The development of papain‐like protease from SARS‐CoV‐2, a potential drug target for antiviral screening: A review

The SARS‐CoV‐2 outbreak caused a global pandemic, claiming numerous lives and becoming this century’s most widespread life‐threatening disease. The virus relies on two specific enzymes to facilitate replication, 3‐chymotrypsin‐like protease (3CLPro) and papain‐like protease (PLpro). These enzymes are crucial in breaking down nonstructural polypeptides into functional proteins. PLpro with LXGG↓X recognition and cleavage sites also play a role in deubiquitylase (DUB) and delSGylase by cleaving after the double glycine residue of ubiquitin (Ub) and ISG15 as a mechanism to suppress the host’s innate immune response. Despite its important role in the viral infection cycle and the potential for drug discovery, no antivirals have been approved as PLpro inhibitors. Therefore, this review focuses on PLpro protein, its recombinant product development and purification, and its application as a protein target in drug discovery for COVID‐19 screening to develop effective COVID‐19 drugs.


Introduction
In December 2019, a new type of coronavirus called Se vere Acute Respiratory Syndrome Coronavirus 2 (SARS CoV2) was discovered in Wuhan, China (Wang et al. 2020).In the past, certain strains of Coronaviruses have led to worldwide epidemics, including MERSCoV in 2012and SARSCoV in 2002(Goldsmith et al. 2004; de Groot et al. 2013; Lu et al. 2020).SARSCoV2 is a virus with a crownlike shape that causes influenza like illness, highly pathogenic, and widespread in vari ous countries (Cao 2020; Hartenian et al. 2020; Kakod kar et al. 2020; Naqvi et al. 2020).According to the World Health Organization (WHO), as of February 21, 2023, the confirmed numbered of COVID19 cases glob ally had reached around 757 million, with approximately 6.85 million reported deaths attributable to COVID19 (WHO 2023).
The primary way that SARSCoV2 spreads is by res piratory droplets that are released when an infected per son talks, coughs, or sneezes.Droplets containing the virus may enter people's noses or mouths, resulting in COVID19 infection.Additionally, COVID19 can be spread through contact with surfaces contaminated with the virus, followed by touching one's mouth, nose, or eyes (Harrison et al. 2020; Rando et al. 2021; Sachs et al. 2022).
The SARSCoV2 genome RNA contains information to produce two proteases, PLpro and 3CLpro (Hartenian et al. 2020).Those proteases enable viral protein matu ration through the cleavage of the nonstructural proteins (nsp) (Amin et al. 2021; Kuo et al. 2021; Yu et al. 2022).PLpro, with a catalytic triad of C111, H272, and D286, has three cleavage sites for cleaving and maturing proteins nsp 1, nsp 2, and nsp 3 (Armstrong et al. 2021; Razali et al. 2021) (Figure 1).On the other hand, 3CLpro, with cat alytic dyads H41 and C145, cuts more with 11 cleavage sites for cleaving and maturing proteins nsp 4 to nsp 16 (Bera et al. 2021; Razali et al. 2021).Interestingly, unlike 3CLpro, which only plays a role in cleaving and maturing nsp, PLpro with LXGG↓X recognition sequence, where X is any amino acid, affects the host's immune response by regulating the removal of a molecule called ISG15 through deubiquitylation.This mechanism will prevent the acti vation of signals from pattern recognition receptors for triggering the host's immune response to viruses (Freitas et al. 2020; Shin et al. 2020; Amin et al. 2021; Lim et al. 2021).However, no antiviral has been used clinically with FIGURE 1 The genome RNA structure of the SARS-CoV-2 virus and PLpro structure (Hartenian et al. 2020;Armstrong et al. 2021;Razali et al. 2021).
a mechanism as a PLpro inhibitor.This review discusses the development of PLpro recombinant protein as a target receptor and its applications in antiviral screening.

SARS-CoV-2 structure
The coronavirus group contains viruses that can infect an imals and humans, such as SARSCoV and MERSCoV.In humans, these viruses are capable to causing respira tory infections that range in severity from mild to severe (Hu et al. 2021).Until now, there have been three out breaks due to coronavirus, namely SARS, MERS, and a most recently COVID19 (Dlamini et al. 2020).Lock downs were imposed in nearly every country in response to the COVID19 pandemic (Zheng 2020; Onyeaka et al. 2021).
