Discovery of Clinical Candidate PF-06648671: A Potent γ-Secretase Modulator for the Treatment of Alzheimer's Disease.

Herein, we describe the design and synthesis of γ-secretase modulator (GSM) clinical candidate PF-06648671 (22) for the treatment of Alzheimer's disease. A key component of the design involved a 2,5-cis-tetrahydrofuran (THF) linker to impart conformational rigidity and lock the compound into a putative bioactive conformation. This effort was guided using a pharmacophore model since crystallographic information was not available for the membrane-bound γ-secretase protein complex at the time of this work. PF-06648671 achieved excellent alignment of whole cell in vitro potency (Aß42 IC50 = 9.8 nM) and absorption, distribution, metabolism, and excretion (ADME) parameters. This resulted in favorable in vivo pharmacokinetic (PK) profile in preclinical species, and PF-06648671 achieved a human PK profile suitable for once-a-day dosing. Furthermore, PF-06648671 was found to have favorable brain availability in rodent, which translated into excellent central exposure in human and robust reduction of amyloid ß (Aß) 42 in cerebrospinal fluid (CSF).

A Second-Generation Oral SARS-CoV-2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19.

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.

Designing small molecules for therapeutic success: A contemporary perspective.

Successful small-molecule drug design requires a molecular target with inherent therapeutic potential and a molecule with the right properties to unlock its potential. Present-day drug design strategies have evolved to leave little room for improvement in drug-like properties. As a result, inadequate safety or efficacy associated with molecular targets now constitutes the primary cause of attrition in preclinical development through Phase II. This finding has led to a deeper focus on target selection. In this current reality, design tactics that enable rapid identification of risk-balanced clinical candidates, translation of clinical experience into meaningful differentiation strategies, and expansion of the druggable proteome represent significant levers by which drug designers can accelerate the discovery of the next generation of medicines.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Innovation in breakthrough drugs and vaccines: Development risk, patient impact, and value.

Innovation has a crucial role in developing breakthrough drugs and vaccines that can change patients' lives. To better understand this role, we evaluated recent outcomes for assets developed using different types of innovation. Although all approaches have delivered breakthroughs, assets that modulate established biological targets with innovative scientific or technological designs provide a unique combination of reduced development risk, high patient impact, and high commercial value. This type of asset currently represents a relatively small proportion of approved drugs and vaccines, but we anticipate that an increasing body of scientific knowledge and ongoing technological advancements could offer opportunities to grow this category in the future.

Harnessing Preclinical Data as a Predictive Tool for Human Brain Tissue Targeting.

One of the objectives within the medicinal chemistry discipline is to design tissue targeting molecules. The objective of tissue specificity can be either to gain drug access to the compartment of interest (e.g., the CNS) for Neuroscience targets or to restrict drug access to the CNS for all other therapeutic areas. Both neuroscience and non-neuroscience therapeutic areas have struggled to quantitatively estimate brain penetration or the lack thereof with compounds that are substrates of efflux transport proteins such as P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP) that are key components of the blood-brain barrier (BBB). It has been well established that drug candidates with high efflux ratios (ER) of these transporters have poor penetration into brain tissue. In the current work, we outline a parallel analysis to previously published models for the prediction of brain penetration that utilize an alternate MDR1-MDCK cell line as a better predictor of brain penetration and whether a correlation between in vitro, rodent data, non-human primate (NHP), and human in vivo brain penetration data could be established. Analysis of structural and physicochemical properties in conjunction with in vitro parameters and preclinical in vivo data has been highlighted in this manuscript as a continuation of the previously published work.

Photoaffinity Labeling and Quantitative Chemical Proteomics Identify LXRß as the Functional Target of Enhancers of Astrocytic apoE.

