tumor cell lines, and CD8+ T cells (Pmel-1). Following PI3Ki, we determined the activation status of the PI3K pathway (p-AKT level), transcriptional profile, and cellular function of these cells. We found both in vitro KD and pharmacological inhibition of either PI3Kα or PI3Kβ displayed a dramatic reduction of the PI3K pathway in tumor cells but moderate or no reduction in T cells, whereas the PI3K pathway significantly decreased in T cells with PI3Kγ or PI3Kδ inhibition. KD of PI3Kα or β isoforms drastically sensitized both D4M and MC38 tumors to αPD1 in vivo. We also observed that only PI3Kγ or PI3Kδ inhibition profoundly suppressed cytokine production and cytotoxicity of CD8+ T cell, suggesting that PI3Kα or PI3Kβ isoform inhibition can achieve tumor specific PI3Ki with limited impacts on T cell function. Furthermore, we used multiple syngeneic melanoma models to determine whether PI3K isoform inhibition can synergize the antitumor activity of ICB in vivo. In PTEN-present tumors, BYL719 (BYL, a PI3Kα inhibitor) synergized with αPD1 to delay tumor growth and extend survival (median survival of MC38-bearing mice in control (Ctrl), BYL, αPD1, and combination (Comb) groups: 30, 36, 33, and >45 respectively; p<0.05: Ctrl/BYL/αPD1 vs Comb). However, a limited combinatorial effect between GSK2636771(a PI3Kβ inhibitor) and αPD1 was observed in PTEN-present tumor models. Moreover, the combination of BYL and αPD1 exhibits superior antitumor activity in a spontaneous Braf-mutant, PTEN-loss melanoma model when compared with either reagent. Mechanistically, the combination of BYL and αPD1 improved CD8+ T cells tumor infiltration (14 days treatment, mean CD8+ number/mg of the tumor, Ctrl:1392.9, BYL:2073.9, αPD1:1545.2, Comb:4691.8; p<0.01: Ctrl/BYL/αPD1 vs Comb) and reduced MDSCs in MC38 tumors (p<0.05: Ctrl vs Comb). Multi-omics profiling of tumor cells with in vitro and in vivo PI3K isoform inhibition is ongoing. Collectively, our results demonstrate that PI3Kα inhibitor can potentiate T cell-mediated antitumor immune responses regardless of PTEN status, providing a strong rationale for the clinical development of the BYL-based IO combination. In collaboration with Novartis, MD Anderson Cancer Center will launch a Phase I/II trial of the FDA-approved BYL in combination with αPD1 in advanced melanoma and breast cancer patients. Citation Format: Ritu Bohat, Xiaofang Liang, Chunyu Xu, Yitao Tang, Jiakai Hou, Nicholas A. Egan, Leilei Shi, Ashley Guerrero, Roshni Jaffery, Elizabeth M. Burton, Han Liang, Hussein Tawbi, Michael A. Davies, Weiyi Peng. Targeting PI3K isoforms to improve the effectiveness of T cell mediated immunotherapy. [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 4444.

Abstract 3097: Prodrugs of a 1-hydroxy-2-oxopiperidin-3-yl phosphonate enolase inhibitor for the treatment of ENO1-deleted cancers

