Adaptor protein 2-associated kinase 1 (AAK1) is a member of the Ark1/Prk1 family of serine/threonine kinases and plays a role in modulating receptor endocytosis. AAK1 was identified as a potential therapeutic target for the treatment of neuropathic pain when it was shown that AAK1 knock out (KO) mice had a normal response to the acute pain phase of the mouse formalin model, but a reduced response to the persistent pain phase. Herein we report our early work investigating a series of pyrrolo[2,1-f][1,2,4]triazines as part of our efforts to recapitulate this KO phenotype with a potent, small molecule inhibitor of AAK1. The synthesis, structure–activity relationships (SAR), and in vivo evaluation of these AAK1 inhibitors is described.

Tau is a microtubule-associated protein (MAPT, tau) implicated in the pathogenesis of tauopathies, a spectrum of neuro-degenerative disorders characterized by accumulation of hyper-phosphorylated, aggregated tau. Because tau pathology can be distinct across diseases, a pragmatic therapeutic approach may be to intervene at the level of the tau transcript, as it makes no assumptions to mechanisms of tau toxicity. Here we per-formed a large library screen of locked-nucleic-acid (LNA)-modified antisense oligonucleotides (ASOs), where careful tiling of the MAPT locus resulted in the identification of hot spots for activity in the 30 UTR. Further modifications to the LNA design resulted in the generation of ASO-001933, which selectively and potently reduces tau in primary cultures from hTau mice, monkey, and human neurons. ASO-001933 was well tolerated and produced a robust, long-lasting reduction in tau protein in both mouse and cynomolgus monkey brain. In monkey, tau protein reduction was maintained in brain for 20 weeks post injection and corresponded with tau protein reduction in the cerebrospinal fluid (CSF). Our results demonstrate that LNA-ASOs exhibit excellent drug-like properties and sustained efficacy likely translating to infrequent, intrathecal dosing in patients. These data further support the development of LNA-ASOs against tau for the treatment of tauopathies.

M cells interspersed in the follicle-associated epithelium of Peyer's patches represent the major antigen sampling cells of the intestinal mucosa providing immune surveillance for particulate antigens. Despite their crucial role in immune defense our knowledge about these elusive cells is still only rudimentary. A Caco-2 co-culture model for the induction of M cell-like cells and DNA microarray analysis for differential gene expression profiling were employed to identify (a) putative suitable surface marker(s). Induction of M cell-like cells was demonstrated morphologically by electron microscopy, evaluated by infection with Yersinia enterocolitica and enteropathogenic Escherichia coli strain E2348/69 and further monitored by changes in binding of the lectin UEA-1. The differentiation of Caco-2 cells was found to be reversible, dependent on (a) lymphocyte-derived soluble factor(s) and accompanied by the up-regulation of the glycoprotein lectin galectin-9, which was specifically expressed on these cells as well as on human follicle-associated epithelial (FAE) cells. Galectin-9 represents a novel surface marker which might be employed for molecular targeting to the Peyer's patches thereby opening new opportunities for drug and vaccine development.

Background Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) has been demonstrated as a negative intracellular immune checkpoint in mediating antitumor immunity in studies with HPK1 knockout and kinase dead mice. Pharmacological inhibition of HPK1 is desirable to investigate the role of HPK1 in human immune cells with therapeutic implications. However, a significant challenge remains to identify a small molecule inhibitor of HPK1 with sufficient potency, selectivity, and other drug-like properties suitable for proof-of-concept studies. In this report, we identified a novel, potent, and selective HPK1 small molecule kinase inhibitor, compound K (CompK). A series of studies were conducted to investigate the mechanism of action of CompK, aiming to understand its potential application in cancer immunotherapy. Methods Human primary T cells and dendritic cells (DCs) were investigated with CompK treatment under conditions relevant to tumor microenvironment (TME). Syngeneic tumor models were used to assess the in vivo pharmacology of CompK followed by human tumor interrogation ex vivo. Results CompK treatment demonstrated markedly enhanced human T-cell immune responses under immunosuppressive conditions relevant to the TME and an increased avidity of the T-cell receptor (TCR) to recognize viral and tumor-associated antigens (TAAs) in significant synergy with anti-PD1. Animal model studies, including 1956 sarcoma and MC38 syngeneic models, revealed improved immune responses and superb antitumor efficacy in combination of CompK with anti-PD-1. An elevated immune response induced by CompK was observed with fresh tumor samples from multiple patients with colorectal carcinoma, suggesting a mechanistic translation from mouse model to human disease. Conclusion CompK treatment significantly improved human T-cell functions, with enhanced TCR avidity to recognize TAAs and tumor cytolytic activity by CD8+ T cells. Additional benefits include DC maturation and priming facilitation in tumor draining lymph node. CompK represents a novel pharmacological agent to address cancer treatment resistance.

