In the preceding work in this issue (10.1021/acsmedchemlett.6c00103), we described our initial structure-activity relationship (SAR) optimization that led to a pan-Cbl inhibitor (6) that demonstrated efficacy in a mouse CT26 syngeneic model. Unfortunately, attempts to improve TGI with higher doses of 6 resulted in poor tolerability which we attributed to a lack of selectivity between Cbl-b and c-Cbl (∼2× by surface plasmon resonance (SPR)). Herein, we report our continued efforts that led to a breakthrough in achieving Cbl-b selectivity (up to 37×). The lead compound 33 demonstrated 14× selectivity against c-Cbl by SPR, was potent in a PBMC cell assay, and showed good oral exposure in mice. When tested in a CT26 model, 33 displayed improved tumor growth inhibition compared to our previously reported pan-Cbl inhibitor 6 (TGI 145% vs 82%). More importantly, 33 was better tolerated than 6, supporting our hypothesis that a selective Cbl-b inhibitor could be advantageous relative to a pan-Cbl inhibitor.

Casitas B-lineage lymphoma-b (Cbl-b), an E3 ubiquitin ligase, is a key negative regulator of immune function, and its inhibition is a promising strategy for cancer immunotherapy. Here, we show the optimization of a series of inactive-state Cbl-b inhibitors to improve their potency and pharmacokinetic properties. Through systematic modification of a benzylic amine and a linker region, compound 16 was identified, which demonstrates a favorable balance of biochemical potency, cellular activity, and in vitro ADME properties. Despite exhibiting high IV clearance in vivo, compound 16 achieved oral exposures sufficient to demonstrate significant tumor growth inhibition in a murine CT26 colon-cancer model.

The oral route of administration continues to be the preferred and most convenient method for drug delivery among both patients and healthcare professionals. This preference is based not only on its user-friendliness but also on the practicality it provides regarding formulation flexibility and dosage adjustability. Nevertheless, the effectiveness of oral drug delivery is significantly influenced by the ADME (Absorption, Distribution, Metabolism, and Excretion) characteristics of the drug. Among these characteristics, permeability is a key factor affecting oral bioavailability, making the exploration of permeability enhancers a vital research focus to boost oral absorption. Recent investigations have highlighted sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC) as a promising permeability enhancer, demonstrating its potential to increase the absorption rates of various substances. Despite its proven effectiveness, the precise mechanisms by which SNAC promotes this enhancement are not yet fully understood. Furthermore, the specific dosage of SNAC required to improve drug absorption remains unclear. This study aims to examine the dose-dependent effects of SNAC on the model compound cyanocobalamin (Vitamin B12) in rats. The in vivo findings were integrated with modeling and simulation to determine the critical concentration of SNAC and the minimum molar ratio, and the overall enhancement effect at various SNAC concentrations for cyanocobalamin needed to achieve permeation enhancement. This knowledge could assist formulators in more effectively incorporating SNAC into formulations for enhanced therapeutic outcomes.