Abstract Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). We previously identified and characterized a competitive, small molecule phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX), and its lipophilic bis-ester prodrug (POMHEX) in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma. Treatment with either HEX (150 mg/kg IV/IP) or POMHEX (20 mg/kg IV/IP) yielded tumor regression even after drug discontinuation. However, due to the poor pharmacokinetics of esterase-labile POMHEX, we synthesized a library of novel phosphonate prodrugs with distinct mechanisms of bioactivation and assessed their potency in D423 (ENO1-/-) cells. By conducting a prodrug structure activity relationship (SAR) study, we found that phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, while canonical McGuigan (ProTide) prodrugs were not. Other strategies, including salicylic alcohol (cycloSal) and lipid prodrugs of HEX, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over POMHEX. En route, we developed a novel class of aliphatic amine/ester prodrugs that can be broadly applied to efficiently deliver phosph(on)ate pharmacophores in cells. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation. In sum, we have developed a novel class of phosph(on)ate prodrugs that is efficiently bioactivated in cells in vitro. Our prodrug SAR study disputes the common notion that ProTides are universally advantageous promoieties on phosph(on)ate pharmacophores and we provide mechanistic rationale for this observation with HEX. Finally, we show that the cycloSal prodrug yields efficient intracellular delivery of HEX in vitro, with a mechanism of bioactivation consistent with the GBM microenvironment, making this promoiety promising for further evaluation in vivo. Citation Format: Victoria C. Yan, Cong-Dat Pham, Elliot S. Ballato, Kristine L. Yang, Sunada Khadka, Yasaman Barektain, Prakriti Shrestha, Theresa Tran, Anton H. Poral, Mykia Washington, Sudhir Raghavan, Barbara Czako, Federica Pisaneschi, Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Jeffrey J. Ackroyd, Dimitra K. Georgiou, Steven W. Millward, Florian L. Muller. Prodrugs of a 1-hydroxy-2-oxopiperidin-3-yl phosphonate enolase inhibitor for the treatment of ENO1-deleted cancers [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 3097.

Abstract 4928: Gedatolisib, a well-tolerated pan-PI3K/mTOR inhibitor, exhibits potent therapeutic effects on endometrial cancer models regardless of their PI3K pathway mutational status

Abstract Background: The PI3K, AKT, and mTOR (PAM) pathway is one of the most commonly activated oncogenic pathways in gynecological cancers. Loss of PTEN function and PIK3CA activating mutations are especially frequent in endometrial cancer (EC). Targeting the PAM pathway, in combination with other cooperative oncogenic pathways (e.g., the estrogen pathway), is a promising EC treatment strategy. Currently, everolimus, an mTORC1 inhibitor, in combination with letrozole, an aromatase inhibitor, is one of the available hormonal therapy options for patients with advanced endometrioid EC. Most PAM inhibitors (PAM-i) selectively spare (or weakly inhibit) one or more key PAM pathway components, which can lead to drug resistance. To minimize drug resistance, a more comprehensive inhibition of the PI3K isoforms and downstream mTOR complexes may be required. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, mTORC1, and mTORC2, can potentially be an effective and well tolerated therapy for patients with EC. Methods: A panel of endometrial, ovarian and cervical cancer cell lines with different PI3K pathway mutational status were assayed for their sensitivity to gedatolisib and other PAM inhibitors (PI3K-α: alpelisib, pan-PI3K: copanlisib; AKT: capivasertib; mTORC1: everolimus; pan-PI3K/mTOR: gedatolisib). Cell viability, cell proliferation, and flow cytometry analytical assays were conducted. Xenograft studies evaluating gedatolisib in EC cell lines were also performed. Results: Gedatolisib strongly inhibited PAM pathway activation, and reduced cell cycle progression and cell viability in the endometrial, ovarian, and cervical cancer cell lines. Compared to the other PAM-i, Gedatolisib exhibited superior anti-proliferative potency and efficacy in almost all the cell lines tested, regardless of the PI3K pathway mutational status or cancer type. In EC xenografts, gedatolisib substantively inhibited tumor growth irrespective of the cell line models’ PI3K pathway mutational status. Conclusions: We demonstrated that, gedatolisib induces superior anti-proliferative activity compared to the other PAM pathway inhibitors, regardless of the PI3K pathway mutational status of the cancer cell lines evaluated. Gedatolisib as a single agent and in combination with other therapies has previously reported promising preliminary clinical efficacy and safety in various solid tumor types, including EC. In summary, nonclinical and preliminary clinical data supports the development of gedatolisib in combination with hormonal therapy for treatment of patients with EC. Citation Format: Stefano Rossetti, Aaron Broege, Ian MacNeil, Ben Rich, Jhomary Molden, Brian Sullivan, Lance Laing. Gedatolisib, a well-tolerated pan-PI3K/mTOR inhibitor, exhibits potent therapeutic effects on endometrial cancer models regardless of their PI3K pathway mutational status. [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 4928.

