The COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is considered the greatest challenge to the global health community of the century as it continues to expand. This has prompted immediate urgency to discover promising drug targets for the treatment of COVID-19. The SARS-CoV-2 viral proteases, 3-chymotrypsin-like protease (3CLpro) and papain-like cysteine protease (PLpro), have become the promising target to study due to their essential functions in spreading the virus by RNA transcription, translation, protein synthesis, processing and modification, virus replication, and infection of the host. As such, understanding of the structure and function of these two proteases is unavoidable as platforms for the development of inhibitors targeting this protein which further arrest the infection and spread of the virus. While the abundance of reports on the screening of natural compounds such as SARS-CoV-2 proteases inhibitors are available, the microorganisms-based compounds (peptides and non-peptides) remain less studied. Indeed, microorganisms-based compounds are also one of the potent antiviral candidates against COVID-19. Microbes, especially bacteria and fungi, are other resources to produce new drugs as well as nucleosides, nucleotides, and nucleic acids. Thus, we have compiled various reported literature in detail on the structures, functions of the SARS-CoV-2 proteases, and potential inhibitors from microbial sources as assistance to other researchers working with COVID-19. The compounds are also compared to HIV protease inhibitors which suggested the microorganisms-based compounds are advantageous as SARS-CoV2 proteases inhibitors. The information should serve as a platform for further development of COVID-19 drug design strategies.

The SARS-CoV-2 coronavirus expresses two essential proteases: firstly, the 3Chymotrypsin-like protease (3CLpro) or main protease (Mpro), and secondly, the papain-like protease (PLpro), both of which are considered as viable drug targets for the inhibition of viral replication. In order to perform drug discovery assays for SARS-CoV-2, it is imperative that efficient methods are established for the production and purification of 3CLpro and PLpro of SARS-CoV-2, designated as 3CLpro-CoV2 and PLpro-CoV2, respectively. This article expands the data collected in the attempts to express SARS-CoV-2 proteases under different conditions and purify them under single-step chromatography. Data showed that the use of E. coli BL21(DE3) strain was sufficient to express 3CLpro-CoV2 in a fully soluble form. Nevertheless, the single affinity chromatography step was only applicable for 3CLpro-CoV2 expressed at 18 °C, with a yield and purification fold of 92% and 49, respectively. Meanwhile, PLpro-CoV2 was successfully expressed in a fully soluble form in either BL21(DE3) or BL21-CodonPlus(DE3) strains. In contrast, the single affinity chromatography step was only applicable for PLpro-CoV2 expressed using E. coli BL21-CodonPlus(DE3) at 18 or 37 °C, with a yield and purification fold of 86% (18 °C) or 83.36% (37 °C) and 112 (18 °C) or 71 (37 °C), respectively. The findings provide a guide for optimizing the production of SARS-CoV-2 proteases of E. coli host cells.

Bromelain is a unique enzyme-based bioactive complex containing a mixture of cysteine proteases specifically found in the stems and fruits of pineapple (Ananas comosus) with a wide range of applications. MD2 pineapple harbors a gene encoding a small bromelain cysteine protease with the size of about 19 kDa, which might possess unique properties compared to the other cysteine protease bromelain. This study aims to determine the expressibility and catalytic properties of small-sized (19 kDa) bromelain from MD2 pineapple (MD2-SBro). Accordingly, the gene encoding MD2-SBro was firstly optimized in its codon profile, synthesized, and inserted into the pGS-21a vector. The insolubly expressed MD2-SBro was then resolubilized and refolded using urea treatment, followed by purification by glutathione S-transferase (GST) affinity chromatography, yielding 14 mg of pure MD2-SBro from 1 L of culture. The specific activity and catalytic efficiency (k_cat/K_m) of MD2-SBro were 3.56 ± 0.08 U mg⁻¹ and 4.75 ± 0.23 × 10⁻³ µM⁻¹ s⁻¹, respectively, where optimally active at 50 °C and pH 8.0, and modulated by divalent ions. The MD2-SBro also exhibited the ability to scavenge the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) with an IC₅₀ of 0.022 mg mL⁻¹. Altogether, this study provides the production feasibility of active and functional MD2-Bro as a bioactive compound.

