Cysteine-related Libraries for Anti-coronavirus Research

Expert-driven In Silico Drug Discovery Solutions
9 May 2020
Andrew Golub
Group Leader, Molecular Design

The mechanism of action of targeted covalent inhibitors implies the covalent bond formation with nucleophilic groups of proteins, which in most cases are cysteine residues. The traditional design approach to cysteine targeting covalent inhibitors consists in the introduction of Michael acceptors (Michael addition) into a ligand with already known high binding affinity. Besides that, there are other nucleophilic addition, addition-elimination, nucleophilic substitution, and oxidation reactions that are appropriate for specific covalent alterations of proteins. The covalent inhibitors specificity is crucial and requires careful optimization of the incorporated warheads, especially taking into account that cysteine residues are abundant in the proteome.

It is well known that cysteine proteases are able to react with a variety of electrophilic (or “warhead”) moieties within a covalent inhibitor. These warhead-containing molecules usually form a noncovalent interaction complex first within an active site of cysteine protease. At that, the warhead group of the inhibitor is located in the close vicinity of the reactive cysteine nucleophilic group. Then a covalently modified enzyme–inhibitor complex forms via a nucleophilic attack of the thiolate on the electrophilic carbon of the warhead group. This reaction subsequently inactivates the protease enzyme. Some of known reactive warhead groups that inhibit cysteine proteases include Michael acceptors, aldehydes, activated ketones, epoxy-ketones, activated esters, vinyl sulfones, acrylamides, alkylhalides, nitriles, and alkynes.

One of the best-characterized drug targets among coronaviruses, including current SARS-CoV2causing coronavirus disease 2019 (COVID-19), is the main protease (3CLpro, or Mpro). Along with the papain-like protease (PLpro) this enzyme is essential for processing the polyproteins that are translated from the viral RNA. Both enzymes belong to a class of cysteine proteases. They play a crucial role in the coronaviral life cycle and, hence, are attractive antiviral drug targets. Inhibiting the activity of these enzymes would block viral replication. It is worth pointing out that such inhibitors are unlikely to be toxic since no human proteases with a similar cleavage specificity are known.

Recently, the crystal structure of COVID-19 virus main protease in complex with an inhibitor N3 has been resolved (PDB ID: 6LU7). The N3 is a warhead-containing compound that covalently binds to Cys145 in the 3CLpro active site (Fig. 1). This X-ray data is a basis for the rational design of the anti-COVID-19 drugs targeting cysteine protease.

Our efforts to contribute to fighting the COVID-19 resulted in a number of compound libraries, including those specifically focused on cysteine:

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Fig. 1. The crystal structure of COVID-19 virus Mpro in complex with N3. a) Cartoon representation of one protomer of the dimeric Mpro-inhibitor complex. b) Surface representation of the homodimer of Mpro. Protomer A is in blue, protomer B is in salmon, N3 is presented as green sticks. c) A zoomed view of the substrate-binding pocket. The key residues forming the binding pocket are shown in sticks, the two water molecules, assigned as W1 and W2, are shown as red spheres. P1, P1′, P2, P3, P4, and P5 sites of N3 are indicated. Hydrogen bonds that help to lock the inhibitor are shown in black dashed lines. d) The C-S covalent bond.

9 May 2020, 10:03 Andrew Golub Computational Chemistry

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