The scientific community classifies SARSCoV2 as a member of the Betacoronavirus genus, which belongs to the Coronaviridae family and the Coronavirinae subfam ily (Hartenian et al. 2020).According to the Baltimore classification, SARSCoV2 belongs to group IV, char acterized by a singlestranded RNA genome with a rela tively extensive size of approximately 30 kb (Ryu 2017; Kim et al. 2020).It is composed of a singlestranded RNA genome and four major structural proteins, i.e., the trimeric spike glycoprotein (S), nucleocapsid (N), mem brane protein (M), and small envelope membrane protein (E) (Yao et al. 2020; Rando et al. 2021) (Figure 2).The genomic RNA coding for structural, nonstructural, and ac cessory proteins plays a critical role in cellular viral reg ulation, including viral infection and replication of new virion (Kim et al. 2020; Naqvi et al. 2020; Osipiuk et al. 2021; Zhang et al. 2022).
Spike glycoprotein (S) will recognize and attach to the angiotensinconverting enzyme 2 (ACE2) receptor on the surface of human cells.This binding event is the entry point for the virus to penetrate and infect the host (Harrison et al. 2020; Lan et al. 2020; Yao et al. 2020).The N pro tein protects the viral genome from damage and is packed into a ribonucleoprotein complex during virion assembly (Zeng et al. 2020; Bai et al. 2021).The M protein is a com ponent of the viral envelope, essential in several aspects of viral assembly.In particular, it aids in binding other struc tural proteins, stabilizes the nucleocapsid, and facilitates the final stages of assembly (Zeng et al. 2020; Bai et al. 2021).Small envelope membrane protein (E) is the small est structural protein that releases the viral genome, viral assembly, budding, and cell lysis (Harrison et al. 2020; Yao et al. 2020).Due to the presence of lipids and proteins on the surface of SARSCoV2, the virus is susceptible to elimination through the application of detergent, whether it is present on the human body or various surfaces (Ryu 2017; Hardison et al. 2022).
The SARSCoV2 genome sequence shares a signifi cant amount of similarity with the SARSCoV genome se FIGURE 2 The arrangement of the SARS-CoV-2 virus (Yao et al. 2020;Rando et al. 2021).
quence (ranging from 79% to 96%), and with the MERS CoV genome sequence, with a similarity of 50% (Yang et al. 2020; Hu et al. 2021).As a result, the amino acid composition and structure of protein SARSCoV2 are comparable to those of SARSCoV, with both viruses hav ing an open reading frame (ORF) arranged in the order of 5′cap to 3′poly(A) tail, namely ORF1ab, which encodes 16 nsp and four structural proteins (Yang et al. 2020; Hu et al. 2021).Nonetheless, there is little homology between the spike (S) protein, ORF8, ORF3b, and ORF10 genome sequences (Yang et al. 2020).While both SARSCoV and SARSCoV2 recognize and bind to the ACE2 receptor, their S1 subunits differ, resulting in a difference in the lo cation of the receptor binding domain (RBD) (Hu et al. 2020).

Mechanism of infection
The spike protein found on the surface of the virion is re sponsible for initiating receptor binding and membrane fu sion (Lan et al. 2020).The spike protein is composed of two subunit proteins, S1 and S2.The S1 subunit contains the RBD, which straight interacts with the ACE2 recep tor on human cell surfaces.Multiple hydrogen bonds and other interactions facilitate the binding of the RBD to the receptor.Once the binding occurs, the spike protein un dergoes a structural change that exposes a site between two subunits (S1 and S2).This site can be cleaved by ei ther the cellular enzyme cathepsin L or the transmembrane protease serine 2 (TMPRSS2).The cleavage is necessary for the virus to enter the host cell and release its genetic material (Harrison et al. 2020; Jackson et al. 2022).
Upon entry into the host cell, the virus will discharge the viral genetic material.The singlestrand positive RNA genome has two open reading frames (ORF1a and ORF1b) that will be used directly as a template to synthesize polyproteins by the host cell ribosome.The polyprotein will then be processed by two proteases, PLpro on nsp3 and 3CLpro on nsp5, to produce 16 nonstructural polypro teins essential for viral replication.The RNAdependent RNA polymerase (RdRp) that originates from the nsp12 is responsible for both copying and transcribing the viral RNA into both the viral RNA genome and a sequence of subgenomic RNAs (Kim et al. 2020; Yao et al. 2020; Amin et al. 2021; Kuo et al. 2021).