Prostate cancer is the second most commonly diagnosed cancer in men, with 1 in 8 men being diagnosed with prostate cancer in his lifetime. AR is a hormone-activated transcription factor that promotes cell growth and survival in the normal prostate, and AR signaling is a key driver of cell proliferation in prostate cancer. Inhibition of AR signaling is a mainstay of current prostate cancer treatment, however, patients often develop resistance to these therapies through mechanisms that retain dependency on AR signaling. GDC-2992 (also known as RO7656594) is a potent, orally bioavailable, heterobifunctional molecule that inhibits AR signaling by binding to both AR and the E3 ubiquitin ligase cereblon (CRBN), resulting in ubiquitination and subsequent degradation of AR. GDC-2992 inhibits AR signaling in the context of wild-type AR and AR proteins with mutations associated with resistance to standard-of-care AR signaling inhibitors (ARSIs). Unlike ARSIs, GDC-2992 does not display evidence of agonism against any AR variants evaluated. Co-treatment of GDC-2992 with the CRBN ligand pomalidomide prevents AR degradation mediated by GDC-2992 in vitro, supporting the role of CRBN in GDC-2992-mediated AR degradation. Importantly however, anti-proliferative potential of GDC-2992 is maintained even when degradation is attenuated, suggesting that the mechanism of GDC-2992 includes competitive AR antagonism in addition to degradation. In vivo, GDC-2992 decreases circulating PSA and inhibits prostate tumor growth in a dose responsive manner. The totality of in vitro and in vivo preclinical data supports that GDC-2992 represents a compelling advance over standard-of-care ARSIs. An ongoing Phase I dose-escalation and expansion study will assess the safety, tolerability, pharmacokinetics, and preliminary anti-tumor activity of GDC-2992 in patients with advanced or metastatic prostate cancer who have previously received AR-targeted therapy [NCT05800665]. By providing more complete and sustained AR inhibition, GDC-2992 has the potential to reduce the occurrence of treatment-resistance and disease relapse in prostate cancer. Ciara Metcalfe, Wei Zhou, Darlene Dela Cruz, Thomas Hunsaker, Pablo Saenz-Lopez Larrocha, Elizabeth Levy, Bu-Er Wang, Tonia Hafner, Nayan Chaudhary, Marc Hafner, Kalpit Shah, Elisia Villemure, Yuxiang Zheng, Jodie Pang, Udi Segal. GDC-2992: A heterobifunctional Androgen Receptor (AR) antagonist and degrader for the treatment of AR wild-type and mutant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr ND02.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults. Available treatments have not markedly improved patient survival in the last twenty years. However, genomic investigations have showed that the PI3K pathway is frequently altered in this glioma, making it a potential therapeutic target.Paxalisib is a brain penetrant PI3K/mTOR inhibitor (mouse Kp,uu 0.31) specifically developed for the treatment of GBM. We characterised the preclinical pharmacokinetics and efficacy of paxalisib and predicted its pharmacokinetics and efficacious dose in humans.Plasma protein binding of paxalisib was low, with the fraction unbound ranging from 0.25 to 0.43 across species. The hepatic clearance of paxalisib was predicted to be low in mice, rats, dogs and humans, and high in monkeys, from hepatocytes incubations. The plasma clearance was low in mice, moderate in rats and high in dogs and monkeys. Oral bioavailability ranged from 6% in monkeys to 76% in rats.The parameters estimated from the pharmacokinetic/pharmacodynamic modelling of the efficacy in the subcutaneous U87 xenograft model combined with the human pharmacokinetics profile predicted by PBPK modelling suggested that a dose of 56 mg may be efficacious in humans. Paxalisib is currently tested in Phase III clinical trials.

Early stage chemical development presents numerous challenges, and achieving a functional balance is a major hurdle, with many early compounds not meeting the clinical requirements for advancement benchmarks due to issues like poor oral bioavailability. There is a need to develop strategies for achieving the desired systemic concentration for these compounds. This will enable further evaluation of the biological response upon a compound–target interaction, providing deeper insight into the postulated biological pathways. Our study elucidates alternative drug delivery paradigms by comparing formulation strategies across oral (PO), intraperitoneal (IP), subcutaneous (SC), and intravenous (IV) routes. While each modality boasts its own set of merits and constraints, it is the drug’s formulation that crucially influences its pharmacokinetic (PK) trajectory and the maintenance of its therapeutic levels. Our examination of model compounds G7883 and G6893 highlighted their distinct physio-chemical attributes. By harnessing varied formulation methods, we sought to fine-tune their PK profiles. PK studies showcased G7883′s extended half-life using an SC oil formulation, resulting in a 4.5-fold and 2.5-fold enhancement compared with the IP and PO routes, respectively. In contrast, with G6893, we achieved a prolonged systemic coverage time above the desired target concentration through a different approach using an IV infusion pump. These outcomes underscore the need for tailored formulation strategies, which are dictated by the compound’s innate properties, to reach the optimal in vivo systemic concentrations. Prioritizing formulation and delivery optimization early on is pivotal for effective systemic uptake, thereby facilitating a deeper understanding of biological pathways and expediting the overall clinical drug development timeline.

