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[Preprint]. 2024 Aug 23:2024.08.23.609196.
doi: 10.1101/2024.08.23.609196.

Identifying novel chemical matter against the Chikungunya virus nsP3 macrodomain through crystallographic fragment screening

Jasmin C Aschenbrenner  1   2 , Andre Schutzer de Godoy  3 , Michael Fairhead  4 , Charles W E Tomlinson  1   2 , Max Winokan  1   2 , Blake H Balcomb  1   2 , Eda Capkin  1   2 , Anu V Chandran  1   2 , Mathew Golding  1   2 , Lizbe Koekemoer  4 , Ryan M Lithgo  1   2 , Peter G Marples  1   2 , Xiaomin Ni  4 , Warren Thompson  1   2 , Conor Wild  1   2 , Mary-Ann E Xavier  1   2 , Daren Fearon  1   2 , Frank von Delft  1   2   4   5
Affiliations

Identifying novel chemical matter against the Chikungunya virus nsP3 macrodomain through crystallographic fragment screening

Jasmin C Aschenbrenner et al. bioRxiv. .

Abstract

Chikungunya virus (CHIKV) causes severe fever, rash and debilitating joint pain that can last for months1,2or even years. Millions of people have been infected with CHIKV, mostly in low and middle-income countries, and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts. Its genome encodes a macrodomain, which functions as an ADP-ribosyl hydrolase, removing ADPr from viral and host-cell proteins interfering with the innate immune response. Mutational studies have shown that the CHIKV nsP3 macrodomain is necessary for viral replication, making it a potential target for the development of antiviral therapeutics. We, therefore, performed a high-throughput crystallographic fragment screen against the CHIKV nsP3 macrodomain, yielding 109 fragment hits covering the ADPr-binding site and two adjacent subsites that are absent in the homologous macrodomain of SARS-CoV-2 but may be present in other alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV). Finally, a subset of overlapping fragments was used to manually design three fragment merges covering the adenine and oxyanion subsites. The rich dataset of chemical matter and structural information discovered from this fragment screen is publicly available and can be used as a starting point for developing a CHIKV nsP3 macrodomain inhibitor.