The subgenomic RNAs serve as a blueprint for pro ducing various components of the SARSCoV2 virus, in cluding the structural and accessory proteins that regulate innate immunity, viral replication, and disease severity.All of these components are created using the host cell's ribosomes during the translation process (Harrison et al. 2020; Hartenian et al. 2020).
New virus particles are formed within the host cell by the assembly of both the newly synthesized structural pro teins and the genome RNA.The assembly process begins with the formation of the ribonucleoprotein complex by the nucleocapsid and genome RNA.The other structural proteins form the envelope, including the ribonucleopro tein complex as the core.This process can trap a place in the endoplasmic reticulum (RE) and utilizes lipids from the ER as part of the viral particle membrane.The new virus will be transported to the cell surface and released from the host cell by budding from the cell membrane into the extracellular space, where it can infect other host cells and continue the cycle (Hartenian et al. 2020; Naqvi et al. 2020).

Antiviral
Ritonavir/Nirmatrelvir (Paxlovid) is a 3CLpro inhibitor that has obtained Emergency Use Authorizations (EUA).The EUA is a permit for using certain drugs and vac cines in emergencies threatening public health.Regula tory authorities grant EUA based on whether the product has sufficient scientific evidence of safety and efficacy and whether there are no other options or therapies that are suf ficient and officially authorized to treat the illness.Riton avir/Nirmatrelvir is recommended as a firstline treatment for nonsevere cases of COVID19 by NIH guidelines and WHO.Nirmatrelvir acts as a 3CLpro inhibitor, combined with Ritonavir as a boosting agent by inhibiting CYP3A4, and increases the halflife of Nirmatrelvir (Cully 2022; Na tional Institute of Health (NIH) 2022; Tan et al. 2022).
The FDA has approved Remdesivir as the sole antivi ral medication that works as an inhibitor of RdRp.This antiviral, administrated by injection, acts as a chain termi nator that inhibits RdRp in the elongation process of RNA and stops the virus's replication.Remdesivir is a broad spectrum antiviral discovered by drug repurposing with the first indication for the Ebola virus.For COVID19, this drug effectively reduces illness duration (Cully 2022; National Institute of Health (NIH) 2022).
Another RdRp inhibitor, Molnupiravir, has already obtained EUA.Healthcare providers can use Molnupiravir as an alternative regimen for COVID19.Unlike Remde sivir, Molnupiravir's mechanism did not terminate the elongation process but induced mutagenesis that will lead to virus death (Kabinger et al. 2021; Cully 2022; National Institute of Health (NIH) 2022).However, this drug can potentially be mutagenic and less safe than other COVID 19 drugs.Molnupiravir is also an antiviral discovered by drug repurposing with the first indication for Venezuelan equine encephalitis virus antiviral (Cully 2022).

Monoclonal antibody and convalescent plasma
Monoclonal antibodies contain four protein chains held to gether by several disulfide bridges.These chains consist of two heavy chains and also two light chains.Monoclonal antibodies are molecules the immune system produces specifically to identify and destroy foreign molecules (antigens) derived from viruses or bacteria that invade the body.The Fab region on monoclonal antibody has an anti gen binding site or a part that will recognize and bind to the epitope of an antigen.Each monoclonal antibody has a different sequence in the Fab region and recognizes a particular antigen explicitly.The Fc region is part of the heavy chain that can attach to B or T cells.The Fc re gion has the same sequence in each monoclonal antibody (SanchezTrincado et al. 2017).
Developing monoclonal antibodies (mAbs) for COVID19 begins with searching for sources from the antibodies.The source can be obtained from the blood plasma of a COVID19 survivor or a humanized mouse that was immunized using antigens from SARSCoV2.Furthermore, searching for B Lymphocyte cells that produce specific antibodies against RBD and mAb identi fication was conducted.In the following process, cloning and expression of mAb were carried out to increase mAb yield, which would then be validated and characterized.The mAb with the best stability and affinity will be selected and formulated as a mAb drug (Taylor et al. 2021).