Utilizing a phenotypic screen, we identified chemical matter that increased astrocytic apoE secretion in vitro. We designed a clickable photoaffinity probe based on a pyrrolidine lead compound and carried out probe-based quantitative chemical proteomics in human astrocytoma CCF-STTG1 cells to identify liver x receptor ß (LXRß) as the target. Binding of the small molecule ligand stabilized LXRß, as shown by cellular thermal shift assay (CETSA). In addition, we identified a probe-modified peptide by mass spectrometry and proposed a model where the photoaffinity probe is bound in the ligand-binding pocket of LXRß. Taken together, our findings demonstrated that the lead chemical matter bound directly to LXRß, and our results highlight the power of chemical proteomic approaches to identify the target of a phenotypic screening hit. Additionally, the LXR photoaffinity probe and lead compound described herein may serve as valuable tools to further evaluate the LXR pathway.

Quantitative Translational Analysis of Brain Kynurenic Acid Modulation via Irreversible Kynurenine Aminotransferase II Inhibition.

Kynurenic acid (KYNA) plays a significant role in maintaining normal brain function, and abnormalities in KYNA levels have been associated with various central nervous system disorders. Confirmation of its causality in human diseases requires safe and effective modulation of central KYNA levels in the clinic. The kynurenine aminotransferases (KAT) II enzyme represents an attractive target for pharmacologic modulation of central KYNA levels; however, KAT II and KYNA turnover kinetics, which could contribute to the duration of pharmacologic effect, have not been reported. In this study, the kinetics of central KYNA-lowering effect in rats and nonhuman primates (NHPs, Cynomolgus macaques) was investigated using multiple KAT II irreversible inhibitors as pharmacologic probes. Mechanistic pharmacokinetic-pharmacodynamic analysis of in vivo responses to irreversible inhibition quantitatively revealed that 1) KAT II turnover is relatively slow [16-76 hours' half-life (t1/2)], whereas KYNA is cleared more rapidly from the brain (<1 hour t1/2) in both rats and NHPs, 2) KAT II turnover is slower in NHPs than in rats (76 hours vs. 16 hours t1/2, respectively), and 3) the percent contribution of KAT II to KYNA formation is constant (∼80%) across rats and NHPs. Additionally, modeling results enabled establishment of in vitro-in vivo correlation for both enzyme turnover rates and drug potencies. In summary, quantitative translational analysis confirmed the feasibility of central KYNA modulation in humans. Model-based analysis, where system-specific properties and drug-specific properties are mechanistically separated from in vivo responses, enabled quantitative understanding of the KAT II-KYNA pathway, as well as assisted development of promising candidates to test KYNA hypothesis in humans.

Late-Stage Microsomal Oxidation Reduces Drug-Drug Interaction and Identifies Phosphodiesterase 2A Inhibitor PF-06815189.

Late-stage oxidation using liver microsomes was applied to phosphodiesterase 2 inhibitor 1 to reduce its clearance by cytochrome P450 enzymes, introduce renal clearance, and minimize the risk for victim drug-drug interactions. This approach yielded PF-06815189 (2) with improved physicochemical properties and a mixed metabolic profile. This example highlights the importance of C-H diversification methods to drug discovery.

Identification of a Potent, Highly Selective, and Brain Penetrant Phosphodiesterase 2A Inhibitor Clinical Candidate.

Computational modeling was used to direct the synthesis of analogs of previously reported phosphodiesterase 2A (PDE2A) inhibitor 1 with an imidazotriazine core to yield compounds of significantly enhanced potency. The analog PF-05180999 (30) was subsequently identified as a preclinical candidate targeting cognitive impairment associated with schizophrenia. Compound 30 demonstrated potent binding to PDE2A in brain tissue, dose responsive mouse brain cGMP increases, and reversal of N-methyl-d-aspartate (NMDA) antagonist-induced (MK-801, ketamine) effects in electrophysiology and working memory models in rats. Preclinical pharmacokinetics revealed unbound brain/unbound plasma levels approaching unity and good oral bioavailability resulting in an average concentration at steady state (Cav,ss) predicted human dose of 30 mg once daily (q.d.). Modeling of a modified release formulation suggested that 25 mg twice daily (b.i.d.) could maintain plasma levels of 30 at or above targeted efficacious plasma levels for 24 h, which became part of the human clinical plan.

The Discovery of a Novel Phosphodiesterase (PDE) 4B-Preferring Radioligand for Positron Emission Tomography (PET) Imaging.