We describe our efforts to introduce structural diversity to a previously described triazole-containing N1-carboline series of bromodomain and extra-terminal (BET) inhibitors. N9 carbolines were designed to retain favorable binding interactions that the N1-carbolines possess. A convergent synthetic route enabled modifications to reduce clearance, enhance physicochemical properties, and improve the overall in vitro profile. This work led to the identification of a potent BET inhibitor, (S)-2-{8-fluoro-5-[(3-fluoropyridin-2-yl)(oxan-4-yl)methyl]-7-[4-(2H3)methyl-1-methyl-1H-1,2,3-triazol-5-yl]-5H-pyrido[3,2-b]indol-3-yl}propan-2-ol (10), a compound with enhanced oral exposure in mice. Subsequent evaluation in a mouse triple-negative breast cancer tumor model revealed efficacy at 4 mg/kg of N9-carboline 10.

We describe our efforts to identify structurally diverse leads in the triazole-containing N1-carboline series of bromodomain and extra-terminal inhibitors. Replacement of the N5 "cap" phenyl moiety with various heteroaryls, coupled with additional modifications to the carboline core, provided analogs with similar potency, improved pharmacokinetic properties, and increased solubility compared to our backup lead, BMS-986225 (2). Rapid SAR exploration was enabled by a convergent, synthetic route. These efforts provided a potent BET inhibitor, 3-fluoropyridyl 12, that demonstrated robust efficacy in a multiple myeloma mouse tumor model at 1 mg/kg.

While the discovery of immune checkpoint inhibitors has led to robust, durable responses in a range of cancers, many patients do not respond to currently available therapeutics. Therefore, an urgent need exists to identify alternative mechanisms to augment the immune-mediated clearance of tumors. Hematopoetic progenitor kinase 1 (HPK1) is a serine-threonine kinase that acts as a negative regulator of T-cell receptor (TCR) signaling, to dampen the immune response. Herein we describe the structure-based discovery of isofuranones as inhibitors of HPK1. Optimization of the chemotype led to improvements in potency, selectivity, plasma protein binding, and metabolic stability, culminating in the identification of compound 24. Oral administration of 24, in combination with an anti-PD1 antibody, demonstrated robust enhancement of anti-PD1 efficacy in a syngeneic tumor model of colorectal cancer.

A triazine hit identified from a screen of the BMS compound collection was optimized for potency, in vivo activity, and off-target profile to produce the bicyclic pyrimidine γ-secretase modulator BMS-932481. The compound showed robust reductions of Aβ1-42 and Aβ1-40 in the plasma, brain, and cerebrospinal fluid of mice and rats. Consistent with the γ-secretase modulator mechanism, increases in Aβ1-37 and Aβ1-38 were observed, with no change in the total amount of Aβ1-x produced. No Notch-based toxicity was observed, and the overall preclinical profile of BMS-932481 supported its further evaluation in human clinical trials.