RegenCOV is an antibody cocktail, or a combina tion of two monoclonal antibodies (casirivimab and imde vimab) used to treat SARSCoV2 infection (Copin et al. 2021).This mAb will bind to the RBD, a particular spike protein segment responsible for recognizing and bind ing to the ACE2 receptor, thus preventing viral infection.Casirivimab and imdevimab have different binding sites on RBD and chose based on the affinity to the region in RBD that is rarely mutated.This mAb works as a ′neutral izer′ for the virus, so it is included in passive immunother apy to minimize virulence (Baum et al. 2020; Hansen et al. 2020).
The Omicron variant has exacerbated the COVID19 pandemic.This new variant is tremendously contagious because it has a greater affinity for binding to ACE2 re ceptors.Molecular dynamics simulations reveal that the Omicron variant can bind more tightly to ACE2 recep tors than the wild type, leading to a higher infection rate.Mutations N440K, T478K, E484A, Q493R, and Q498R in RBD cause an increase in RBD charge, thus increasing RBD's electrostatic interaction with ACE2 (Nguyen et al. 2022).Those mutations caused most of the mAb prod ucts for COVID19, including RegenCOV, to lose their neutralizing ability against the Omicron variant (Fan et al. 2022).
Convalescent plasma is a medical treatment option used to treat COVID19, which involves administering blood plasma obtained from individuals who have success fully fought off a SARSCoV2 infection.Blood plasma is the fluid part of blood comprising antibodies and other proteins.People who have recuperated from COVID19 have generated antibodies to counteract the SARSCoV 2 virus.Their blood plasma is rich in these antibodies (Lee et al. 2020).Transferring these antibodies that specif ically attack the SARSCoV2 virus from recovered pa tients to those currently infected potentially assists the re cipient's immune system in combating the virus.Conva lescent plasma therapy can rapidly reduce the viral load and improve clinical outcomes, reducing disease sever ity and mortality.There are also potential risks associ ated with convalescent plasma therapy, including trans mitting other pathogens.Therefore, the use of conva lescent plasma should be carefully considered in a case when other treatment options are not available (Duan et al. 2020).

Vaccines
The primary strategy to combat COVID19 is prevention through vaccination to generate herd immunity.A vaccine is a substance designed to trigger the immune system to synthesize antibodies and offers protection against a par ticular disease.It is typically created using a weakened or inactivated version of the diseasecausing agent, which functions as an antigen without causing illness.Multiple COVID19 vaccine platforms are currently in use, each utilizing a unique approach to generate an immune re sponse.The mRNA platform was developed by Pfizer Biontech and Moderna using mRNA technology to intro duce genetic instructions to human cells, prompting them to produce the spike protein found on the surface of the SARSCoV2 virus.This protein is then recognized by the body as an antigen, triggering an immune response and the production of antibodies.This vaccine platform was first approved clinically for COVID19 (Frederiksen et al. 2020; Corti et al. 2021).
AstraZeneca and Johnson & Johnson have created an other approach utilizing viral vectors.The vaccine utilizes a harmless adenovirus as a vector to deliver genetic mate rial that contains the virus's protein to be introduced into human cells.The vaccine employs nonreplicating aden ovirus or replicationdeficient adenovirus as a vector, fa cilitating the entry of the adenovirus into host cells, stim ulating antigen production, and subsequently eliciting an immune response.However, the viral vector lacks the ca pacity for selfreplication, ensuring the adenovirus's in ability to replicate (Frederiksen et al. 2020; Corti et al. 2021).
The subunit protein platform developed by Novavax contains the spike protein produced with recombinant DNA technology, purified, and formulated into a vaccine with adjuvant.Inactivated virus platforms such as Sino vac and Synopharm use the SARSCoV2 whole virus, ac tivated or deactivated by chemical processes to trigger the human body's immune system (Corti et al. 2021; Jackson et al. 2022).Some vaccines can provide longterm solu tions to COVID19, but virus mutations can reduce vac cine effectiveness.

Recombinant PLpro for AntiCOVID-19 Development
The SARSCoV2 infection process involves multiple pro teins and enzymes.The subunit S1 located in the spike (S), contains RBD at the open state that can recognize the human ACE2 receptor (Ni et al. 2020).Thus, inhibit ing ACE2 can avert the virus from entering cells (Ahmad et al. 2021).Another attractive target is TMPRSS2, which also has a role in viral entry by cleaving the spike pro tein to bind to ACE2 (Mantzourani et al. 2022; Shirzad et al. 2022).Aside from the antientry target, one of the main proteases of coronaviruses, PLpro has a dual role that participates in the viral replication process by processing polypeptide chains and interfering with the host cell's in nate immune response (Fu et al. 2021; Zhou et al. 2021).
