An efficient treatment against a COVID-19 disease, caused by the novel coronavirus SARS-CoV-2 (CoV2), remains a challenge. The papain-like protease (PLpro) from the human coronavirus is a protease that plays a critical role in virus replication. Moreover, CoV2 uses this enzyme to modulate the host’s immune system to its own benefit. Therefore, it represents a highly promising target for the development of antiviral drugs. We used Approximate Bayesian Computation tools, molecular modelling and enzyme activity studies to identify highly active inhibitors of the PLpro. We discovered organoselenium compounds, ebselen and its structural analogues, as a novel approach for inhibiting the activity of PLproCoV2. Furthermore, we identified, for the first time, inhibitors of PLproCoV2 showing potency in the nanomolar range. Moreover, we found a difference between PLpro from SARS and CoV2 that can be correlated with the diverse dynamics of their replication, and, putatively to disease progression.
Since December 2019 a novel a coronavirus identified as SARS-CoV-2 or COV2 has been spreading around the world. On the 16th of May around 4.5 million people got infected and over 300,000 died due to the infection of COV2. The effective treatment remains a challenge. Targeted therapeutics are still under investigation. The papain-like protease (PL Pro ) from the human SARS-CoV-2 coronavirus is a cysteine protease that plays a critical role in virus replication. Its activity is required to process the viral polyprotein into functional, mature subunits. Moreover, COV2 uses this enzyme to modulate the host's immune system to its own benefit. Therefore, it represents a highly promising target for the development of antiviral drugs. In this work, we discovered that ebselen, a synthetic organoselenium drug molecule with anti-inflammatory, anti-oxidant and cytoprotective activity in mammalian cells and cytotoxicity in lower organisms, is a highly active inhibitor of PL Pro CoV2. We proved that ebselen is a covalent, fast-binding inhibitor of PL Pro CoV2 exhibiting a low micromolar potency. Furthermore, we identified a difference between PL Pro from SARS-CoV-1 (the corona virus which caused the 2002-2004 outbreak, SARS) and SARS-CoV-2 that allows to explain the difference in dynamics of the replication, and, thus, the disease progression. Namely, we present that they show differences in the binding affinity of substrates that we observed through kinetics and molecular docking studies. Using a novel Approximate Bayesian Computation method we were able to find kinetic constants for both enzymes. Molecular modeling study on the structure of the active site and binding mode of the ebselen with SARS and COV2 showed also significant differences that could explain our observation that ebselen is less active and slower bounding with SARS than COV2. In conclusion, we show that ebselen inhibits the activity of the essential viral enzyme papain-like protease (PLpro) from SARS-COV-2 in low micromolar range.
Seven crystal structures of alanyl
aminopeptidase from Neisseria meningitides (the etiological
agent of meningitis, NmAPN) complexed with organophosphorus
compounds were resolved
to determine the optimal inhibitor–enzyme interactions. The
enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond
to the P1 amino acid residues of well-processed substrates, were used
to assess the impact of the absolute configuration and the stereospecific
hydrogen bond network formed between the aminophosphonate polar head
and the active site residues on the binding affinity. For the hPhe
analog, an imperfect stereochemical complementarity could be overcome
by incorporating an appropriate P1 side chain. The constitution of
P1′-extended structures was rationally designed and the lead,
phosphinic dipeptide hPhePψ[CH2]Phe, was modified
in a single position. Introducing a heteroatom/heteroatom-based fragment
to either the P1 or P1′ residue required new synthetic pathways.
The compounds in the refined structure were low nanomolar and subnanomolar
inhibitors of N. meningitides, porcine and human
APNs, and the reference leucine aminopeptidase (LAP). The unnatural
phosphinic dipeptide analogs exhibited a high affinity for monozinc
APNs associated with a reasonable selectivity versus dizinc LAP. Another
set of crystal structures containing the NmAPN dipeptide
ligand were used to verify and to confirm the predicted binding modes;
furthermore, novel contacts, which were promising for inhibitor development,
were identified, including a π–π stacking interaction
between a pyridine ring and Tyr372.
Neisseria meningitides is a gram-negative diplococcus bacterium and is the main causative agent of meningitis and other meningococcal diseases. Alanine aminopeptidase from N. meningitides (NmAPN) belongs to the family of metallo-exopeptidase enzymes, which catalyze the removal of amino acids from the N-terminus of peptides and proteins, and are found among all the kingdoms of life. NmAPN is suggested to be mostly responsible for proteolysis and nutrition delivery, similar to the orthologs from other bacteria.
To explore the possibility of NmAPN being a potential drug target for inhibition and development of novel therapeutic agents, the specificity of the S1 and S1′ binding sites were explored using an integrated approach. Initially, an extensive library consisting of almost 100 fluorogenic substrates derived from both natural and unnatural amino acids, were used to obtain a detailed substrate fingerprint of the S1 pocket of NmAPN. A broad substrate tolerance of NmAPN was revealed, with bulky basic and hydrophobic ligands being the most favored substrates. Additionally, the potency of a set of organophosphorus inhibitors of neutral aminopeptidases, amino acid and dipeptide analogues was determined. Inhibition constants in the nanomolar range, determined for phosphinic dipeptides, proves the positive increase in inhibition impact of the P1′ ligand elongation. The results were further verified via molecular modeling and docking of canonical aminopeptidase phosphinic dipeptide inhibitors in the NmAPN active site. These studies present comprehensive characterization of interactions responsible for specific ligand binding. This knowledge provides invaluable insight into understanding of the enzyme and development of novel NmAPN inhibitors.
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