Development of Glucose Regularted Protein 94-Selective Inhibitors Based on the Bnlm and Radamide Scaffold

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum resident of the heat shock protein 90 kDa (Hsp90) family of molecular chaperones. Grp94 associates with many proteins involved in cell adhesion and signaling, including integrins, Toll-like receptors, immunoglobulins, and mutant myocilin. Grp94 has been implicated as a target for several therapeutic areas including glaucoma, cancer metastasis, and multiple myeloma. While 85% identical to other Hsp90 isoforms, the N-terminal ATP-binding site of Grp94 possesses a unique hydrophobic pocket that was used to design isoform-selective inhibitors. Incorporation of a cis-amide bioisostere into the radamide scaffold led to development of the original Grp94-selective inhibitor, BnIm. Structure–activity relationship studies have now been performed on the aryl side chain of BnIm, which resulted in improved analogues that exhibit better potency and selectivity for Grp94. These analogues also manifest superior antimigratory activity in a metastasis model as well as enhanced mutant myocilin degradation in a glaucoma model compared to BnIm

Trifunctional High-Throughput Screen Identifies Promising Scaffold To Inhibit Grp94 and Treat Myocilin-Associated Glaucoma

Gain-of-function mutations within the olfactomedin (OLF) domain of myocilin result in its toxic intracellular accumulation and hasten the onset of open-angle glaucoma. The absence of myocilin does not cause disease; therefore, strategies aimed at eliminating myocilin could lead to a successful glaucoma treatment. The endoplasmic reticulum Hsp90 paralog Grp94 accelerates OLF aggregation. Knockdown or pharmacological inhibition of Grp94 in cells facilitates clearance of mutant myocilin via a non-proteasomal pathway. Here, we expanded our support for targeting Grp94 over cytosolic paralogs Hsp90α and Hsp90β. We then developed a high-throughput screening assay to identify new chemical matter capable of disrupting the Grp94/OLF interaction. When applied to a blind, focused library of 17 Hsp90 inhibitors, our miniaturized single-read in vitro thioflavin T -based kinetics aggregation assay exclusively identified compounds that target the chaperone N-terminal nucleotide binding site. In follow up studies, one compound (2) decreased the extent of co-aggregation of Grp94 with OLF in a dose-dependent manner in vitro, and enabled clearance of the aggregation-prone full-length myocilin variant I477N in cells without inducing the heat shock response or causing cytotoxicity. Comparison of the co-crystal structure of compound 2 and another non-selective hit in complex with the N-terminal domain of Grp94 reveals a docking mode tailored to Grp94 and explains its selectivity. A new lead compound has been identified, supporting a targeted chemical biology assay approach to develop a protein degradation-based therapy for myocilin-associated glaucoma by selectively inhibiting Grp94

Simulations and Experiments Delineate Amyloid Fibrilization by Peptides Derived from Glaucoma-Associated Myocilin

Mutant myocilin aggregation is associated with inherited open angle glaucoma, a prevalent optic neuropathy leading to blindness. Comprehension of mutant myocilin aggregation is of fundamental importance to glaucoma pathogenesis and ties glaucoma to amyloid diseases such as Alzheimer’s. Here, we probe the aggregation properties of peptides derived from the myocilin olfactomedin domain. Peptides P1 (residues 326–337) and P3 (residues 426–442) were identified previously to form amyloids. Coarse-grained discontinuous molecular dynamics simulations using the PRIME20 force field (DMD/PRIME20) predict that P1 and P3 are aggregation-prone; P1 consistently forms fibrillar aggregates with parallel in-register β-sheets, whereas P3 forms β-sheet-containing aggregates without distinct order. Natural abundance ¹³C solid-state NMR spectra validate that aggregated P1 exhibits amyloid signatures and is more homogeneous than aggregated P3. DMD/PRIME20 simulations provide a viable method to predict peptide aggregation propensities and aggregate structure/order which cannot be accessed by bioinformatics or readily attained experimentally

Discovery of AMG 925, a FLT3 and CDK4 Dual Kinase Inhibitor with Preferential Affinity for the Activated State of FLT3

We describe the structural optimization of a lead compound <b>1</b> that exhibits dual inhibitory activities against FLT3 and CDK4. A series of pyrido­[4′,3′:4,5]­pyrrolo­[2,3-<i>d</i>]­pyrimidine derivatives was synthesized, and SAR analysis, using cell-based assays, led to the discovery of <b>28</b> (<b>AMG 925</b>), a potent and orally bioavailable dual inhibitor of CDK4 and FLT3, including many FLT3 mutants reported to date. Compound <b>28</b> inhibits the proliferation of a panel of human tumor cell lines including Colo205 (Rb⁺) and U937 (FLT3^WT) and induced cell death in MOLM13 (FLT3^ITD) and even in MOLM13 (FLT3^ITD, D835Y), which exhibits resistance to a number of FLT3 inhibitors currently under clinical development. At well-tolerated doses, compound <b>28</b> leads to significant growth inhibition of MOLM13 xenografts in nude mice, and the activity correlates with inhibition of STAT5 and Rb phosphorylation