Many studies have attempted to produce PLpro as an ingredient for antiviral drug discovery.PLpro is a part of nsp3 located in the ORF1a region.The gene encoding PL pro consists of 948 bases, corresponding to 316 amino acid residues with approximately 36 kDa.PLpro is a small part of nsp3 in the open reading frames 1a (ORF1a) region.The PLpro coding gene consists of 948 bp encoding 316 amino acid residues with a theoretical molecular weight of 36 kDa (Arya et al. 2021; Lim et al. 2021).This protein resembles the righthand fold, which consists of "thumb palmfingers", and the structure resembles the ubiquitin specific protease (USPs).The palm and thumb domains of PLpro have a catalytic triad of C111, H272, and D286 residues as their active site (Klemm et al. 2020; Razali et al. 2021; Shen et al. 2022).Within the PLpro structure, several studies identified a binding site for a ligand that has good potential, specifically the BL2 loop (Shen et al. 2022).This loop is located near the active site, which can change its shape when substrates or inhibitors are present.This loop is adjacent to the active site that can undergo a conformational change in the presence of substrates or in hibitors (Gao et al. 2021; Osipiuk et al. 2021).Several in hibitors have been studied, such as GRL0617 and VIR251 (Gao et al. 2021; Shen et al. 2022).

Construction of recombinant PLpro
Several studies have attempted to produce PLpro recom binants, shown in Table 1.
The function of PLpro as a protease enzyme makes it a potential agent for COVID19 drug receptor targets.Sev eral studies have attempted to produce these proteins with recombinant technology and various modifications.The most frequently used expression system is the pET sys tem, especially pET28a, expressed by E.coli BL21(DE3) (Shilling et al. 2020).The pET28a system is recombinant with codon optimization and encodes the Nterminal or C terminal Histag to assist the purification process with Ni NTA (Arya et al. 2021; Fu et al. 2021; Gao et al. 2021; Patchett et al. 2021; Xu et al. 2021).In addition, the production process with this construction also inserts the SUMOtag, leading to a more significant amount of solu ble protein (Fu et al. 2021; Gao et al. 2021).
Several researchers have attempted to address the low yield issue in producing soluble PLpro.One approach is to use fusion proteins that incorporate a highly soluble pro tein, which can increase the solubility of the protein inter est.Recently, Lim et al. (2021) demonstrated that fusing the protein of interest with a solubilityenhancing protein, such as Small Ubiquitinlike Modifier (SUMO), led to a higher yield of soluble PLpro compared to gene constructs without fusion with SUMO.Additionally, other studies have shown that fusion with Glutathione Stransferases (GST) is another effective strategy for increasing the sol ubility of PLpro (Rut et al. 2020; Armstrong et al. 2021) (Figure 3).
Codon optimization is an advantageous technique for expressing proteins in organisms that do not naturally ex press these genes (Lipinszki et al. 2018).Since the choices of synonym codons, codon bias can alter between the host and the original organism (Lipinszki et al. 2018).In the process of producing eukaryotic proteins using E. coli, there may be a problem that causes errors in protein trans lation and decreases the levels of tRNA (Choi and Geletu 2018; Lipinszki et al. 2018).Codon optimization can be used, which has been found to increase both protein ex pression and tRNA abundance (Lozano Terol et al. 2021).In addition, this strategy can minimize protein folding er rors that lead to increased protein solubility (Rosano and  Ceccarelli 2014; Lipinszki et al. 2018; Fu et al. 2020).

Expression of recombinant PLpro
The E. coli system is a popular and adaptable method for producing recombinant proteins.Specifically, the E. coli BL21 (DE3) strain commonly utilized with T7 RNA poly merase (T7 RNAP) can perform recombinant protein tran scription eight times faster than the RNAP strain, which clarifies it as a strong promoter (Wang et al. 2014; Lozano Terol et al. 2021).It also has a deficiency of the pro tease Lon and OmpT making it suitable to produce het erologous proteins in large quantities to prevent degrada tion of the heterologous proteins produced.BL21 (DE3) RIPL and Rosetta are also strains derived from BL21 with the additional ability to increase gene expression with rare codons (Mergulhão et al. 2005; Kayser and Warzecha 2012; Schlegel et al. 2013).