Endometrial hyperplasia and progesterone resistance: a complex relationship

The endometrium is one of the most dynamic tissues that constantly undergoes changes during the menstrual cycle in women of the reproductive period. All these processes take place mainly under the influence of steroid hormones that are produced in the woman’s body. However, it is important to remember that throughout life the endometrial tissue undergoes changes under the influence of various factors that lead to imbalances in hormonal regulation. All these changes can lead to the development of endometrial hyperplasia, which has a high risk of both recurrence and malignization. Over the past few decades, the incidence of endometrial cancer has increased in many countries. This trend is thought to be related to the increasing prevalence of obesity, as well as to changing female reproductive patterns. Although there are currently no well-established screening programmers for endometrial cancer, endometrial hyperplasia is a recognized precursor, and its detection provides an opportunity for prevention. Studying the pathogenesis and risk factors will give a great advantage in the future to prevent possible complications. At this point, the activity and inhibition of the different hormone isoforms can lead to different hyperplastic processes. The management of patients depends on many factors: age, species, reproductive potential and other factors. Therefore, a comprehensive approach to treatment is always necessary. In recent years, interest in the study of endometrial hyperplasia has increased dramatically due to the increase in endometrial cancer. Therefore, the issue of early diagnosis and prevention is most urgent in modern gynecology and requires further study. This review reflects the current understanding of the disruption of progesterone signaling mechanisms in endometrial hyperplasia according to domestic and foreign literature.

Design of new reversible and selective inhibitors of monoamine oxidase A and a comparison with drugs already approved

BackgroundMonoamine oxidase (MAO) is an enzyme that has been targeted pharmacologically for the treatment of depression and neurodegenerative diseases such as Parkinson's disease. To avoid side effects, drugs currently in use must selectively target either of the enzyme's two isoforms, A or B. In this study, we designed molecules derived from chalcone as potential reversible and selective inhibitors of isoform A of the MAO enzyme.ResultsTen thousand one hundred compounds were designed and screened using molecular docking, considering the pharmacokinetic processes of chemical absorption, distribution, metabolism, and excretion. Density functional theory calculations were performed for the main ligands to evaluate their reactivity. Six drugs qualified as reversible and irreversible inhibitors of both isoform A and isoform B. Among these, molecule 356 was found to be a reversible inhibitor with the best performance in selectively targeting isoform A of the MAO enzyme. The interaction stability of ligand 356 in the isoform A binding site was confirmed by molecular dynamics. One hydrogen bond was found between the ligand and the cofactor, and up to six hydrogen bonds were formed between the ligand and the protein.ConclusionsWe selected a drug model (molecule 356) for its high affinity to isoform A over isoform B of the MAO enzyme. This proposal should decrease experimental costs in drug testing for neurodegenerative diseases. Therefore, our silico design of a reversible inhibitor of isoform A of enzyme monoamine oxidase can be used in further experimental designs of novel drugs with minimal side effects.Graphical

Development of novel anilinoquinazoline-based carboxylic acids as non-classical carbonic anhydrase IX and XII inhibitors

As part of our ongoing endeavour to identify novel inhibitors of cancer-associated CA isoforms IX and XII as possible anticancer candidates, here we describe the design and synthesis of small library of 2-aryl-quinazolin-4-yl aminobenzoic acid derivatives (6a–c, 7a–c, and 8a–c) as new non-classical CA inhibitors. On account of its significance in the anticancer drug discovery and in the development of effective CAIs, the 4-anilinoquinazoline privileged scaffold was exploited in this study. Thereafter, the free carboxylic acid functionality was appended in the ortho (6a–c), meta (7a–c), or para-positon (8a–c) of the anilino motif to furnish the target inhibitors. All compounds were assessed for their inhibitory activities against the hCA I, II (cytosolic), IX, and XII (trans-membrane, tumour-associated) isoforms. Moreover, six quinazolines (6a–c, 7b, and 8a–b) were chosen by the NCI-USA for in vitro anti-proliferative activity evaluation against 59 human cancer cell lines representing nine tumour subpanels.

