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Fragment-based design of small molecule PCSK9 inhibitors using simulated annealing of chemical potential simulations.

ABSTRACT: PCSK9 is a protein secreted by the liver that binds to the low-density lipoprotein receptor (LDLR), causing LDLR internalization, decreasing the clearance of circulating LDL particles. Mutations in PCSK9 that strengthen its interactions with LDLR result in familial hypercholesterolemia (FH) and early onset atherosclerosis, while nonsense mutations of PCSK9 result in cardio-protective hypocholesterolemia. These observations led to PCSK9 inhibition for cholesterol lowering becoming a high-interest therapeutic target, with antibody drugs reaching the market. An orally-available small molecule drug is highly desirable, but inhibiting the PCSK9/LDLR protein-protein interaction (PPI) has proven challenging. Alternate approaches to finding good lead candidates are needed. Motivated by the FH mutation data on PCSK9, we found that modeling the PCSK9/LDLR interface revealed extensive electron delocalization between and within the protein partners. Based on this, we hypothesized that compounds assembled from chemical fragments could achieve the affinity required to inhibit the PCSK9/LDLR PPI if they were selected to interact with PCSK9 in a way that, like LDLR, also involves significant fractional charge transfer to form partially covalent bonds. To identify such fragments, Simulated Annealing of Chemical Potential (SACP) fragment simulations were run on multiple PCSK9 structures, using optimized partial charges for the protein. We designed a small molecule, composed of several fragments, predicted to interact at two sites on the PCSK9. This compound inhibits the PPI with 1 ?M affinity. Further, we designed two similar small molecules where one allows charge delocalization though a linker and the other doesn't. The first inhibitor with charge delocalization enhances LDLR surface expression by 60% at 10 nM, two orders of magnitude more potent than the EGF domain of LDLR. The other enhances LDLR expression by only 50% at 1 ?M. This supports our conjecture that fragments can have surprisingly outsized efficacy in breaking PPI's by achieving fractional charge transfer leading to partially covalent bonding.

SUBMITTER: Guarnieri F

PROVIDER: S-EPMC6894869 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Fragment-based design of small molecule PCSK9 inhibitors using simulated annealing of chemical potential simulations.

Guarnieri Frank F Kulp John L JL Kulp John L JL Cloudsdale Ian S IS

PloS one 20191205 12

PCSK9 is a protein secreted by the liver that binds to the low-density lipoprotein receptor (LDLR), causing LDLR internalization, decreasing the clearance of circulating LDL particles. Mutations in PCSK9 that strengthen its interactions with LDLR result in familial hypercholesterolemia (FH) and early onset atherosclerosis, while nonsense mutations of PCSK9 result in cardio-protective hypocholesterolemia. These observations led to PCSK9 inhibition for cholesterol lowering becoming a high-interest ...[more]

PMID: 31805108

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Project description:The chemokine CXCL12 and its G protein-coupled receptor (GPCR) CXCR4 are high-priority clinical targets because of their involvement in metastatic cancers (also implicated in autoimmune disease and cardiovascular disease). Because chemokines interact with two distinct sites to bind and activate their receptors, both the GPCRs and chemokines are potential targets for small molecule inhibition. A number of chemokines have been validated as targets for drug development, but virtually all drug discovery efforts focus on the GPCRs. However, all CXCR4 receptor antagonists with the exception of MSX-122 have failed in clinical trials due to unmanageable toxicities, emphasizing the need for alternative strategies to interfere with CXCL12/CXCR4-guided metastatic homing. Although targeting the relatively featureless surface of CXCL12 was presumed to be challenging, focusing efforts at the sulfotyrosine (sY) binding pockets proved successful for procuring initial hits. Using a hybrid structure-based in silico/NMR screening strategy, we recently identified a ligand that occludes the receptor recognition site. From this initial hit, we designed a small fragment library containing only nine tetrazole derivatives using a fragment-based and bioisostere approach to target the sY binding sites of CXCL12. Compound binding modes and affinities were studied by 2D NMR spectroscopy, X-ray crystallography, molecular docking and cell-based functional assays. Our results demonstrate that the sY binding sites are conducive to the development of high affinity inhibitors with better ligand efficiency (LE) than typical protein-protein interaction inhibitors (LE ? 0.24). Our novel tetrazole-based fragment 18 was identified to bind the sY21 site with a K(d) of 24 ?M (LE = 0.30). Optimization of 18 yielded compound 25 which specifically inhibits CXCL12-induced migration with an improvement in potency over the initial hit 9. The fragment from this library that exhibited the highest affinity and ligand efficiency (11: K(d) = 13 ?M, LE = 0.33) may serve as a starting point for development of inhibitors targeting the sY12 site.

Dataset Information

Fragment-based design of small molecule PCSK9 inhibitors using simulated annealing of chemical potential simulations.

Publications

Fragment-based design of small molecule PCSK9 inhibitors using simulated annealing of chemical potential simulations.

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