Although the E. coli expression system has many ad vantages for PLpro production, such as growing high speed and low production costs because E. coli can grow to high densities on cheap substrates, most PLpro formed inclusion bodies, protein aggregates with no activity.Spe cific procedures are necessary to process protein as in clusion bodies, ensuring its eventual refolding into func tional soluble protein.Nevertheless, the downstream steps involved in this process are intricate and laborious, thus making the expression of soluble proteins a more favorable option (Kayser andWarzecha 2012; Ulfah et al. 2022).
Furthermore, using chaperone molecules can assist in refolding protein (Khow andSuntrarachun 2012; Rosano andCeccarelli 2014).Coexpression of this chaperone will prevent the formation of inclusion bodies and in crease the solubility of recombinant protein (Khow and Suntrarachun 2012).Not only using chaperone molecules, but the recovery of soluble protein can also increase with lowtemperature incubation (Song et al. 2012).Hence the Arctic Express strains, a BL21 strain derivate, apply to these conditions (Francis and Page 2010).These cells express chaperones that can withstand low temperatures (4-12 °C), namely Cpn10 and Cpn60.Two of them can improve the formation of protein structures and increase the yield of soluble and active PLpro (Rosano et al. 2019; Bhatwa et al. 2021).
Besides using the E. coli expression system, the bac Indonesian Journal of Biotechnology 28(3), 2023, 158-172 ulovirus expression system, like S. frugiperda, can pro duce large amounts of soluble PLpro.Unlike E. coli with limited posttranslational modifications (PTM), the bac ulovirus expression system permits multiple PTMs such as glycosylation, phosphorylation, acylation, and disulfide bond formation (Trowitzsch et al. 2010; Lim et al. 2021).PTM in the baculovirus system can alter the structure of PLpro.PLpro produced by E. coli has a higher activity than insect cells (Trowitzsch et al. 2010; Lim et al. 2021).

Recombinant PLpro Purification and Downstream Processing
To purify recombinant PLpro, affinity chromatography is typically used, which separates proteins based on their in teraction with specific immobilized ligands on a station ary phase.For instance, a ligand such as NiNTA or glutathione selectively binds to a protein of interest that contains polyhistidine or GST, respectively.The station ary phase typically comprises resins or gels packed into columns.Upon loading a protein mixture sample onto the column, the immobilized ligand selectively binds to the protein of interest while allowing other proteins to pass through.The subsequent washing of the column elimi nates nonspecifically bound proteins.The bound proteins are eluted by using specific buffers containing imidazole for NiNTA, which disrupts the binding interactions (Jan son 2011; Rut et al. 2020; Lim et al. 2021).Six histidines or 14 histidines are used either at the C terminal or the Nterminal of PLpro for purification (Rut et al. 2020; Lim et al. 2021).When fusing with poly histidine or other tagged proteins, adding spacers, such as glycine residues, to prevent steric hindrance that could reduce the protease's ability to cleave the fusion protein (Waugh 2011).
Proteases are used in PLpro purification to cleavage protein tags needed only in expression steps and some parts of the purification process.It should be eliminated because it could alter the PLpro structure.A few stud ies indicate that polyhistidine tags have a negligible ef fect; hence they can be disregarded.Some proteases, such as Thrombin (LVPR↓GS), TEV protease (ENLYFQ↓G), and Ulp1 Protease (specifically can recognize and cleave SUMO tags), are commonly used for PLpro and other re combinant proteins (Waugh 2011; Gao et al. 2021; Lim et al. 2021).
During the purification process of recombinant PL pro, various techniques are employed during the polish ing process, including ultrafiltration, ion exchange chro matography, and gel filtration.Ultrafiltration involves us ing a semipermeable membrane to filter out particles and solutes larger than the membrane's pore size.This pro cess effectively separates small molecules like salts and concentrates the desired proteins (Janson 2011; Lim et al. 2021).
Ion exchange chromatography works based on the principle that protein is charged molecules that can be separated based on their electrostatic interactions with charged resins.Weakly bound proteins can be removed by adding salt concentration into the buffer.Protein has a unique isoelectric point.Changes in pH will affect the charge of the protein and the strength of its interaction with the resin (Jungbauer and Hahn 2009; Janson 2011; Lim et al. 2021).
