Mechanistic and inhibition studies on a nucleophilic cysteine protease and transpeptidases

Enzymes with a nucleophilic cysteine residue catalyse important reactions in biology, including proteolysis, redox reactions, and radical transfer reactions. Nucleophilic cysteine enzymes are validated medicinal chemistry targets, e.g. thioredoxin reductase and glutathione reductase. The work presented in this thesis describes biochemical and inhibition studies on viral and bacterial nucleophilic cysteine enzymes. Chapters 2-6 focus on inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro), which is a therapeutic target to treat coronavirus disease 2019 (COVID-19). Chapter 7 describes studies focusing on the mycobacterial L, D-transpeptidases, which are investigational targets for tuberculosis treatment.
Antiviral therapies to treat infections caused by SARS-CoV-2 were lacking at the onset of the COVID-19 pandemic in December 2019. In March 2020, I initiated a collaborative research program in the Schofield group to identify efficient and selective inhibitors of SARS-CoV-2 Mpro, which is essential for viral replication and is structurally distinct from human proteases, making it a potential target for antiviral therapies. Note that at the end of 2021, a small molecule Mpro inhibitor, i.e. nirmatrelvir, was approved for clinical use in humans.
Initially, a mass spectrometry (MS)-based label-free SARS-CoV-2 Mpro assay was developed to investigate the effect of small molecules on catalysis and to identify efficient inhibitors. Screens of commercial small molecules and compounds available in the Schofield group revealed that β-lactams can be effective Mpro inhibitors, a finding of interest considering the established safety profiles of β-lactams. Structure-activity relationship (SAR) studies, in combination with MS and crystallographic studies, helped to improve the potency of the β-lactams as Mpro inhibitors and provided evidence for their covalent reaction with the active site cysteine. Apart from β-lactams, compounds containing reactive sulfur (e.g., ebsulfur derivatives), substrate-competitive linear peptides, and heterocyclic molecules (characterised as part of the COVID Moonshot Consortium) were identified as promising Mpro inhibitors.
Macrocyclic peptide Mpro inhibitors, which tightly bind the Mpro active site (e.g., TMGM4-05), were developed in collaboration with Prof. Suga’s group (University of Tokyo). SAR studies led to the identification of residues and intrapeptide interactions crucial for potent Mpro inhibition. Efforts to develop cell-permeable macrocyclic peptides are ongoing and are expected to enable their use as potential antivirals for the treatment of COVID-19.
In addition to the SARS-CoV-2 Mpro inhibition work, the research investigated the fragmentation of β-lactam antibiotics by mycobacterial L,D-transpeptidase nucleophilic cysteine enzymes, and analysed the extent to which these pathways resemble those catalysed by serine-β-lactamases and metallo-βlactamases. In the case of LdtMt2 and aminopenicillins, contrary to the expected hydrolytic mechanism, non-hydrolytic C5-C6 fragmentation and cyclisation via rearrangement to give a diketopiperazine were observed. Faropenem undergoes C5-C6 fragmentation on reaction with LdtMts, whereas an intact hydrolysed product is formed upon reaction of faropenem with metallo-β-lactamases (MBLs) and serine-β-lactamases. Interestingly, a degradation product in which the faropenem tetrahydrofuran ring is opened is observed with serine-β-lactamases; the THF ring remains closed in the cases of the metallo-β-lactamases, VIM-1 and VIM-2, but not NDM-1 and L1.
In summary, the results have identified new types of inhibitors of nucleophilic cysteine enzymes and have provided insight into their mechanism of action.

Keywords: mass spec
NMR
COVID-19
assay development
tuberculosis
antivirals
penicillins
Mpro
macrocyclic peptides
antibiotics
LdtMt2
beta-lactams
TB
RapidFire
Advisor: Schofield, C
Lohans, C