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.

Design, Optimization, and Study of Small Molecules That Target Tau Pre-mRNA and Affect Splicing.

Approximately 95% of human genes are alternatively spliced, and aberrant splicing events can cause disease. One pre-mRNA that is alternatively spliced and linked to neurodegenerative diseases is tau (microtubule-associated protein tau), which can cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and can contribute to Alzheimer's disease. Here, we describe the design of structure-specific lead small molecules that directly target tau pre-mRNA from sequence. This was followed by hit expansion and analogue synthesis to further improve upon these initial lead molecules. The emergent compounds were assessed for functional activity in a battery of assays, including binding assays and an assay that mimics molecular recognition of tau pre-mRNA by a U1 small nuclear ribonucleoprotein (snRNP) splicing factor. Compounds that emerged from these studies had enhanced potency and selectivity for the target RNA relative to the initial hits, while also having significantly improved drug-like properties. The compounds are shown to directly target tau pre-mRNA in cells, via chemical cross-linking and isolation by pull-down target profiling, and to rescue disease-relevant splicing of tau pre-mRNA in a variety of cellular systems, including primary neurons. More broadly, this study shows that lead, structure-specific compounds can be designed from sequence and then further optimized for their physicochemical properties while at the same time enhancing their activity.

Author Correction: Dopamine D3 receptor antagonist reveals a cryptic pocket in aminergic GPCRs.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

In Vitro-In Vivo Extrapolation of Key Transporter Activity at the Blood-Brain Barrier.

Understanding the quantitative implications of P-glycoprotein and breast cancer resistance protein efflux is a key hurdle in the design of effective, centrally acting or centrally restricted therapeutics. Previously, a comprehensive physiologically based pharmacokinetic model was developed to describe the in vivo unbound brain-to-plasma concentration ratio as a function of efflux activity measured in vitro. In the present work, the predictive utility of this framework was examined through application to in vitro and in vivo data generated on 133 unique compounds across three preclinical species. Two approaches were examined for the scaling of efflux activity to in vivo, namely relative expression as determined by independent proteomics measurements and relative activity as determined via fitting the in vivo neuropharmacokinetic data. The results with both approaches indicate that in vitro efflux data can be used to accurately predict the degree of brain penetration across species within the context of the proposed physiologically based pharmacokinetic framework.

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.

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 and Profiling of a Selective and Brain Penetrant Radioligand for in Vivo Target Occupancy Measurement of Casein Kinase 1 (CK1) Inhibitors.

To enable the clinical development of our CNS casein kinase 1 delta/epsilon (CK1δ/ε) inhibitor project, we investigated the possibility of developing a CNS positron emission tomography (PET) radioligand. For this effort, we focused our design and synthesis efforts on the initial CK1δ/ε inhibitor HTS hits with the goal of identifying a compound that would fulfill a set of recommended PET ligand criteria. We identified [3H]PF-5236216 (9) as a tool ligand that meets most of the key CNS PET attributes including high CNS MPO PET desirability score and kinase selectivity, CNS penetration, and low nonspecific binding. We further used [3H]-9 to determine the binding affinity for PF-670462, a literature CK1δ/ε inhibitor tool compound. Lastly, [3H]-9 was used to measure in vivo target occupancy (TO) of PF-670462 in mouse and correlated TO with CK1δ/ε in vivo pharmacology (circadian rhythm modulation).

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.

Targeting of the circadian clock via CK1δ/ε to improve glucose homeostasis in obesity.

