Fragment-Based Discovery of a Series of Allosteric-Binding Site Modulators of ß-Glucocerebrosidase.

ß-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher's disease and are a major genetic risk factor for Parkinson's disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.

Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Fragment-based discovery of type I inhibitors of maternal embryonic leucine zipper kinase.

Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 µM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology.

Structure-Based Design of Type II Inhibitors Applied to Maternal Embryonic Leucine Zipper Kinase.

A novel Type II kinase inhibitor chemotype has been identified for maternal embryonic leucine zipper kinase (MELK) using structure-based ligand design. The strategy involved structural characterization of an induced DFG-out pocket by protein-ligand X-ray crystallography and incorporation of a slender linkage capable of bypassing a large gate-keeper residue, thus enabling design of molecules accessing both hinge and induced pocket regions. Optimization of an initial hit led to the identification of a low-nanomolar, cell-penetrant Type II inhibitor suitable for use as a chemical probe for MELK.

Heteroalicyclic carboxamidines as inhibitors of inducible nitric oxide synthase; the identification of (2R)-2-pyrrolidinecarboxamidine as a potent and selective haem-co-ordinating inhibitor.

Heteroalicyclic carboxamidines were synthesised and evaluated as inhibitors of nitric oxide synthases. (2R)-2-Pyrrolidinecarboxamidine, in particular, was shown to be a highly potent in vitro (IC(50)=0.12 µM) and selective iNOS inhibitor (>100-fold vs both eNOS and nNOS), with probable binding to the key anchoring glutamate residue and co-ordination to the haem iron.

A structural comparison of inhibitor binding to PKB, PKA and PKA-PKB chimera.

Although the crystal structure of the anti-cancer target protein kinase B (PKBbeta/Akt-2) has been useful in guiding inhibitor design, the closely related kinase PKA has generally been used as a structural mimic due to its facile crystallization with a range of ligands. The use of PKB-inhibitor crystallography would bring important benefits, including a more rigorous understanding of factors dictating PKA/PKB selectivity, and the opportunity to validate the utility of PKA-based surrogates. We present a "back-soaking" method for obtaining PKBbeta-ligand crystal structures, and provide a structural comparison of inhibitor binding to PKB, PKA, and PKA-PKB chimera. One inhibitor presented here exhibits no PKB/PKA selectivity, and the compound adopts a similar binding mode in all three systems. By contrast, the PKB-selective inhibitor A-443654 adopts a conformation in PKB and PKA-PKB that differs from that with PKA. We provide a structural explanation for this difference, and highlight the ability of PKA-PKB to mimic the true PKB binding mode in this case.