Selenium-analogs based on natural sources as cancer-associated carbonic anhydrase isoforms IX and XII inhibitors

In the relentless search for new cancer treatments, organoselenium compounds, and carbonic anhydrase (CA) inhibitors have emerged as promising drug candidates. CA isoforms IX and XII are overexpressed in many types of cancer, and their inhibition is associated with potent antitumor/antimetastatic effects. Selenium-containing compounds, particularly selenols, have been shown to inhibit tumour-associated CA isoforms in the nanomolar range since the properties of the selenium atom favour binding to the active site of the enzyme. In this work, two series of selenoesters (1a–19a and 1b–19b), which gathered NSAIDs, carbo/heterocycles, and fragments from natural products, were evaluated against hCA I, II, IX, and XII. Indomethacin (17b) and flufenamic acid (19b) analogs exhibited selectivity for tumour-associated isoform IX in the low micromolar range. In summary, selenoesters that combine NSAIDs with fragments derived from natural sources have been developed as promising nonclassical inhibitors of the tumour-associated CA isoforms.

Design, Synthesis and Biological Evaluation of Quinoline-8-Sulfonamides as Inhibitors of the Tumor Cell-Specific M2 Isoform of Pyruvate Kinase: Preliminary Study.

Cancer cells need to carefully regulate their metabolism to keep them growing and dividing under the influence of different nutrients and oxygen levels. Muscle isoform 2 of pyruvate kinase (PKM2) is a key glycolytic enzyme involved in the generation of ATP and is critical for cancer metabolism. PKM2 is expressed in many human tumors and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various modulators regulate PKM2, shifting it between highly active and less active states. In the presented work, a series of 8-quinolinesulfonamide derivatives of PKM2 modulators were designed using molecular docking and molecular dynamics techniques. New compounds were synthesized using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Compound 9a was identified in in silico studies as a potent modulator of muscle isoform 2 of pyruvate kinase. The results obtained from in vitro experiments confirmed the ability of compound 9a to reduce the intracellular pyruvate level in A549 lung cancer cells with simultaneous impact on cancer cell viability and cell-cycle phase distribution. Moreover, compound 9a exhibited more cytotoxicity on cancer cells than normal cells, pointing to high selectivity in the mode of action. These findings indicate that the introduction of another quinolinyl fragment to the modulator molecule may have a significant impact on pyruvate levels in cancer cells and provides further directions for future research to find novel analogs suitable for clinical applications in cancer treatment.

Roles of PI3Kγ and PI3Kδ in mantle cell lymphoma proliferation and migration contributing to efficacy of the PI3Kγ/δ inhibitor duvelisib

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma that is incurable with existing therapies, and therefore presents a significant unmet clinical need. The ability of this disease to overcome therapy, including those that target the B cell receptor pathway which has a pathogenic role in MCL, highlights the need to develop new treatment strategies. Herein, we demonstrate that a distinguishing feature of lymph node resident MCL cells is the expression of phosphatidylinositol 3-kinase γ (PI3Kγ), a PI3K isoform that is not highly expressed in other B cells or B-cell malignancies. By exploring the role of PI3K in MCL using different PI3K isoform inhibitors, we provide evidence that duvelisib, a dual PI3Kδ/γ inhibitor, has a greater effect than PI3Kδ- and PI3Kγ-selective inhibitors in blocking the proliferation of primary MCL cells and MCL cell lines, and in inhibiting tumour growth in a mouse xenograft model. In addition, we demonstrated that PI3Kδ/γ signalling is critical for migration of primary MCL cells and cell lines. Our data indicates that aberrant expression of PI3Kγ is a critical feature of MCL pathogenesis. Thus, we suggest that the dual PI3Kδ/γ duvelisib would be effective for the treatment of mantle cell lymphoma.

Synthesis and biological evaluation of new 3-substituted coumarin derivatives as selective inhibitors of human carbonic anhydrase IX and XII

The Carbonic anhydrase isoforms IX and XII play a significant role in regulating the intracellular and extracellular pH in hypoxic tumours abetting the metastasis of solid tumours. Selective and potent inhibitors targeting carbonic anhydrase IX and XII reduce the activity of these isoforms in hypoxic tumours, representing an antitumor and antimetastatic mechanism. Coumarin-based derivatives are selective inhibitors of CA isoforms IX and XII. In this study, we report the design and synthesis of new 3-substituted coumarin derivatives with different functional moieties and their inhibitory activity against various carbonic anhydrase isoforms. We found that the tertiary sulphonamide derivative 6c showed selective inhibition against CA IX with IC50 of 4.1 µM. Similarly, the carbothioamides 7c, 7b and oxime ether derivative 20a exhibited good inhibition against CA IX and CA XII. Additionally, the binding mode was predicted and validated using molecular docking and dynamic simulations.