This commentary reflects the collective view of pharmaceutical scientists from four different organizations with extensive experience in the field of drug discovery support. Herein, engaging discussion is presented on the current and future approaches for the selection of the most optimal and developable drug candidates. Over the past two decades, developability assessment programs have been implemented with the intention of improving physicochemical and metabolic properties. However, the complexity of both new drug targets and non-traditional drug candidates provides continuing challenges for developing formulations for optimal drug delivery. The need for more enabled technologies to deliver drug candidates has necessitated an even more active role for pharmaceutical scientists to influence many key molecular parameters during compound optimization and selection. This enhanced role begins at the early in vitro screening stages, where key learnings regarding the interplay of molecular structure and pharmaceutical property relationships can be derived. Performance of the drug candidates in formulations intended to support key in vivo studies provides important information on chemotype-formulation compatibility relationships. Structure modifications to support the selection of the solid form are also important to consider, and predictive in silico models are being rapidly developed in this area. Ultimately, the role of pharmaceutical scientists in drug discovery now extends beyond rapid solubility screening, early form assessment, and data delivery. This multidisciplinary role has evolved to include the practice of proactively taking part in the molecular design to better align solid form and formulation requirements to enhance developability potential.

The nature of the pharmaceutical industry is constantly evolving. There is currently a strong demand for new therapeutics to be simultaneously safer, more effective, and less expensive. These seemingly incompatible expectations will likely increase over time, and balancing them will require continuous innovation, such as novel technologies to expand the so-called "druggable" chemical space and new insights to reduce the number and severity of costly clinical failures. As a result, the industry has begun pivoting away from the traditional therapeutic product profile: once-a-day, orally administered small molecule drugs intended for large patient populations. Instead, there is a renewed focus toward more niche or specialty areas, using alternative molecular therapeutic modalities administered with novel delivery technologies, often via non-oral routes to smaller and more specific patient populations.The shift from traditional to specialty drug candidates has also resulted in larger organizations focusing less on establishing new internal drug discovery expertise and more on leveraging the existing clinical expertise toward new therapies discovered by external partners. Such partners include specialized smaller pharmaceutical organizations or academic groups with novel molecular assets, targeting capabilities or even whole drug discovery platforms. The size and lack of experience of these small entities often necessitate that their technologies be developed with the assistance of larger, more established pharmaceutical organizations. The successful execution of this approach requires a collaborative mind-set to collectively overcome the interconnected drug discovery and development challenges. This includes assessing the progressability and developability of promising drug candidates as well as ensuring pertinent clinical information is translated upstream into continuing discovery efforts.Despite implementing significant changes, a number of challenges continue to impede the industry's productivity improvement efforts. These include significant knowledge gaps, effective decision-making in an uncertain environment, and competing stakeholder interests. This chapter explores how these issues were, are, and may be addressed in the past, present, and future.

Thoughtfully designed early clinical formulations not only meet the needs of the study at hand and inform the development of the commercial product, but can influence the direction of the clinical program as well as provide further guidance to potential backups still in exploratory stages. This chapter focuses on the various types of early clinical formulations, why they are developed, and how the preclinical formulation space helps to guide initial clinical formulation selection. Impacts on clinical program development will be presented through case studies and examples in context of the types of clinical studies being supported: what is the goal of the clinical study, what questions need to be answered, and how will the information be used in subsequent development? Additional factors influencing formulation selection such as patient needs, the practicality of switching between dosage forms, and commercialization potential will be reviewed.

Variability in oral absorption in pre-clinical species makes human dose projection challenging. In this study, we investigated the mechanistic basis of variability in oral absorption of a model hydrophobic compound with pH-dependent solubility, BMS-955829, after oral dosing in rats, dogs, and cynomolgus monkeys. The contribution of regional absorption to pharmacokinetic variability was assessed in ported monkeys by direct intraduodenal and intraileal administration. The effect of BMS-955829 on gastric emptying and intestinal motility was investigated by radiography after co-administration of barium. BMS-955829 exhibited species dependent oral bioavailability, with high variability in monkeys. During regional absorption studies, highest rate of drug absorption was observed after direct intraduodenal administration. Radiography studies indicated that BMS-955829 slowed gastric emptying and intestinal motility. The effect of rate and site of drug release on oral exposure was studied using different drug product formulations. Reducing the rate of drug release reduced oral exposure variability without compromising exposure in cynomolgus monkeys. This effect was likely mediated by avoidance of rapid initial absorption and drug effect on gastric emptying and intestinal transit within the biorelevant timeframe. Thus, drug release rate can modulate the effect of physiological factors on variability in the oral absorption of sensitive compounds.

