Biology subjects

Marples, P. G.

Publications and source records attributed to Marples, P. G..

5 recordsLinked to original sources

bioRxiv2024

Crystallographic fragment screen of Enterovirus D68 3C protease and iterative design of lead-like compounds using structure-guided expansions

The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics. One cause for concern is the currently emerging pathogen Enterovirus D68 (EV-D68) which primarily spreads through respiratory routes causing mostly mild to severe respiratory illness but, in severe cases, acute flaccid myelitis. The 3C protease of EV-D68 (3Cpro) is a potential target for the development of antiviral drugs due to its essential role in the viral life cycle and high sequence conservation amongst family members. In this study, we describe the identification of fragments which bind to3Cpro using crystallographic screening and the expansion of these into more lead-like compounds. The hits revealed interesting directions for hit-to-lead progression, specifically the importance of the pocket occupied by the conserved glutamine sidechain of the substrates and the interactions formed. Additionally, two pockets could be joined by not following the backbone of the native substrates, thus circumventing the screening issues arising from the flexibility of the catalytic triad. These observations of the novel binding modes of the chemical matter found by this screen can help shape future drug design campaigns against 3C proteases.

biochemistry↗

bioRxiv2024

High-throughput crystallographic fragment screening of Zika virus NS3 Helicase

The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barre syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. Here we applied high-throughput crystallographic fragment screening on ZIKV NS3Hel, which yielded structures that reveal 3D binding poses of 46 fragments at multiple sites of the protein, including 11 unique fragments in the RNA-cleft site. These fragment structures provide templates for direct design of hit compounds and should thus assist the development of novel direct-acting antivirals against ZIKV and related flaviviruses, thus opening a promising avenue for combating future outbreaks.

biophysics↗

bioRxiv2024

Crystallographic fragment screening delivers diverse chemical scaffolds for Zika virus NS2B-NS3 protease inhibitor development

The Zika viral protease NS2B-NS3 is essential for the cleavage of viral polyprotein precursor into individual structural and non-structural (NS) proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 47 fragments with diverse scaffolds were identified to bind in the active site of the protease, with another 6 fragments observed in a potential allosteric site. To identify binding sites that are intolerant to mutation and thus suppress the outgrowth of viruses resistant to inhibitors developed from bound fragments, we performed deep mutational scanning of NS2B-NS3 protease. Merging fragment hits yields an extensive set of mergers, defined as synthetically accessible compounds that recapitulate constellations of observed fragment-protein interactions. In addition, the highly sociable fragment hits enable rapid exploration of chemical space via algorithmic calculation and thus yield diverse possible starting points that maximally explore the binding opportunities to NS2B-NS3 protease, facilitating its resistance-resilient antiviral development.

biochemistry↗

bioRxiv2024

Crystallographic Fragment Screen of Coxsackievirus A16 2A Protease identifies new opportunities for the development of broad-spectrum anti-enterovirals

Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of these viruses is responsible for the self-cleavage of the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. These 2A proteases are highly conserved between Enterovirus species, such as Enterovirus A71 and Coxsackievirus A16. Inhibition of the 2A protease deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. Herein, we describe a crystallographic fragment screening campaign that identified 75 fragments which bind to the 2A protease including 38 unique compounds shown to bind within the active site. These fragments reveal a path for the development of non-peptidomimetic inhibitors of the 2A protease with broad-spectrum anti-enteroviral activity.

biochemistry↗

bioRxiv2023

Targeting LC3/GABARAP for degrader development and autophagy modulation

Recent successes in developing small-molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the target recruitment to LC3/GABARAP, a family of membrane-bound proteins that tether autophagy receptors to the autophagosome. In order to validate the existing ligands, we rigorously tested target engagement of reported ATTEC ligands and handles. Surprisingly, using various biophysical methods, most available ligands did not interact with their designated target LC3. Intrigued by the idea of developing ATTECs, we evaluated the druggability of LC3/GABARAP by in silico docking and large scale crystallographic fragment screening. The data revealed that most fragments bound to the HP2, but not the HP1 pocket of the LC3-interacting region (LIR) docking site, suggesting favorable druggability of this binding pocket. Here, we present diverse comprehensively validated ligands for future ATTEC development.

biochemistry↗