Abstract OT3-22-01: First-in-human global multi-center study of RLY-2608, a pan mutant and isoform selective PI3Kα inhibitor, as a single agent in advanced solid tumor patients and in combination with fulvestrant in patients with advanced breast cancer

Abstract Background: Targeting constitutively active mutant kinases with selective small molecule inhibitors is a key therapeutic pillar of precision oncology. Phosphatidylinositol-4,5bisphosphate-3 kinase, catalytic subunit alpha (PIK3CA) mutations leading to oncogenic activation of PI3Kα represent the largest opportunity for this approach in solid tumors. However, there is no selective inhibitor that targets mutant PI3Kα in the clinic. Toxicity related to non-selective inhibition of WT PI3Kα (hyperglycemia) and other PI3K isoforms limits the tolerability, dosing and efficacy of the orthosteric inhibitor, alpelisib, the only approved solid tumor PI3K inhibitor. RLY-2608, a novel oral allosteric PI3Kα inhibitor, is uniquely designed to overcome these limitations via mutant- and isoform-selective PI3Kα inhibition for greater target coverage, improved tolerability and antitumor activity. We initiated a first-in-human (FIH), study to evaluate the clinical activity of RLY-2608 as a single agent in advanced solid tumor patients (pts) with PI3KCA mutations and in combination with fulvestrant in pts with PIK3CA mutant, HR+, HER2- metastatic breast cancer (MBC). Methods: This is a global, multi-center, dose escalation/expansion study (NCT05216432) of RLY2608 as a single agent in adults who have advanced solid tumors and are refractory, intolerant, or declined standard therapy and RLY-2608 in combination with fulvestrant in previously treated pts with HR+/HER2- MBC. Eligibility criteria include presence of PI3KCA mutation (blood or tumor) per local assessment, ECOG performance status 0-1, measurable or evaluable disease per RECIST 1.1 and no prior PI3K inhibitor (except combination group 2). RLY-2608 is administered on a continuous schedule with 4-week cycles. Adverse events (AEs) per CTCAE v5, PK, biomarkers (mutant ctDNAs and insulin pathway markers) and anti-tumor activity are assessed serially. Dose escalation employs a Bayesian Optimal Interval design to identify MTD and RP2D. Following dose escalation, pts will be treated with RLY-2608 at the MTD/RP2D in a monotherapy dose expansion with 5 groups (N=75, 15 each): 1. Clear cell ovarian carcinoma 2. Head and neck squamous cell carcinoma 3. Cervical cancer 4. Other solid tumors 5. PI3KCA double mutations. In addition, two expansion cohorts will enroll patients with HR+/HER2- MBC treated with RLY-2608 and fulvestrant combination (N = 30, 15 each): 1. No prior PI3K therapy 2. Intolerant to PI3K inhibitors. The primary endpoints are MTD/RP2D and AE profile for single agent and combination; key secondary endpoints are PI3KCA genotype in blood and tumor, PK, biomarkers, and overall response rate. US enrollment began December 2021 and ex-USA startup is under way. Citation Format: Andreas Varkaris, Erika Hamilton, Jason Henry, Alexander I. Spira, Alison M. Schram, Julia E. McGuinness, Gege Tan, Xiaoyan Li, Tamieka Hunter, Ramin Samadani, Alison Timm, Djuro Karanovic, Vivek Subbiah, Cesar A. Perez. First-in-human global multi-center study of RLY-2608, a pan mutant and isoform selective PI3Kα inhibitor, as a single agent in advanced solid tumor patients and in combination with fulvestrant in patients with advanced breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-22-01.

Therapeutic effect of gedatolisib, a pan-PI3K/mTOR inhibitor, on prostate cancer models with PI3K or PTEN mutational status.