Growing evidence indicates that disruption of our internal timing system contributes to the incidence and severity of metabolic diseases, including obesity and type 2 diabetes. This is perhaps not surprising since components of the circadian clockwork are tightly coupled to metabolic processes across the body. In the current study, we assessed the impact of obesity on the circadian system in mice at a behavioural and molecular level, and determined whether pharmacological targeting of casein kinase 1δ and ε (CK1δ/ε), key regulators of the circadian clock, can confer metabolic benefit. We demonstrate that although behavioural rhythmicity was maintained in diet-induced obesity (DIO), gene expression profiling revealed tissue-specific alteration to the phase and amplitude of the molecular clockwork. Clock function was most significantly attenuated in visceral white adipose tissue (WAT) of DIO mice, and was coincident with elevated tissue inflammation, and dysregulation of clock-coupled metabolic regulators PPARα/γ. Further, we show that daily administration of a CK1δ/ε inhibitor (PF-5006739) improved glucose tolerance in both DIO and genetic (ob/ob) models of obesity. These data further implicate circadian clock disruption in obesity and associated metabolic disturbance, and suggest that targeting of the clock represents a therapeutic avenue for the treatment of metabolic disorders.

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.

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 Parkinson's 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.

Casein kinase 1δ/ε inhibitor PF-5006739 attenuates opioid drug-seeking behavior.

Casein kinase 1 delta (CK1δ) and casein kinase 1 epsilon (CK1ε) inhibitors are potential therapeutic agents for a range of psychiatric disorders. The feasibility of developing a CNS kinase inhibitor has been limited by an inability to identify safe brain-penetrant compounds with high kinome selectivity. Guided by structure-based drug design, potent and selective CK1δ/ε inhibitors have now been identified that address this gap, through the design and synthesis of novel 4-[4-(4-fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl]pyrimidin-2-amine derivatives. PF-5006739 (6) possesses a desirable profile, with low nanomolar in vitro potency for CK1δ/ε (IC50 = 3.9 and 17.0 nM, respectively) and high kinome selectivity. In vivo, 6 demonstrated robust centrally mediated circadian rhythm phase-delaying effects in both nocturnal and diurnal animal models. Further, 6 dose-dependently attenuated opioid drug-seeking behavior in a rodent operant reinstatement model in animals trained to self-administer fentanyl. Collectively, our data supports further development of 6 as a promising candidate to test the hypothesis of CK1δ/ε inhibition in treating multiple indications in the clinic.

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 Parkinson's 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.

A novel mechanism controlling resetting speed of the circadian clock to environmental stimuli.

Many aspects of mammalian physiology are driven through the coordinated action of internal circadian clocks. Clock speed (period) and phase (temporal alignment) are fundamental to an organism's ability to synchronize with its environment. In humans, lifestyles that disturb these clocks, such as shift work, increase the incidence of diseases such as cancer and diabetes. Casein kinases 1δ and ε are closely related clock components implicated in period determination. However, CK1δ is so dominant in this regard that it remains unclear what function CK1ε normally serves. Here, we reveal that CK1ε dictates how rapidly the clock is reset by environmental stimuli. Genetic disruption of CK1ε in mice enhances phase resetting of behavioral rhythms to acute light pulses and shifts in light cycle. This impact of CK1ε targeting is recapitulated in isolated brain suprachiasmatic nucleus and peripheral (lung) clocks during NMDA- or temperature-induced phase shift in association with altered PERIOD (PER) protein dynamics. Importantly, accelerated re-entrainment of the circadian system in vivo and in vitro can be achieved in wild-type animals through pharmacological inhibition of CK1ε. These studies therefore reveal a role for CK1ε in stabilizing the circadian clock against phase shift and highlight it as a novel target for minimizing physiological disturbance in shift workers.

Improving the odds of success in drug discovery: choosing the best compounds for in vivo toxicology studies.

A set of molecules that advanced into exploratory animal toxicology studies (two species) was examined to determine what properties contributed to success in these safety studies. Compounds were rigorously evaluated across numerous safety end points and classified as "pass" if a suitable in vivo therapeutic index (TI) was achieved for advancement into regulatory toxicology studies. The most predictive end point contributing to compound survival was a predicted human efficacious concentration (Ceff) of ≤250 nM (total drug) and ≤40 nM (free drug). This trend held across a wide range of CNS modes of action, encompassing targets such as enzymes, G-protein-coupled receptors, ion channels, and transporters.

Design and selection parameters to accelerate the discovery of novel central nervous system positron emission tomography (PET) ligands and their application in the development of a novel phosphodiesterase 2A PET ligand.