As part of our effort in identifying phosphodiesterase (PDE) 4B-preferring inhibitors for the treatment of central nervous system (CNS) disorders, we sought to identify a positron emission tomography (PET) ligand to enable target occupancy measurement in vivo. Through a systematic and cost-effective PET discovery process, involving expression level (Bmax) and biodistribution determination, a PET-specific structure-activity relationship (SAR) effort, and specific binding assessment using a LC-MS/MS "cold tracer" method, we have identified 8 (PF-06445974) as a promising PET lead. Compound 8 has exquisite potency at PDE4B, good selectivity over PDE4D, excellent brain permeability, and a high level of specific binding in the "cold tracer" study. In subsequent non-human primate (NHP) PET imaging studies, [18F]8 showed rapid brain uptake and high target specificity, indicating that [18F]8 is a promising PDE4B-preferring radioligand for clinical PET imaging.

Discovery and Characterization of (R)-6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates.

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance.

Application of Structure-Based Design and Parallel Chemistry to Identify a Potent, Selective, and Brain Penetrant Phosphodiesterase 2A Inhibitor.

Phosphodiesterase 2A (PDE2A) inhibitors have been reported to demonstrate in vivo activity in preclinical models of cognition. To more fully explore the biology of PDE2A inhibition, we sought to identify potent PDE2A inhibitors with improved brain penetration as compared to current literature compounds. Applying estimated human dose calculations while simultaneously leveraging synthetically enabled chemistry and structure-based drug design has resulted in a highly potent, selective, brain penetrant compound 71 (PF-05085727) that effects in vivo biochemical changes commensurate with PDE2A inhibition along with behavioral and electrophysiological reversal of the effects of NMDA antagonists in rodents. This data supports the ability of PDE2A inhibitors to potentiate NMDA signaling and their further development for clinical cognition indications.

Discovery of cyclopropyl chromane-derived pyridopyrazine-1,6-dione γ-secretase modulators with robust central efficacy.

Herein we describe the discovery of a novel series of cyclopropyl chromane-derived pyridopyrazine-1,6-dione γ-secretase modulators for the treatment of Alzheimer's disease (AD). Using ligand-based design tactics such as conformational analysis and molecular modeling, a cyclopropyl chromane unit was identified as a suitable heterocyclic replacement for a naphthyl moiety that was present in the preliminary lead 4. The optimized lead molecule 44 achieved good central exposure resulting in robust and sustained reduction of brain amyloid-ß42 (Aß42) when dosed orally at 10 mg kg-1 in a rat time-course study. Application of the unpaced isolated heart Langendorff model enabled efficient differentiation of compounds with respect to cardiovascular safety, highlighting how minor structural changes can greatly impact the safety profile within a series of compounds.

Quantitative Assessment of the Impact of Fluorine Substitution on P-Glycoprotein (P-gp) Mediated Efflux, Permeability, Lipophilicity, and Metabolic Stability.

Strategic replacement of one or more hydrogen atoms with fluorine atom(s) is a common tactic to improve potency at a given target and/or to modulate parameters such as metabolic stability and pKa. Molecular weight (MW) is a key parameter in design, and incorporation of fluorine is associated with a disproportionate increase in MW considering the van der Waals radius of fluorine versus hydrogen. Herein we examine a large compound data set to understand the effect of introducing fluorine on the risk of encountering P-glycoprotein mediated efflux (as measured by MDR efflux ratio), passive permeability, lipophilicity, and metabolic stability. Statistical modeling of the MDR ER data demonstrated that an increase in MW as a result of introducing fluorine atoms does not lead to higher risk of P-gp mediated efflux. Fluorine-corrected molecular weight (MWFC), where the molecular weight of fluorine has been subtracted, was found to be a more relevant descriptor.

Central Nervous System Multiparameter Optimization Desirability: Application in Drug Discovery.