149 Background: Metastatic castration-resistant prostate cancer (mCRPrC) is characterized by a loss of androgen receptor (AR) sensitivity and oncogenic activation of the PI3K, AKT, and mTOR (PAM) pathway. Loss of the PI3K regulator, PTEN, is frequent during prostate cancer (PrC) initiation, progression, and therapeutic resistance. Co-targeting PAM/AR pathways is a promising PrC treatment strategy but hampered by complex PAM-AR pathway crosstalk driving either reciprocal negative feedback inhibition or feedback relief. Most PAM inhibitors (PAM-i) selectively spare (or weakly inhibit) one or more key PAM pathway components, potentiating drug resistance depending on the PAM mutation status of patients. AKT-i demonstrated limited efficacy in PTEN-deficient PrC and even less efficacy in PTEN-wild type (wt) in recent clinical studies, suggesting that more comprehensive inhibition of the PI3K isoforms and mTOR may be required. We posited that gedatolisib, a well-tolerated and potent inhibitor of all Class I PI3K isoforms, mTORC1, and mTORC2, would be effective in both PTEN-wt and PTEN-deficient PrC. Methods: A panel of PrC cell lines with different PI3K or PTEN status ( wt: 22RV1, MDA-PCa-2b, DU145; null: LNCaP, PC3, C4-2) were assayed under different conditions for their sensitivity to gedatolisib and other PAM-I ( PI3K: alpelisib, copanlisib; AKT: capivasertib, ipatasertib; mTOR: everolimus; pan-PI3K/mTOR: samotolisib, gedatolisib). A combination of cell viability, growth rate, cytotoxicity and phospho-FACS analytical assays were used. Xenograft studies evaluating gedatolisib with a subset of these models were also performed in castrated mice. Results: We confirmed that AKT-i is more effective in PTEN-null than PTEN-wt PrC. In contrast, the two pan-PI3K/mTOR-i evaluated were effective independent of PI3K or PTEN status. Gedatolisib exhibited superior potency and efficacy compared to each of the other PAM-i for cell proliferation, death, or PAM pathway activation. In vivo, gedatolisib substantively inhibited tumor growth regardless of PTEN or PI3K status. Conclusions: We demonstrated that gedatolisib exerts superior activity regardless of PI3K or PTEN status in all PrC cell lines evaluated compared to the other PAM pathway inhibitors evaluated. Our results indicate potent and simultaneous blockade of all Class I PI3K isoforms, mTORC1, and mTORC2 could circumvent PTEN dependent resistance. Gedatolisib as a single agent and in combination with other therapies reported promising preliminary efficacy and safety in various solid tumor types. In light of the PrC findings reported here, development of gedatolisib may be warranted in PrC patients regardless of their PTEN or PI3K status.

Distinct protein kinase C isoforms drive the cell cycle re-entry of two separate populations of neonatal rat ventricular cardiomyocytes.