To accelerate the discovery of novel small molecule central nervous system (CNS) positron emission tomography (PET) ligands, we aimed to define a property space that would facilitate ligand design and prioritization, thereby providing a higher probability of success for novel PET ligand development. Toward this end, we built a database consisting of 62 PET ligands that have successfully reached the clinic and 15 radioligands that failed in late-stage development as negative controls. A systematic analysis of these ligands identified a set of preferred parameters for physicochemical properties, brain permeability, and nonspecific binding (NSB). These preferred parameters have subsequently been applied to several programs and have led to the successful development of novel PET ligands with reduced resources and timelines. This strategy is illustrated here by the discovery of the novel phosphodiesterase 2A (PDE2A) PET ligand 4-(3-[(18)F]fluoroazetidin-1-yl)-7-methyl-5-{1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}imidazo[5,1-f][1,2,4]triazine, [(18)F]PF-05270430 (5).

Strategies to minimize CNS toxicity: in vitro high-throughput assays and computational modeling.

INTRODUCTION: Healthy functioning of the brain is dependent on the ability of the blood-brain barrier (BBB) and other central nervous system (CNS) barriers to protect the neurocompartments from potential disruptive and damaging xenobiotic agents. In vitro high-throughput (HT) screens and computational models that assess a compound's ability to pass through or disrupt the BBB have become important tools in the identification of new well-tolerated peripheral drugs and safer chemical products such as pesticides. Leveraging these HT in vitro assays and computational BBB tools together with the current understanding of brain penetration may enable the drug discovery community to minimize access of drug candidates into the CNS compartment. AREAS COVERED: This article reviews aspects of the most recent in vitro and computational approaches designed to provide an early assessment of a compound's ability to access the neurocompartment. This article also provides insight into using these tools to identify compounds that have restricted access to the neurocompartment. EXPERT OPINION: The development of safer peripheral-acting medicines and chemical products can be achieved through prospective design and early assessment with HT assays of the BBB in conjunction with computational models. Exclusion or significantly reduced access of a compound to the neurocompartment will increase the odds of identifying a compound with reduced CNS-related adverse drug reactions. A holistic approach to compound design and evaluation that incorporates prospective design principles (e.g., optimization of physicochemical properties), leverages HT in vitro assays and integrates the use of BBB computational models may yield the 'best-in-class' peripherally acting product.

Csnk1e is a genetic regulator of sensitivity to psychostimulants and opioids.

Csnk1e, the gene encoding casein kinase 1-epsilon, has been implicated in sensitivity to amphetamines. Additionally, a polymorphism in CSNK1E was associated with heroin addiction, suggesting that this gene may also affect opioid sensitivity. In this study, we first conducted genome-wide quantitative trait locus (QTL) mapping of methamphetamine (MA)-induced locomotor activity in C57BL/6J (B6) × DBA/2J (D2)-F(2) mice and a more highly recombinant F(8) advanced intercross line. We identified a QTL on chromosome 15 that contained Csnk1e (63-86 Mb; Csnk1e=79.25 Mb). We replicated this result and further narrowed the locus using B6.D2(Csnk1e) and D2.B6(Csnk1e) reciprocal congenic lines (78-86.8 and 78.7-81.6 Mb, respectively). This locus also affected sensitivity to the µ-opioid receptor agonist fentanyl. Next, we directly tested the hypothesis that Csnk1e is a genetic regulator of sensitivity to psychostimulants and opioids. Mice harboring a null allele of Csnk1e showed an increase in locomotor activity following MA administration. Consistent with this result, coadministration of a selective pharmacological inhibitor of Csnk1e (PF-4800567) increased the locomotor stimulant response to both MA and fentanyl. These results show that a narrow genetic locus that contains Csnk1e is associated with differences in sensitivity to MA and fentanyl. Furthermore, gene knockout and selective pharmacological inhibition of Csnk1e define its role as a negative regulator of sensitivity to psychostimulants and opioids.