The caseinolytic protease (Clp) system plays an essential role in the protein homeostasis of the malaria parasite, particularly at the stage of apicoplast development. The inhibition of this protein is known to have a lethal effect on the parasite and is therefore considered an interesting avenue for antimalaria drugs discovery. The catalytic activity of the Clp system is modulated by its proteolytic subunit (ClpP), which belongs to the serine protease family member and is therefore extensively studied for further inhibitors development. Among many inhibitors, the group of β-lactone is known to be a specific inhibitor for ClpP. Nevertheless, other groups of lactones have never been studied. This study aims to characterize the catalytic properties of ClpP of Plasmodium knowlesi (Pk-ClpP) and the inhibition properties of a δ-lactone hyptolide against this protein. Accordingly, a codon-optimized synthetic gene encoding Pk-ClpP was expressed in Escherichia coli BL21(DE3) and purified under a single step of Ni²⁺-affinity chromatography, yielding a 2.20 mg from 1 L culture. Meanwhile, size-exclusion chromatography indicated that Pk-ClpP migrated primarily as homoheptameric with a size of 205 kDa. The specific activity of pure Pk-ClpP was 0.73 U µg⁻¹, with a catalytic efficiency k_cat/K_M of 0.05 µM⁻¹ s⁻¹, with optimum temperature and pH of 50 °C and 7.0–7.5, respectively. Interestingly, hyptolide, a member of δ-lactone, was shown to inhibit Pk-ClpP with an IC₅₀ value of 17.36 ± 1.44 nM. Structural homology modelling, secondary structure prediction, and far-UV CD spectra revealed that helical structures dominate this protein. In addition, the structural homology modeling showed that this protein forms a barrel-shaped homoheptamer. Docking simulation revealed that the inhibition was found to be a competitive inhibition in which hyptolide was able to dock into the catalytic site and block the substrate. The competitiveness of hyptolide is due to the higher binding affinity of this molecule than the substrate.

Proteolytic subunit of the caseinolytic protease system of Plasmodium knowlesi (Pk-ClpP; EC 3.4.21.92) is considered a viable target for antimalarial drug development to eradicate P. knowlesi malaria infection in Malaysia and Southeast Asian region. Inhibition of this system leads to a disruption in the protein homeostasis molecular machinery and therefore be lethal for the parasite. While plants are considered excellent sources of bioactive compounds exhibiting inhibition activity towards Pk-ClpP, many local medicinal plants remain unexplored. This article expands the data collected from the inhibition properties of the methanolic extract of Asystasia gangetica (Chinese Violet), Alstonia scholaris (Pulai Tree), Piper retrofractum (Javanese Long Pepper) and Smallanthus sonchifolius (Yacon) towards Pk-ClpP. These plants are widely found in Malaysia and Indonesia and have been traditionally used in various medical treatments. The present dataset showed that the extracts contained phenolic and flavonoid compounds in various concentrations, whereby S. sonchifolius was found to have the lowest content of phenolic and flavonoid contents, while A. gangetica and A. scholaris were statistically comparable, yet higher than P. retrofactum and S. sonchifolus. Further inhibition data assay towards Pk-ClpP revealed that A. gangetica, A. scholaris and P. retrofactum demonstrated remarkable inhibition activity with IC₅₀ values of 39.06 ± 1.98, 48.92 ± 1.52, and 87.63 ± 3.55, respectively. However, the inhibition activity of these extracts was significantly lower than a serine protease inhibitor of phenylmethylsulfonyl fluoridenone (PMSF). Meanwhile, S. sonchifolus did not exhibit significant inhibition activity towards Pk-ClpP. In addition, Pk-ClpP was not inhibited by a cysteine protease inhibitor of E64.

The spike glycoprotein (S protein), 3-chymotrypsin-like protease (3CL-Pro), and papain-like protease (PL-Pro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus are widely targeted for the discovery of therapeutic compounds against this virus. Dietary flavonoid compounds were proposed as a candidate for safe therapy for COVID-19 patients. Nevertheless, wet lab experiments for high-throughput screening of the compounds are undoubtedly time and cost consuming. This study aims to screen dietary flavonoid compounds that bind to S protein, 3CL-Pro, and PL-Pro of SARS-CoV-2. For this purpose, protein structures of the receptor-binding domain (RBD) of S protein (6M0J), 3CL-Pro (6LU7), and PL-Pro (6W9C) were retrieved from the RCSB Protein Data Bank (PDB). Twelve dietary flavonoid compounds were selected for the studies on their binding affinity to the targeted proteins by global and local docking. The docking and molecular dynamic (MD) simulations were performed using YASARA software. Out of 12 compounds, the highest binding score was observed between hesperidin against RBD S protein (−9.98 kcal/mol), 3CL-Pro (−9.43 kcal/mol), and PL-Pro (−8.89 kcal/mol) in global docking. Interestingly, MD simulation revealed that the complex between 3CL-Pro and RBD S protein has better stability than PL-Pro. This study suggests that hesperidin might have versatile inhibitory properties against several essential proteins of SARS-CoV-2. This study, nevertheless, remains to be confirmed through in vitro and in vivo assays.