Publications by authors named "Olga Ilyichova"

7 Publications

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Rapid Elaboration of Fragments into Leads by X-ray Crystallographic Screening of Parallel Chemical Libraries (REFiL).

J Med Chem 2020 07 24;63(13):6863-6875. Epub 2020 Jun 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.

A bottleneck in fragment-based lead development is the lack of systematic approaches to elaborate the initial fragment hits, which usually bind with low affinity to their target. Herein, we describe an analysis using X-ray crystallography of a diverse library of compounds prepared using microscale parallel synthesis. This approach yielded an 8-fold increase in affinity and detailed structural information for the resulting complex, providing an efficient and broadly applicable approach to early fragment development.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00111DOI Listing
July 2020

The Fragment-Based Development of a Benzofuran Hit as a New Class of DsbA Inhibitors.

Molecules 2019 Oct 18;24(20). Epub 2019 Oct 18.

Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia.

A fragment-based drug discovery approach was taken to target the thiol-disulfide oxidoreductase enzyme DsbA from (DsbA). This enzyme is critical for the correct folding of virulence factors in many pathogenic Gram-negative bacteria, and small molecule inhibitors can potentially be developed as anti-virulence compounds. Biophysical screening of a library of fragments identified several classes of fragments with affinity to DsbA. One hit with high mM affinity, 2-(6-bromobenzofuran-3-yl)acetic acid (), was chemically elaborated at several positions around the scaffold. X-ray crystal structures of the elaborated analogues showed binding in the hydrophobic binding groove adjacent to the catalytic disulfide bond of DsbA. Binding affinity was calculated based on NMR studies and compounds and were identified as the highest affinity binders with dissociation constants () of 326 ± 25 and 341 ± 57 µM respectively. This work suggests the potential to develop benzofuran fragments into a novel class of DsbA inhibitors.
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http://dx.doi.org/10.3390/molecules24203756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832960PMC
October 2019

KLK4 Inhibition by Cyclic and Acyclic Peptides: Structural and Dynamical Insights into Standard-Mechanism Protease Inhibitors.

Biochemistry 2019 05 13;58(21):2524-2533. Epub 2019 May 13.

Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute , Monash University , Clayton , Victoria 3800 , Australia.

Sunflower trypsin inhibitor (SFTI-1) is a 14 amino acid serine protease inhibitor. The dual antiparallel β-sheet arrangement of SFTI-1 is stabilized by an N-terminal-C-terminal backbone cyclization and a further disulfide bridge to form a final bicyclic structure. This constrained structure is further rigidified by an extensive network of internal hydrogen bonds. Thus, the structure of SFTI-1 in solution resembles the protease-bound structure, reducing the entropic penalty upon protease binding. When cleaved at the scissile bond, it is thought that the rigidifying features of SFTI-1 maintain its structure, allowing the scissile bond to be reformed. The lack of structural plasticity for SFTI-1 is proposed to favor initial protease binding and continued occupancy in the protease active site, resulting in an equilibrium between the cleaved and uncleaved inhibitor in the presence of a protease. We have determined, at 1.15 Å resolution, the X-ray crystal structures of complexes between human kallikrein-related peptidase 4 (KLK4) and SFTI-FCQR(Asn14) and between KLK4 and an acyclic form of the same inhibitor, SFTI-FCQR(Asn14)[1,14], with the latter displaying a cleaved scissile bond. Structural analysis and MD simulations together reveal the roles of the altered contact sequence, intramolecular hydrogen bonding network, and backbone cyclization in altering the state of SFTI's scissile bond ligation at the protease active site. Taken together, the data presented reveal insights into the role of dynamics in the standard-mechanism inhibition and suggest that modifications on the non-contact strand may be a useful, underexplored approach for generating further potent or selective SFTI-based inhibitors against members of the serine protease family.
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http://dx.doi.org/10.1021/acs.biochem.9b00191DOI Listing
May 2019

A ligand-induced structural change in fatty acid-binding protein 1 is associated with potentiation of peroxisome proliferator-activated receptor α agonists.

J Biol Chem 2019 03 31;294(10):3720-3734. Epub 2018 Dec 31.

From Medicinal Chemistry,

Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of lipid metabolism. GW7647 is a potent PPARα agonist that must reach the nucleus to activate this receptor. In cells expressing human fatty acid-binding protein 1 (FABP1), GW7647 treatment increases FABP1's nuclear localization and potentiates GW7647-mediated PPARα activation; GW7647 is less effective in cells that do not express FABP1. To elucidate the underlying mechanism, here we substituted residues in FABP1 known to dictate lipid signaling by other intracellular lipid-binding proteins. Substitutions of Lys-20 and Lys-31 to Ala in the FABP1 helical cap affected neither its nuclear localization nor PPARα activation. In contrast, Ala substitution of Lys-57, Glu-77, and Lys-96, located in the loops adjacent to the ligand-binding portal region, abolished both FABP1 nuclear localization and GW7647-induced PPARα activation but had little effect on GW7647-FABP1 binding affinity. Using solution NMR spectroscopy, we determined the WT FABP1 structure and analyzed the dynamics in the apo and GW7647-bound structures of both the WT and the K57A/E77A/K96A triple mutant. We found that GW7647 binding causes little change in the FABP1 backbone, but solvent exposes several residues in the loops around the portal region, including Lys-57, Glu-77, and Lys-96. These residues also become more solvent-exposed upon binding of FABP1 with the endogenous PPARα agonist oleic acid. Together with previous observations, our findings suggest that GW7647 binding stabilizes a FABP1 conformation that promotes its interaction with PPARα. We conclude that full PPARα agonist activity of GW7647 requires FABP1-dependent transport and nuclear localization processes.
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http://dx.doi.org/10.1074/jbc.RA118.006848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416440PMC
March 2019

Determination of ligand binding modes in weak protein-ligand complexes using sparse NMR data.

J Biomol NMR 2016 11 24;66(3):195-208. Epub 2016 Oct 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia.

We describe a general approach to determine the binding pose of small molecules in weakly bound protein-ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met CH assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-C,N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein-ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.
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http://dx.doi.org/10.1007/s10858-016-0067-4DOI Listing
November 2016

Direct and indirect mechanisms of KLK4 inhibition revealed by structure and dynamics.

Sci Rep 2016 10 21;6:35385. Epub 2016 Oct 21.

Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia.

The kallikrein-related peptidase (KLK) family of proteases is involved in many aspects of human health and disease. One member of this family, KLK4, has been implicated in cancer development and metastasis. Understanding mechanisms of inactivation are critical to developing selective KLK4 inhibitors. We have determined the X-ray crystal structures of KLK4 in complex with both sunflower trypsin inhibitor-1 (SFTI-1) and a rationally designed SFTI-1 derivative to atomic (~1 Å) resolution, as well as with bound nickel. These structures offer a structural rationalization for the potency and selectivity of these inhibitors, and together with MD simulation and computational analysis, reveal a dynamic pathway between the metal binding exosite and the active site, providing key details of a previously proposed allosteric mode of inhibition. Collectively, this work provides insight into both direct and indirect mechanisms of inhibition for KLK4 that have broad implications for the enzymology of the serine protease superfamily, and may potentially be exploited for the design of therapeutic inhibitors.
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http://dx.doi.org/10.1038/srep35385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5073354PMC
October 2016

Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA.

Angew Chem Int Ed Engl 2015 Feb 30;54(7):2179-84. Epub 2014 Dec 30.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052 (Australia) http://www.pharm.monash.edu.au.

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.
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http://dx.doi.org/10.1002/anie.201410341DOI Listing
February 2015