The present study tested the hypothesis that protein kinase C-α (PKC-α) recruitment in the presence of the p38α/β MAPK inhibitor SB203580 facilitated the appearance and cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes (NNVMs) and induced a transcript profile delineating a proliferative phenotype. Phorbol 12,13-dibutyrate (PDBu) treatment did not induce de novo nestin expression or increase the cell cycle re-entry of 1-day-old NNVMs but significantly increased runt-related transcription factor 1 (Runx1) and p16 cell cycle inhibitor (CDKN2a) mRNA levels and downregulated epithelial cell transforming 2 (ECT2) mRNA expression. SB203580 administration to PDBu-treated NNVMs induced de novo nestin expression, preferentially increased the density (normalized to 500 NNVMs) of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu, 1.4 ± 3 vs. PDBu/SB203580, 128 ± 34; n = 5 independent litters), significantly inhibited CDKN2a and Runx1 mRNA upregulation and reversed ECT2 mRNA downregulation. PDBu treatment of NNVMs reduced PKC-α, protein kinase-δ (PKC-δ) and protein kinase-ε (PKC-ε) protein levels and GF109203X (conventional PKC isoform inhibitor) selectively attenuated PKC-α protein downregulation. GF109203X administration to PDBu/SB203580-treated NNVMs significantly reduced the density of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 40 ± 46; n = 5). Moreover, GF109203X/PDBu/SB203580 treatment unmasked the predominant appearance of a separate NNVM population that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 192 ± 42; n = 5) delineated by the absence of de novo nestin expression. Sotrastaurin (conventional/novel PKC isoform inhibitor) administration to PDBu/SB203580-treated NNVMs significantly attenuated the density of nestin(+)-NNVMs (PDBu/SB203580/sotrastaurin, 8 ± 10; n = 4) and nestin(-)-NNVMs (PDBu/SB203580/sotrastaurin, 64 ± 30; n = 4) that incorporated 5-bromo-2'-deoxyuridine. These data reveal that the neonatal rat heart contains at least two separate populations of NNVMs that re-enter the cell cycle and the preferential appearance of nestin(+)- or nestin(-)-NNVMs is driven by distinct PKC isoforms in the presence of SB203580.NEW & NOTEWORTHY The appearance of nestin(+)-neonatal rat ventricular cardiomyocytes that re-entered the cell cycle following phorbol ester stimulation in the presence of p38α/β MAPK inhibitor SB203580 was associated with the inhibition of Runx1 and CDKN2a mRNA upregulation. PKC-α selectively induced the cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes. Pharmacological inhibition of PKC-α with concomitant p38α/β MAPK suppression unmasked the cell cycle re-entry of a second population of neonatal rat ventricular cardiomyocytes in the absence of nestin expression.

Adagrasib: First Approval.

Adagrasib (KRAZATI™) is an orally available, potent, irreversible, small molecule inhibitor of KRAS G12C mutant isoform being developed by Mirati Therapeutics for the treatment of solid tumours harbouring KRAS G12C oncogenic driver mutation, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). Adagrasib covalently binds to the mutant cysteine in KRAS G12C and locks the mutant KRAS protein in its inactive state, thereby preventing downstream signalling without affecting wild-type KRAS protein. In December 2022, adagrasib received its first approval in the USA for the treatment of adults with KRAS G12C-mutated locally advanced or metastatic NSCLC (as determined by an FDA approved test) who have received ≥1 prior systemic therapy. It was approved under accelerated approval based on objective response rate and duration of response, and its continued approval for this indication may be contingent upon verification and description of a clinical benefit in a confirmatory trial(s). The drug is under regulatory review for NSCLC in the European Union and is in development for CRC in the US. Clinical studies of adagrasib in solid tumours, including CRC, are underway in several countries. This article summarizes the milestones in the development of adagrasib leading to this first approval for KRAS G12C-mutated locally advanced or metastatic NSCLC.

Pyrrolyl and Indolyl α-γ-Diketo Acid Derivatives Acting as Selective Inhibitors of Human Carbonic Anhydrases IX and XII.

Solid tumors are active tissues containing hypoxic regions and producing metabolic acids. By decreasing pH, cancer cells create a hostile environment for surrounding host cells and foster tumor growth and progression. By governing acid/base regulation, carbonic anhydrases (CAs) are involved in several physiological/pathological processes, including tumors. Indeed, CAs are clinically relevant in cancer therapy as among the fifteen human isoforms, two of them, namely CA IX (overexpressed in solid tumors and associated with increased metastasis and poor prognosis) and CA XII (overexpressed in some tumors) are involved in tumorigenesis. Targeting these two isoforms is considered as a pertinent approach to develop new cancer therapeutics. Several CA inhibitors (CAIs) have been described, even though they are unselective inhibitors of different isoforms. Thus, efforts are needed to find new selective CAIs. In this work, we described new diketo acid derivatives as CAIs, with the best acting compounds 1c and 5 as nanomolar inhibitors of CA IX and XII, being also two orders of magnitude selective over CAs I and II. Molecular modeling studies showed the different binding poses of the best acting CAIs within CA II and IX, highlighting the key structural features that could confer the ability to establish specific interactions within the enzymes. In different tumor cell lines overexpressing CA IX and XII, the tested compounds showed antiproliferative activity already at 24 h treatment, with no effects on somatic not transformed cells.

Carborane Analogues of Fenoprofen Exhibit Improved Antitumor Activity.

