Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

Abstract

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

Fig. 1. The crystal structure of ligand free M^pro is amenable to X-ray fragment screening.

a Cartoon representation of the M^pro dimer. The nearmost monomer is shown with secondary structure features coloured to demarcate domains I, II, and III, in orange, cyan, and violet respectively. The active site of the rear monomer is indicated by the presence of a peptide-based inhibitor in green, generated by aligning the ligand-free structure with pdb 6Y2F [10.2210/pdb6y2f/pdb]. A yellow sphere indicates Ser1 from the dimer partner that completes the active site. b Residues of the active site are labelled, and subsites involved in ligand binding are shown with circles. c Active site plasticity is observed when comparing the apo structure to peptide inhibitor bound structures (green—Apo, grey—6Y2F [10.2210/pdb6y2f/pdb], pink 6LU7 [10.2210/pdb6lu7/pdb]). Displacement distances associated with loop movements are indicated.

Fig. 2. Timeline of crystallographic fragment screen.

Progress of the Mpro fragment screening experiment from the start of protein production and purification (9 Feb 2020) to the deposition and release of the high-resolution ligand-free structure of Mpro PDB ID 6YB7[10.2210/pdb6yb7/pdb] and the structures of the 96 fragment hits identified in the fragment screening campaign using the XChem platform at Diamond Light Source.

Fig. 3. Bound fragments sample the active site comprehensively.

The central surface representation is of the M^pro monomer with all fragment hits shown as sticks, and active site subsites highlighted by coloured boxes. Each subsite is expanded along with a selection of hits to demonstrate common features and interactions. S1: Z44592329 (x0434); S1′: Z369936976 (×0397) in aquamarine and PCM-0102372 (×1311) in magenta bound to active site cysteine; S2: Z1220452176 (x0104); S3: Overlay of Z18197050 (×0161), Z1367324110 (×0195) and NCL-00023830 (×0946).

Fig. 4. Plasticity of S1´ is revealed by fragment Z369936976 (×0397).

Comparing the electrostatic surfaces of Z1129283193 (×0107) a The most commonly observed conformation, with that of Z369936976 (x0397). b How the shape of S1 and S1´ can change. c Sidechain movement of catalytic residues Cys145 and His41 upon binding of Z369936976 (×0397, magenta) compared to Z1129283193 (×0197, grey).

Fig. 5. Fragments at dimer interface indicate opportunities for allosteric modulation.

The overview shows the surface of the M^pro dimer, the protomers in grey and cyan. Fragments and surrounding residues are shown as sticks and hydrogen bonds in dashed black lines. a Z1849009686 (×1086). b Z264347221 (×1187). c POB0073 (×0887).

Fig. 6. Covalent fragments are anchored at Cys145 and sample different regions of the orthosteric M^pro binding pocket.

a Fragments containing N-chloroacetyl piperidinyl-4-carboxamide motif. b Fragments containing N-chloroacetyl-N´-sulfonamido-piperazine motif. c Fragments containing N-chloroacetyl-N´-carboxamido- and N-chloroacetyl-N´-heterobenzyl-piperazine in two binding modes. The second order kinetic constants refer to the intrinsic thiol reactivity of these fragment hits as previously measured. d Reaction schema of the unexpected covalent modification to Cys145 by PepLites hits. e Threonine PepLite (NCL-00025058 (x0978)) bound covalently to active site cysteine. f Asparagine PepLite (NCL-00025412 (x0981)) bound to active site cysteine. Labelling of M^pro by 2nd generation compounds proven by intact protein LC-MS: g Labelling by PG-COV-35; h Labelling by PG-COV-34. Covalently bound cyclic electrophiles: i Cov_HetLib 030 (×2097) and j Cov_HetLib 053 (×2119).

Fig. 7. Fragment merging opportunities can be directly inferred from many hits.

Covalently bound fragments are in green shades, and non-covalent fragments in yellow. a Overlay of Z509756472/×1249 and PCM-0102269/×0770. b Overlay of PCM-0102277/x1334 and PCM-0102269/×0770. c Overlay of PCM-0102287/×0830 and Z219104216/×0305. d Overlay of PCM-0102340/×0692, PCM-0102277/×1334 and Z219104216/×0305.