ABSTRACT
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 cKi 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.
Subject(s)
Checkpoint Kinase 1/chemistry , Drug Design , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Checkpoint Kinase 1/metabolism , Crystallography, X-Ray , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Pyrroles/chemical synthesis , Pyrroles/chemistry , Structure-Activity RelationshipABSTRACT
Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson's disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LRRK2-pSer935 IC50 values for 22 in mouse brain and kidney being 1.3 and 5 nM, respectively.
Subject(s)
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Animals , Brain/metabolism , Checkpoint Kinase 1 , Crystallography/methods , HEK293 Cells , Humans , Kidney/metabolism , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Mice , Mutation , Parkinson Disease/genetics , Protein Binding , Protein Domains , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokineticsABSTRACT
Leucine-rich repeat kinase 2 (LRRK2) has attracted considerable interest as a therapeutic target for the treatment of Parkinson's disease. Compounds derived from a 2-aminopyridine screening hit were optimised using a LRRK2 homology model based on mixed lineage kinase 1 (MLK1), such that a 2-aminopyridine-based lead molecule 45, with in vivo activity, was identified.
Subject(s)
Aminopyridines/pharmacology , Drug Design , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/antagonists & inhibitors , Aminopyridines/chemical synthesis , Aminopyridines/chemistry , Animals , Dogs , Dose-Response Relationship, Drug , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Madin Darby Canine Kidney Cells/drug effects , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Molecular Structure , Rats , Structure-Activity RelationshipABSTRACT
Libraries of nonpurified resorcinol amide derivatives were screened by surface plasmon resonance (SPR) to determine the binding dissociation constant (off-rate, kd) for compounds binding to the pyruvate dehydrogenase kinase (PDHK) enzyme. Parallel off-rate measurements against HSP90 and application of structure-based drug design enabled rapid hit to lead progression in a program to identify pan-isoform ATP-competitive inhibitors of PDHK. Lead optimization identified selective sub-100-nM inhibitors of the enzyme which significantly reduced phosphorylation of the E1α subunit in the PC3 cancer cell line in vitro.