Fenoprofen is a widely used nonsteroidal anti-inflammatory drug (NSAID) against rheumatoid arthritis, degenerative joint disease, ankylosing spondylitis and gout. Like other NSAIDs, fenoprofen inhibits the synthesis of prostaglandins by blocking both cyclooxygenase (COX) isoforms, COX-1 the "house-keeping" enzyme and COX-2 the induced isoform from pathological stimuli. Unselective inhibition of both COX isoforms results in many side effects, but off-target effects have also been reported. The steric modifications of the drugs could afford the desired COX-2 selectivity. Furthermore, NSAIDs have shown promising cytotoxic properties. The structural modification of fenoprofen using bulky dicarba-closo-dodecaborane(12) (carborane) clusters and the biological evaluation of the carborane analogues for COX inhibition and antitumor potential showed that the carborane analogues exhibit stronger antitumor potential compared to their respective aryl-based compounds.

Mechanism by Which PF-3758309, a Pan Isoform Inhibitor of p21-Activated Kinases, Blocks Reactivation of HIV-1 Latency.

The "block and lock" strategy is one approach that might elicit a sterilizing cure for HIV-1 infection. The "block" refers to a compound's ability to inhibit latent HIV-1 proviral transcription, while the "lock" refers to its capacity to induce permanent proviral silencing. We previously identified PF-3758309, a pan-isoform inhibitor of p21-activated kinases (PAKs), as a potent inhibitor of HIV-1 latency reversal. The goal of this study was to define the mechanism(s) involved. We found that both 24ST1NLESG cells (a cell line model of HIV-1 latency) and purified CD4+ naïve and central memory T cells express high levels of PAK2 and lower levels of PAK1 and PAK4. Knockdown of PAK1 or PAK2, but not PAK4, in 24ST1NLESG cells resulted in a modest, but statistically significant, decrease in the magnitude of HIV-1 latency reversal. Overexpression of PAK1 significantly increased the magnitude of latency reversal. A phospho-protein array analysis revealed that PF-3758309 down-regulates the NF-κB signaling pathway, which provides the most likely mechanism by which PF-3758309 inhibits latency reversal. Finally, we used cellular thermal shift assays combined with liquid chromatography and mass spectrometry to ascertain whether PF-3758309 off-target binding contributed to its activity. In 24ST1NLESG cells and in peripheral blood mononuclear cells, PF-3758309 bound to mitogen-activated protein kinase 1 and protein kinase A; however, knockdown of either of these kinases did not impact HIV-1 latency reversal. Collectively, our study suggests that PAK1 and PAK2 play a key role in the maintenance of HIV-1 latency.

Selective Inhibition of PI3K Isoforms in Brain Tumors Suppresses Tumor Growth by Increasing Radiosensitivity.

Glioblastoma (GBM) is a malignant brain tumor with poor prognosis. Radioresistance is a major challenge in the treatment of brain tumors. The development of several types of tumors, including GBM, involves the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Upon activation, this pathway induces radioresistance. In this study, we investigated whether additional use of selective inhibitors of PI3K isoforms would enhance radiosensitivity in GBM. We evaluated whether radiation combined with PI3K isoform selective inhibitors can suppress radioresistance in GBM. Glioma 261 expressing luciferase (GL261-luc) and LN229 were used to confirm the effect of combination of radiation and PI3K isoform inhibitors in vitro. Cell viability was confirmed by clonogenic assay, and inhibition of PI3K/AKT signaling activation was observed by Western blot. To confirm radiosensitivity, the expression of phospho-γ-H2AX was observed by immunofluorescence. In addition, to identify the effect of a combination of radiation and PI3K-α isoform inhibitor in vivo, an intracranial mouse model was established by implanting GL261-luc. Tumor growth was observed by IVIS imaging, and survival was analyzed using Kaplan-Meier survival curves. Suppression of the PI3K/AKT signaling pathway increased radiosensitivity, and PI3K-α inhibition had similar effects on PI3K-pan inhibition in vitro. The combination of radiotherapy and PI3K-α isoform inhibitor suppressed tumor growth and extended survival in vivo. This study verified that PI3K-α isoform inhibition improves radiosensitivity, resulting in tumor growth suppression and extended survival in GBM mice.