Hematopoietic progenitor kinase 1 (HPK1) serves a key immunosuppressive role as a negative regulator of T-cell receptor (TCR) signaling. HPK1 loss-of-function is associated with augmentation of immune function and has demonstrated synergy with immune checkpoint inhibitors in syngeneic mouse cancer models. These data offer compelling evidence for the use of selective small molecule inhibitors of HPK1 in cancer immunotherapy. We identified a novel series of isoquinoline HPK1 inhibitors through fragment-based screening that displayed promising levels of biochemical potency and activity in functional cell-based assays. We used structure-based drug design to introduce key selectivity elements while simultaneously addressing pharmacokinetic liabilities. These efforts culminated in a molecule demonstrating subnanomolar biochemical inhibition of HPK1 and strong in vitro augmentation of TCR signaling in primary human T-cells. Further profiling of this molecule revealed excellent kinase selectivity (347/356 kinases <50% inhibition @ 0.1 μM), a favorable in vitro safety profile, and good projected human pharmacokinetics.

The inhibition of emopamil binding protein (EBP), a sterol isomerase within the cholesterol biosynthesis pathway, promotes oligodendrocyte formation, which has been proposed as a potential therapeutic approach for treating multiple sclerosis. Herein, we describe the discovery and optimization of brain-penetrant, orally bioavailable inhibitors of EBP. A structure-based drug design approach from literature compound 1 led to the discovery of a hydantoin-based scaffold, which provided balanced physicochemical properties and potency and an improved in vitro safety profile. The long half-lives of early hydantoin-based EBP inhibitors in rodents prompted an unconventional optimization strategy, focused on increasing metabolic turnover while maintaining potency and a brain-penetrant profile. The resulting EBP inhibitor 11 demonstrated strong in vivo target engagement in the brain, as illustrated by the accumulation of EBP substrate zymostenol after repeated dosing. Furthermore, compound 11 enhanced the formation of oligodendrocytes in human cortical organoids, providing additional support for our therapeutic hypothesis.

1. Small molecule inhibitors of the PI3K pathway have been extensively investigated as potential anticancer agents. Among the effectors in this pathway, PI3K alpha is the kinase most frequently associated with the development of tumours, through mutations and amplifications of the PIK3CA gene encoding the p110 alpha catalytic subunit.2. Inavolisib (GDC-0077) is a potent and PI3K alpha-selective inhibitor that also specifically triggers the degradation of the mutant p110 alpha protein.3. We characterised inavolisib ADME properties in preclinical in vitro and in vivo studies, assessed its efficacy in the PIK3CA mutant KPL-4 breast cancer xenograft model, and predicted its pharmacokinetics and efficacious dose in humans.4. Inavolisib had a moderate permeability (1.9 center dot 10-6 cm/s) in MDCK cells and was a P-gp and Bcrp1 substrate. It appeared metabolically stable in hepatocytes incubations from human and preclinical species. The systemic clearance was low in mouse, monkey and dog and high in rat. Oral bioavailability ranged from 57.5% to 100%. Inavolisib was efficacious in the KPL-4 sub-cutaneous xenograft model.5. The PK/PD model parameters estimated from the efficacy study, combined with PBPK model-predicted human PK profiles, projected that a dose of 3 mg could lead to clinical response. Inavolisib is currently being tested in phase 3 trials.

PDF file - 47K, Anti-proliferative EC50's for GNE-317 against a Panel of Nine Glioma Cell lines

The subcutaneous administration of therapeutic peptides would provide significant benefits to patients. However, subcutaneous injections are limited in dosing volume, potentially resulting in high peptide concentrations that can incur significant challenges with solubility limitations, high viscosity, and stability liabilities. Herein, we report on the discovery that low-shear resonant acoustic mixing can be used as a general method to prepare stable nanoparticles of a number of peptides of diverse molecular weights and structures in water without the need for extensive amounts of organic solvents or lipid excipients. This approach avoids the stability issues observed with typical high-shear, high-intensity milling methods. The resultant peptide nanosuspensions exhibit low viscosity even at high concentrations of >100 mg/mL while remaining chemically and physically stable. An example nanosuspension of cyclosporine nanoparticles was dosed in rats via a subcutaneous injection and exhibited sustained release behavior. This suggests that peptide nanosuspension formulations can be one approach to overcome the challenges with high-concentration peptide formulations.