Significant progress has been made in prospectively designing molecules using the central nervous system multiparameter optimization (CNS MPO) desirability tool, as evidenced by the analysis reported herein of a second wave of drug candidates that originated after the development and implementation of this tool. This simple-to-use design algorithm has expanded design space for CNS candidates and has further demonstrated the advantages of utilizing a flexible, multiparameter approach in drug discovery rather than individual parameters and hard cutoffs of physicochemical properties. The CNS MPO tool has helped to increase the percentage of compounds nominated for clinical development that exhibit alignment of ADME attributes, cross the blood-brain barrier, and reside in lower-risk safety space (low ClogP and high TPSA). The use of this tool has played a role in reducing the number of compounds submitted to exploratory toxicity studies and increasing the survival of our drug candidates through regulatory toxicology into First in Human studies. Overall, the CNS MPO algorithm has helped to improve the prioritization of design ideas and the quality of the compounds nominated for clinical development.

Identification of Phosphorylation Consensus Sequences and Endogenous Neuronal Substrates of the Psychiatric Risk Kinase TNIK.

Traf2- and Nck-interacting kinase (TNIK) is a serine/threonine kinase highly expressed in the brain and enriched in the postsynaptic density of glutamatergic synapses in the mammalian brain. Accumulating genetic evidence and functional data have implicated TNIK as a risk factor for psychiatric disorders. However, the endogenous substrates of TNIK in neurons are unknown. Here, we describe a novel selective small molecule inhibitor of the TNIK kinase family. Using this inhibitor, we report the identification of endogenous neuronal TNIK substrates by immunoprecipitation with a phosphomotif antibody followed by mass spectrometry. Phosphorylation consensus sequences were defined by phosphopeptide sequence analysis. Among the identified substrates were members of the delta-catenin family including p120-catenin, δ-catenin, and armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), each of which is linked to psychiatric or neurologic disorders. Using p120-catenin as a representative substrate, we show TNIK-induced p120-catenin phosphorylation in cells requires intact kinase activity and phosphorylation of TNIK at T181 and T187 in the activation loop. Addition of the small molecule TNIK inhibitor or knocking down TNIK by two shRNAs reduced endogenous p120-catenin phosphorylation in cells. Together, using a TNIK inhibitor and phosphomotif antibody, we identify endogenous substrates of TNIK in neurons, define consensus sequences for TNIK, and suggest signaling pathways by which TNIK influences synaptic development and function linked to psychiatric and neurologic disorders.

Challenges and Opportunities in the Discovery of New Therapeutics Targeting the Kynurenine Pathway.

The kynurenine pathway is responsible for the metabolism of more than 95% of dietary tryptophan (TRP) and produces numerous bioactive metabolites. Recent studies have focused on three enzymes in this pathway: indoleamine dioxygenase (IDO1), kynurenine monooxygenase (KMO), and kynurenine aminotransferase II (KAT II). IDO1 inhibitors are currently in clinical trials for the treatment of cancer, and these agents may also have therapeutic utility in neurological disorders, including multiple sclerosis. KMO inhibitors are being investigated as potential treatments for neurodegenerative diseases, such as Huntington's and Alzheimer's diseases. KAT II inhibitors have been proposed in new therapeutic approaches toward psychiatric and cognitive disorders, including cognitive impairment associated with schizophrenia. Numerous medicinal chemistry studies are currently aimed at the design of novel, potent, and selective inhibitors for each of these enzymes. The emerging opportunities and significant challenges associated with pharmacological modulation of these enzymes will be explored in this review.

Design of Pyridopyrazine-1,6-dione γ-Secretase Modulators that Align Potency, MDR Efflux Ratio, and Metabolic Stability.

Herein we describe the design and synthesis of a series of pyridopyrazine-1,6-dione γ-secretase modulators (GSMs) for Alzheimer's disease (AD) that achieve good alignment of potency, metabolic stability, and low MDR efflux ratios, while also maintaining favorable physicochemical properties. Specifically, incorporation of fluorine enabled design of metabolically less liable lipophilic alkyl substituents to increase potency without compromising the sp(3)-character. The lead compound 21 (PF-06442609) displayed a favorable rodent pharmacokinetic profile, and robust reductions of brain Aß42 and Aß40 were observed in a guinea pig time-course experiment.