Fragment-based discovery of potent ERK2 pyrrolopyrazine inhibitors.

A fragment-based lead discovery approach was used to discover novel ERK2 inhibitors. The crystal structure of N-benzyl-9H-purin-6-amine 1 in complex with ERK2 elucidated its hinge-binding mode. In addition, the simultaneous binding of an imidazole molecule adjacent to 1 suggested a direction for fragment expansion. Structure-based core hopping applied to 1 led to 5H-pyrrolo[3,2-b]pyrazine (3) that afforded direct vectors to probe the pockets of interest while retaining the essential hinge binding elements. Utilizing the new vectors for SAR exploration, the new core 3 was quickly optimized to compound 39 resulting in a greater than 6600-fold improvement in potency.

Discovery of 3,5-substituted 6-azaindazoles as potent pan-Pim inhibitors.

Pim kinase inhibitors are promising cancer therapeutics. Pim-2, among the three Pim isoforms, plays a critical role in multiple myeloma yet inhibition of Pim-2 is challenging due to its high affinity for ATP. A co-crystal structure of a screening hit 1 bound to Pim-1 kinase revealed the key binding interactions of its indazole core within the ATP binding site. Screening of analogous core fragments afforded 1H-pyrazolo[3,4-c]pyridine (6-azaindazole) as a core for the development of pan-Pim inhibitors. Fragment and structure based drug design led to identification of the series with picomolar biochemical potency against all three Pim isoforms. Desirable cellular potency was also achieved.

Conformation-locking antibodies for the discovery and characterization of KRAS inhibitors.

Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRASG12C stabilized by covalent inhibitors. One CLAMP enables the visualization of KRASG12C covalent modification in vivo and can be used to investigate response heterogeneity to KRASG12C inhibitors in patient tumors. A second CLAMP enhances the affinity of weak ligands binding to the KRASG12C switch II region (SWII) by stabilizing a specific conformation of KRASG12C, thereby enabling the discovery of such ligands that could serve as leads for the development of drugs in a high-throughput screen. We show that combining the complementary properties of antibodies and small molecules facilitates the study and drugging of dynamic proteins.

Differential effects of divalent manganese and magnesium on the kinase activity of leucine-rich repeat kinase 2 (LRRK2).

Various mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to susceptibility for both familial and idiopathic late-onset Parkinson's disease (PD). In this study, we have demonstrated that phosphorylation of MBP and LRRKtide by the LRRK2 G2019S mutant was activated by Mn(2+) in vitro. This enhanced G2019S kinase activity was due to the combination of an increase in kinase and a decrease in ATPase activity by Mn(2+). Compared to 10 mM Mg(2+), 1 mM Mn(2+) reduced ATP K(m) for G2019S from 103 to 1.8 muM and only modestly reduced k(cat) (2.5-fold); as a result, the Mn(2+) increased its k(cat)/K(m) by 22-fold. This change in ATP K(m) was due in large part to an increase in nucleotide affinity. While Mn(2+) also increased ATP affinity and had similar effects on k(cat)/K(m) for LRRK2 WT and R1441C enzymes, it reduced their k(cat) values significantly by 13-17-fold. Consequently, the difference in the kinase activity between G2019S and other LRRK2 variants was enhanced from about 2-fold in Mg(2+) to 10-fold in Mn(2+) at saturating ATP concentrations relative to its K(m). Furthermore, while Mg(2+) yielded optimal V(max) values at Mg(2+) concentration greater than 5 mM, the optimal Mn(2+) concentration for activating LRRK2 catalysis was in the micromolar range with increasing Mn(2+) above 1 mM causing a decrease in enzyme activity. Finally, despite the large but expected differences in IC(50) tested at 100 muM ATP, the apparent K(i) values of a small set of LRRK2 ATP-competitive inhibitors were within 5-fold between Mg(2+)- and Mn(2+)-mediated reactions except AMP-CPP, an ATP analogue.