Background & Objective: Compound X is a drug candidate for the treatment of neurodegenerative diseases. Its brain distribution was evaluated as part of the lead identification and optimization activities undertaken in early drug discovery. Methods: The brain distribution of compound X was studied in genetic transporter knockout rodent models, in vivo models with a chemical inhibitor, and in vitro transporter cell systems. Results: Compound X was found to be a substrate for human Breast Cancer-Resistance Protein (BCRP) in vitro (efflux ratio 8.1) and rodent Bcrp in vivo (Kp, uuKO/Kp, uuWT = 0.15/0.057 = 2.7, p< 0.05) but not a substrate for human P-glycoprotein (P-gp) in vitro (efflux ratio 1.0) nor rodent P-gp in vivo (Kp, uuKO/Kp, uuWT = 0.056/ 0.051 = 1.1, p> 0.05). When both transporters were knocked out in vivo, Kp, uu increased to 0.51±0.02. A similar pattern observed across compounds with related chemistry corroborating the structure-activity relationship. Conclusion: While in vitro assays showed compound X to be a substrate for human BCRP and not P-gp, in vivo studies indicated a synergistic effect between rodent efflux transporters. However, this only accounted for ~50% of restricted BBB-transport, suggesting involvement of other efflux transporters. Considering Kp, uu as a key criterion for assessing the technical quality of CNS candidates before progression into clinical development, it is important to identify relevant screening assays for a better understanding of low Kp, uu and brain distribution in pre-clinical models for translation to humans.

Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3-d]pyrimidines underwent optimization using X-ray structures of LRRK2 kinase domain surrogates, based on checkpoint kinase 1 (CHK1) and a CHK1 10-point mutant. (2R)-2-Methylpyrrolidin-1-yl derivative 18 (LRRK2 G2019S cK(i) 0.7 nM, LE 0.66) was identified, with increased potency consistent with an X-ray structure of 18/CHK1 10-pt. mutant showing the 2-methyl substituent proximal to Ala147 (Ala2016 in LRRK2). Further structure-guided elaboration of 18 gave the 2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino] derivative 32. Optimization of 32 afforded diastereomeric oxolan-3-yl derivatives 44 and 45, which demonstrated a favorable in vitro PK profile, although they displayed species disconnects in the in vivo PK profile, and a propensity for P-gp- and/or BCRP-mediated efflux in a mouse model. Compounds 44 and 45 demonstrated high potency and exquisite selectivity for LRRK2 and utility as chemical probes for the study of LRRK2 inhibition.

Drug distribution in the brain is generally associated with an affinity for fatty brain tissues and therefore known to be species- and concentration-independent. We report here the effect of target affinity on brain tissue binding for 10 small molecules designed to inhibit brain heat shock protein 90 (HSP90), a widespread protein whose expression is 1–2% of total cytosolic proteins in eucaryotes. Our results show that increasing the test item concentrations from 0.3 to 100 µM increased the unbound fraction 32-fold for the most potent molecules, with no change for the inactive one (1.1 fold change). Saturation of HSP90 led to normal concentration-independent brain tissue binding. In vivo pharmacokinetics performed in rats showed that the overall volume of distribution of compounds is correlated with their affinity for HSP90. The in vitro binding and in vivo pharmacokinetics (PK) performed in rats showed that small molecule HSP90 inhibitors followed the principle of target-mediated drug disposition. We demonstrate that assessing unbound fractions in brain homogenate was subject to HSP90 target interference; this may challenge the process of linking systemic-free drug concentrations to central nervous system unbound concentrations necessary to establish the proper pharmacokinetics/pharmacodynamics (PK/PD) relation needed for human dose prediction.

HSP90 (Heat shock protein 90) is a molecular chaperone protein ubiquitously expressed throughout all tissues in the body. HSP90 has been proposed as a target to increase turnover of pathological proteins leading to neurodegeneration in Huntington's disease, Parkinson's disease and Alzheimer's disease. The mechanism of how HSP90 inhibition leads to clearance of misfolded proteins is not fully understood. It may involve direct effects of inhibiting ATPase function, indirect effects by inducing the heat-shock-response resulting in upregulation of other chaperone proteins like HSP70 or a combination of both. In the current work we established a methodology to investigate the relationship between HSP90 target occupancy and HSP70 induction in vivo. We also characterized the acute effect of two different HSP90 inhibitors in the rTg4510 transgenic mouse model of Alzheimer's disease which displays a tau-mediated synaptic dysfunction. We show that reversal of synaptic impairments in this model can be obtained with a compound which has a high HSP70 induction capacity. The current developed assay methodologies may thus be of significant use in the further elucidation of the mechanism involved in the in vivo effect of HSP90 inhibition in models of neurodegeneration. Further on, the ability of HSP90 inhibitors to normalize synaptic dysfunction in an in vivo disease model of Alzheimer's disease could have therapeutic relevance and further strengthens the usefulness of this animal model to establish pharmacodynamic effect of HSP90 inhibition.

The identification of the novel and selective GPR3 inverse agonist AF64394, the first small molecule inhibitor of GPR3 receptor function, is described. Structure activity relationships and syntheses based around AF64394 are reported.