The amyloid-β peptide (Aβ)-in particular, the 42-amino acid form, Aβ1-42-is thought to play a key role in the pathogenesis of Alzheimer's disease (AD). Thus, several therapeutic modalities aiming to inhibit Aβ synthesis or increase the clearance of Aβ have entered clinical trials, including γ-secretase inhibitors, anti-Aβ antibodies, and amyloid-β precursor protein cleaving enzyme inhibitors. A unique class of small molecules, γ-secretase modulators (GSMs), selectively reduce Aβ1-42 production, and may also decrease Aβ1-40 while simultaneously increasing one or more shorter Aβ peptides, such as Aβ1-38 and Aβ1-37. GSMs are particularly attractive because they do not alter the total amount of Aβ peptides produced by γ-secretase activity; they spare the processing of other γ-secretase substrates, such as Notch; and they do not cause accumulation of the potentially toxic processing intermediate, β-C-terminal fragment. This report describes the translation of pharmacological activity across species for two novel GSMs, (S)-7-(4-fluorophenyl)-N2-(3-methoxy-4-(3-methyl-1H-1,2,4-triazol-1-yl)phenyl)-N4-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine (BMS-932481) and (S,Z)-17-(4-chloro-2-fluorophenyl)-34-(3-methyl-1H-1,2,4-triazol-1-yl)-16,17-dihydro-15H-4-oxa-2,9-diaza-1(2,4)-cyclopenta[d]pyrimidina-3(1,3)-benzenacyclononaphan-6-ene (BMS-986133). These GSMs are highly potent in vitro, exhibit dose- and time-dependent activity in vivo, and have consistent levels of pharmacological effect across rats, dogs, monkeys, and human subjects. In rats, the two GSMs exhibit similar pharmacokinetics/pharmacodynamics between the brain and cerebrospinal fluid. In all species, GSM treatment decreased Aβ1-42 and Aβ1-40 levels while increasing Aβ1-38 and Aβ1-37 by a corresponding amount. Thus, the GSM mechanism and central activity translate across preclinical species and humans, thereby validating this therapeutic modality for potential utility in AD.

Alzheimer’s disease is the most prevalent cause of dementia and is associated with accumulation of amyloid-βpeptide (Aβ), particularly the 42-amino acid Aβ1-42, in the brain. Aβ1-42 levels can be decreased byγ-secretase modulators (GSM), which are small molecules that modulateγ-secretase, an enzyme essential for Aβproduction. BMS-869780 is a potent GSM that decreased Aβ1-42 and Aβ1-40 and increased Aβ1-37 and Aβ1-38, without inhibiting overall levels of Aβpeptides or other APP processing intermediates. BMS-869780 also did not inhibit Notch processing byγ-secretase and lowered brain Aβ1-42 without evidence of Notch-related side effects in rats. Human pharmacokinetic (PK) parameters were predicted through allometric scaling of PK in rat, dog, and monkey and were combined with the rat pharmacodynamic (PD) parameters to predict the relationship between BMS-869780 dose, exposure and Aβ1-42 levels in human. Off-target and safety margins were then based on comparisons to the predicted exposure required for robust Aβ1-42 lowering. Because of insufficient safety predictions and the relatively high predicted human daily dose of 700 mg, further evaluation of BMS-869780 as a potential clinical candidate was discontinued. Nevertheless, BMS-869780 demonstrates the potential of the GSM approach for robust lowering of brain Aβ1-42 without Notch-related side effects.