Bethany L. Kormos

Discovery and preclinical profiling of 3-[4-(morpholin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]benzonitrile (PF-06447475), a highly potent, selective, brain penetrant, and in vivo active LRRK2 kinase inhibitor.

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinsons disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of 14 (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies.

Structure-inspired design of β-arrestin-biased ligands for aminergic GPCRs

Development of biased ligands targeting G protein-coupled receptors (GPCRs) is a promising approach for current drug discovery. Although structure-based drug design of biased agonists remains challenging even with an abundance of GPCR crystal structures, we present an approach for translating GPCR structural data into β-arrestin-biased ligands for aminergic GPCRs. We identified specific amino acid-ligand contacts at transmembrane helix 5 (TM5) and extracellular loop 2 (EL2) responsible for Gi/o and β-arrestin signaling, respectively, and targeted those residues to develop biased ligands. For these ligands, we found that bias is conserved at other aminergic GPCRs that retain similar residues at TM5 and EL2. Our approach provides a template for generating arrestin-biased ligands by modifying predicted ligand interactions that block TM5 interactions and promote EL2 interactions. This strategy could facilitate the structure-guided design of arrestin-biased ligands at other GPCRs, including polypharmacological biased ligands.

Kinase domain inhibition of leucine rich repeat kinase 2 (LRRK2) using a [1,2,4]triazolo[4,3-b]pyridazine scaffold.

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinsons disease (PD). The most common mutant, G2019S, increases kinase activity, thus LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the structure, potential ligand-protein binding interactions, and pharmacological profiling of potent and highly selective kinase inhibitors based on a triazolopyridazine chemical scaffold.

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.

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.

Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors

Mutations in the leucine-rich repeat kinase 2 (LRRK2) are the most common known cause of autosomal dominant Parkinson’s disease (PD), accounting for approximately 1% of “sporadic” and 4% of familial cases. These mutations either lead directly to an increased kinase activity (G2019S and I2020T are in the kinase activation loop) or to a reduced GTPase activity (R1441C/G and Y1699C), that in turn positively regulate kinase activity. The physiological substrate of the LRRK2 kinase has yet to be definitively identified, yet autophosphorylation is emerging as a relatively robust measure of its activity. LRRK2 has been implicated in a number of diverse cellular processes such as vesicular trafficking, microtubule dynamics, protein translation control, inflammation, and immune function, all of which have been linked to PD. LRRK2 is a large, multi-domain protein; a thorough understanding of the protein domain organization and identification of interacting partners is important to determine the underlying mechanism of LRRK2. Substantial recent effort has been directed towards identifying potent LRRK2 kinase inhibitors, from the repurposed kinase inhibitors to the first through third generation of LRRK2-focused kinase inhibitors, from a range of chemotypes, which are now providing researchers with new tools to better interrogate LRRK2 function.

Dopamine D3/D2 Receptor Antagonist PF-4363467 Attenuates Opioid Drug-Seeking Behavior without Concomitant D2 Side Effects

Dopamine receptor antagonism is a compelling molecular target for the treatment of a range of psychiatric disorders, including substance use disorders. From our corporate compound file, we identified a structurally unique D3 receptor (D3R) antagonist scaffold, 1. Through a hybrid approach, we merged key pharmacophore elements from 1 and D3 agonist 2 to yield the novel D3R/D2R antagonist PF-4363467 (3). Compound 3 was designed to possess CNS drug-like properties as defined by its CNS MPO desirability score (≥4/6). In addition to good physicochemical properties, 3 exhibited low nanomolar affinity for the D3R (D3 Ki = 3.1 nM), good subtype selectivity over D2R (D2 Ki = 692 nM), and high selectivity for D3R versus other biogenic amine receptors. In vivo, 3 dose-dependently attenuated opioid self-administration and opioid drug-seeking behavior in a rat operant reinstatement model using animals trained to self-administer fentanyl. Further, traditional extrapyramidal symptoms (EPS), adverse side effects arising from D2R antagonism, were not observed despite high D2 receptor occupancy (RO) in rodents, suggesting that compound 3 has a unique in vivo profile. Collectively, our data support further investigation of dual D3R and D2R antagonists for the treatment of drug addiction.

Chapter Four - Development of LRRK2 Kinase Inhibitors for Parkinson's Disease

Abstract Parkinsons disease (PD) is the most common movement disorder and the second most common neurodegenerative disorder after Alzheimers disease. The etiology of PD is complex but the most common phenotype is the loss of dopaminergic neurons of the substantia nigra leading to the clinical symptoms of bradykinesia, resting tremors, rigidity, and postural instability. The most common genetic cause of autosomal dominant PD is mutations in leucine-rich repeat kinase 2 (LRRK2) which accounts for 1% of sporadic and 4% of familial cases. The G2019S mutation is the most common in LRRK2 and occurs in the DYG motif of the kinase domain, where it appears to increase its kinase activity. Consequently, a great deal of effort has been directed toward identifying potent LRRK2 kinase inhibitors with a biopharmaceutical profile congruent with clinical evaluation and this report summarizes efforts in this regard.

Dopamine D3 receptor antagonist reveals a cryptic pocket in aminergic GPCRs

The recent increase in the number of X-ray crystal structures of G-protein coupled receptors (GPCRs) has been enabling for structure-based drug design (SBDD) efforts. These structures have revealed that GPCRs are highly dynamic macromolecules whose function is dependent on their intrinsic flexibility. Unfortunately, the use of static structures to understand ligand binding can potentially be misleading, especially in systems with an inherently high degree of conformational flexibility. Here, we show that docking a set of dopamine D3 receptor compounds into the existing eticlopride-bound dopamine D3 receptor (D3R) X-ray crystal structure resulted in poses that were not consistent with results obtained from site-directed mutagenesis experiments. We overcame the limitations of static docking by using large-scale high-throughput molecular dynamics (MD) simulations and Markov state models (MSMs) to determine an alternative pose consistent with the mutation data. The new pose maintains critical interactions observed in the D3R/eticlopride X-ray crystal structure and suggests that a cryptic pocket forms due to the shift of a highly conserved residue, F6.52. Our study highlights the importance of GPCR dynamics to understand ligand binding and provides new opportunities for 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.