PDF file - 58K, Bioluminescence Analysis of U87 Intracranial Tumors in Mice at the Start of Treatment (Day 7 post-surgery) and at the End of Study (Day 28)

PDF file - 60K, Plasma Concentrations, Brain Concentrations and Brain-to-Plasma Ratio Measured at the End of Study (42 days) 2 hours Following the Last Daily PO Administration of GNE-317 (40 mg/kg), GDC-0941 (250 mg/kg) or GDC-0980 (10 mg/kg) to GS2 Tumor-Bearing Mice

PDF file - 57K, Selectivity of GNE-317 against a Panel of 59 Kinases. GNE-317 was Tested at 1 M in the Invitrogen SelectScreen

Taselisib (also known as GDC-0032) is a potent and selective phosphoinositide 3-kinase (PI3K) inhibitor that displays greater selectivity for mutant PI3Kα than wild-type PI3Kα To better understand the absorption, distribution, metabolism, and excretion properties of taselisib, mass balance studies were conducted following single oral doses of [14C]taselisib in rats, dogs, and humans. Absolute bioavailability (ABA) of taselisib in humans was determined by oral administration of taselisib at the therapeutic dose followed by intravenous dosing of [14C]taselisib as a microtracer. The ABA in humans was 57.4%. Absorption of taselisib was rapid in rats and dogs and moderately slow in humans. The recovery of radioactivity in excreta was high (>96%) in the three species where feces was the major route of excretion. Taselisib was the major circulating component in the three species with no metabolite accounting for >10% of the total drug-derived material. The fraction absorbed of taselisib was 35.9% in rats and 71.4% in dogs. In rats, absorbed drug underwent moderate to extensive metabolism and biliary excretion of taselisib was minor. In dog, biliary excretion and metabolism were major clearance pathways. In humans, 84.2% of the dose was recovered as the parent drug in excreta indicating that metabolism played a minor role in the drug's clearance. Major metabolism pathways were oxidation and amide hydrolysis in the three species while methylation was another prominent metabolism pathway in dogs. The site of methylation was identified on the triazole moiety. In vitro experiments characterized that the N-methylation was dog-specific and likely mediated by a thiol methyltransferase. SIGNIFICANCE STATEMENT: This study provides a comprehensive description of the absorption, distribution, and metabolism and pharmacokinetic properties of taselisib in preclinical species and humans. This study demonstrated the importance of oral bioavailability results for understanding taselisib's clearance pathways. The study also describes the identification and characterization of a unique dog-specific N-methylation metabolite of taselisib and the enzyme mediating N-methylation in vitro.

PDF file - 116K, Plot of CNS-MPO score vs. efflux ratio

PDF file - 77K, Cell viability following treatment with GNE-317

The dramatic increase in the prevalence of multi-drug resistant Gram-negative bacterial infections and the simultaneous lack of new classes of antibiotics is projected to result in approximately 10 million deaths per year by 2050. We report on efforts to target the Gram-negative ATP-binding cassette (ABC) transporter MsbA, an essential inner membrane protein that transports lipopolysaccharide from the inner leaflet to the periplasmic face of the inner membrane. We demonstrate the improvement of a high throughput screening hit into compounds with on-target single digit micromolar (μM) minimum inhibitory concentrations against wild-type uropathogenic Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. A 2.98 Å resolution X-ray crystal structure of MsbA complexed with an inhibitor revealed a novel mechanism for inhibition of an ABC transporter. The identification of a fully encapsulated membrane binding site in Gram-negative bacteria led to unique physicochemical property requirements for wild-type activity.