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Figures

Figure 1.
Figure 1.. Structure of the ADP-ribose bound nsP3 macrodomain of CHIKV.
The ADPr binding site of CHIKV nsP3 macrodomain can be divided into smaller pockets according to the chemical groups and interactions found between ADPr and macrodomain into adenine site, oxyanion site, phosphate site and the (distal) ribose site, which is the catalytic site. The CHIKV nsP3 macrodomain structure (6VUQ) with bound ADPr (pale green) is shown in grey. Residues within 4 Å to the bound substrate are shown as sticks.
Figure 2.
Figure 2.. Crystallographic fragment screening of the CHIKV nsP3 macrodomain identified 109 fragments bound to the ADPr binding site and nearby subsites.
(A) Overview of fragments (yellow) bound to ADPr binding site and nearby subsites on the CHIKV nsP3 macrodomain (shown as surface representation). (B) Overview of the fragments screened, the number of processed datasets, the hits found from each library and the calculated hit rate (%). ‘Processed datasets’ refers to the datasets that could successfully be solved by Dimple and were subsequently analysed by PanDDA2. From the 1385 fragments screened, we obtained 1074 processable datasets. 311 datasets (22 %) were not obtained or analysed due to crystal pathologies.
Figure 3.
Figure 3.. Common scaffolds found in fragments binding to the adenine site of CHIKV nsP3 macrodomain.
The majority of the fragments found binding to the adenine site could be grouped into (A) amino benzimidazoles/benzothiazoles, (B) aminoquinolines/benzoxazoles, (C) pyrrolopyridines, (D) amides, (E) aminopyridine-like, (F) benzodioxoles/-dioxanes/-furans.
Figure 4.
Figure 4.. Fragments binding to the adenine site of CHIKV nsP3 macrodomain.
(A) Stick representation of ADPr (pale green) binding to the adenine site. (Bi-xi) Examples of representative fragments bound to the adenine site in CHIKV nsP3 macrodomain. The CHIKV nsP3 macrodomain structure is shown in grey. The bound fragment is shown with yellow sticks surrounded by a blue mesh generated from the PanDDA2 event map. Polar interactions are highlighted by black dashes.
Figure 5.
Figure 5.. Fragments binding to oxyanion site of CHIKV nsP3 macrodomain.
(A) Stick representation of ADPr (pale green) binding to the CHIKV nsP3 macrodomain with view of the water network mediating polar interactions to the proximal ribose of ADPr. (Bi-vi) Examples of representative fragments bound to the oxyanion site in CHIKV nsP3 macrodomain. The CHIKV nsP3 macrodomain structure is shown in grey. The bound fragment is shown with yellow sticks surrounded by a blue mesh generated from the PanDDA2 event map. Polar interactions and π-stacking interactions are highlighted by black and green dashes, respectively.
Figure 6.
Figure 6.. Fragments binding to phosphate site of CHIKV nsP3 macrodomain.
(A) Stick representation of ADPr (pale green) binding to the CHIKV nsP3 macrodomain with view of the water network mediating polar interactions to the diphosphate and the ribose of the adenosine. (Bi-vi) Examples of representative fragments bound to the phosphate site in CHIKV nsP3 macrodomain. The CHIKV nsP3 macrodomain structure is shown in grey. The bound fragment is shown with yellow sticks surrounded by a blue mesh generated from the PanDDA2 event map. Polar interactions and π-stacking interactions are highlighted by black and green dashes, respectively.
Figure 7.
Figure 7.. Fragments binding to ribose site of CHIKV nsP3 macrodomain.
(A) Stick representation of ADPr (pale green) binding to the CHIKV nsP3 macrodomain with view of the water network mediating polar interactions to the distal ribose of the ADPr. (Bi-vi) Examples of representative fragments bound to the ribose site in CHIKV nsP3 macrodomain. The CHIKV nsP3 macrodomain structure is shown in grey. The bound fragment is shown with yellow sticks surrounded by a blue mesh generated from the PanDDA2 event map. Polar interactions and π-stacking interactions are highlighted by black and green dashes, respectively.
Figure 8.
Figure 8.. Fragments binding to the Arg144 pocket of CHIKV nsP3 macrodomain.
(A) Stick representation of ADPr (pale green) binding to the CHIKV nsP3 macrodomain with view of Arg144 next to the adenine site. (B) The same view of ADPr (pale cyan) bound to the SARS-CoV-2 macrodomain which has a phenylalanine (F156) in place of the arginine residue (R144) of CHIKV. (Ci-vi) Examples of representative fragments bound to the Arg144 pocket in CHIKV nsP3 macrodomain. The CHIKV nsP3 macrodomain structure is shown in grey. The bound fragment is shown with yellow sticks surrounded by a blue mesh generated from the PanDDA2 event map. Polar interactions and π-stacking interactions are highlighted by black and green dashes, respectively.
Figure 9.
Figure 9.. Fragments binding to Trp148 subsite of CHIKV nsP3 macrodomain.
(A) Stick representation of ADPr (pale green) bound to the CHIKV nsP3 macrodomain with view of Trp148 below the oxyanion site. (B) The same view of ADPr (pale cyan) bound to the SARS-CoV-2 macrodomain which has a leucine (L160) in place of the tryptophane residue (W148) of CHIKV. (Ci-vi) Examples of representative fragments bound to the oxyanion site in CHIKV nsP3 macrodomain. The CHIKV nsP3 macrodomain structure is shown in grey. The bound fragment is shown with yellow sticks surrounded by a blue mesh generated from the PanDDA2 event map. Polar interactions and π-stacking interactions are highlighted by black and green dashes, respectively.
Figure 10.
Figure 10.. Manual fragment merges spanning the adenine and oxyanion subsite of CHIKV nsP3 macrodomain.
(A) Fragment merge based on fragments Z1162778919 (pale cyan) in the oxyanion hole and Z1262633486 (pale orange) binding to the adenine site. (B) Fragment merge created by merging fragments Z1162778919 (pale cyan) in the oxyanion hole and Z271099378 (pale green) binding to the adenine site. (C) Fragment merge derived from fragments Z1162778919 (pale cyan) in the oxyanion hole and Z104475374 (pale blue) binding to the adenine site.
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