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
78 kDa glucose-regulated protein (Grp78) is a heat shock protein (HSP) involved in protein folding that plays a role in cancer cell proliferation. Binding of adenosine-derived inhibitors to Grp78 was characterized by surface plasmon resonance and isothermal titration calorimetry. The most potent compounds were 13 (VER-155008) with K(D) = 80 nM and 14 with K(D) = 60 nM. X-ray crystal structures of Grp78 bound to ATP, ADPnP, and adenosine derivative 10 revealed differences in the binding site between Grp78 and homologous proteins.
Subject(s)
Adenosine Triphosphatases/antagonists & inhibitors , Furans/chemical synthesis , Heat-Shock Proteins/antagonists & inhibitors , Purines/chemical synthesis , Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/chemistry , Adenylyl Imidodiphosphate/chemistry , Binding Sites , Calorimetry , Crystallography, X-Ray , Endoplasmic Reticulum Chaperone BiP , Furans/chemistry , HSC70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/chemistry , Heat-Shock Proteins/chemistry , Ligands , Models, Molecular , Protein Binding , Protein Conformation , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/chemistry , Purines/chemistry , Stereoisomerism , Structure-Activity Relationship , Surface Plasmon Resonance , ThermodynamicsABSTRACT
The design and synthesis of novel adenosine-derived inhibitors of HSP70, guided by modeling and X-ray crystallographic structures of these compounds in complex with HSC70/BAG-1, is described. Examples exhibited submicromolar affinity for HSP70, were highly selective over HSP90, and some displayed potency against HCT116 cells. Exposure of compound 12 to HCT116 cells caused significant reduction in cellular levels of Raf-1 and Her2 at concentrations similar to that which caused cell growth arrest.