Gel filtration is used to separate and purify proteins according to their size.This chromatography consists of a stationary phase of polymer resin particles that will fil ter protein molecules continuously during the elution pro cess with the mobile phase (Janson 2011).Optimizing the flow rate will affect the sample's interaction with the col umn resin.If the flow rate is low, the sample will dif fuse longitudinally and migrate in all directions.Mean while, if the flow rate is too high, eddy diffusion occurs, in which molecules migrate through the fluid vortex be tween the resins, resulting in nonuniform molecule sepa ration (Thenawidjaja et al. 2017).

In silico and In vitro Studies Designed for PLpro Inhibition Testing
As a protease, short peptide substrates are the leading choice for the design of PLpro inhibition assays.Pep tide substrates can be modified by implementing Fluo rescence Resonance Energy Transfer (FRET) technology.Anthranilate (2amino benzoyl/Abz) was attached to the peptide substrate at the N terminal, and nitroLtyrosine [Y(3NO 2 )R] at the C terminal and connected.The pep tide substrate cleaved by the PLpro will produce a fluo rescence intensity from free Abz that can be measured by spectroscopy.(Lim et al. 2021).Substrate design using the peptide sequences RELNGGAYTRYV, FTLKGGAP TKVT, and IALKGGGKIVNNW had cleavage rates of 0.003 ± 0.001 μM/s, 0.09 ± 0.012 μM/s, and 0.01 ± 0.004 μM/s, respectively.The specificity of the FTLKGGAP TKVT substrate for enzymes is proven to produce differ ent cleavage rates with differences of up to nine to 30 times faster than the other (Kuo et al. 2021; Jeong et al. 2022).
Virtual screening of bioactive compounds derived from Indonesian medicinal plants has identified that some plants have the potential to inhibit PLpro, for instance, Aloe vera, Andrographis paniculata, Phyllanthus niruri L., and Sonchus arvensisi L. Seco4hydroxylintetralin from Phyllanthus niruri L. inhibits PLpro most effectively, with a binding energy of -5,68 kcal/mol.The amino acid residues Tyr 273, Gly 163, Thr 301, and Arg 166 form hy drogen bonds with seco4hydroxylintetralin, allowing it to bind to PLpro (Firdayani et al. 2022).
Another in silico study on Indonesian medicinal plants reported that curcumin found in Curcuma longa Linn.has the potential to inhibit PLpro with a binding energy of -8.45 kcal/mol (Laksmiani et al. 2020).This data aligns with another study that reported that curcumin potentially be a PLpro inhibitor (Rizma et al. 2021).
Indonesian Journal of Biotechnology 28(3), 2023, 158-172 Potential PLpro inhibitors have also been reported from virtual screening of phytochemicals found in In dian medicinal plants.Carvacrol found in black seeds (Nigella sativa) has been found to exhibit a favorable bind ing affinity with the PLpro, as indicated by a binding en ergy value of -5.8 kcal/mol (Debnath et al. 2022).Another study discovered four of 225 phytocompounds in 28 Indian spices: rutin, luteolin7glucoside40neohesperidoside, PDT, and DOC, showing potential inhibition activity against SARSCoV2 proteases (Rudrapal et al. 2022).
A promising drug candidate for treating COVID19 was reported in a study with China's 12 most frequently used herbal medicine to treat COVID19.Liquorice was discovered to possess inhibiting effects against 3CLpro and PLpro among 12, with inhibition rates of 32.85% and 40.93%.Among the 125 compounds from licorice tested, schaftoside has a good affinity with 3Clpro and PLpro, which are -8.4 kcal/mol and -8.5 kcal/mol.Enzymatic assay at eight μmol/L schaftoside has an inhibition rate of 75.9% against 3CLpro and 60% against PLpro.Schafto side exhibits IC 50 values of 1.73 ± 0.22 μmol/L and 3.91 ± 0.19 μmol/L against 3CLpro and PLpro, respectively.Additionally, the results indicate that schaftoside is favor able safety and pharmacokinetic characteristics, making it a potential drug candidate for treating COVID19 (Yi et al. 2022).
Several tanshinone derivatives are potential PLpro in hibitors."Xuebijing", is a traditional Chinese medicine containing Salvia miltiorrhiza, one of the drugs used in China for COVID19.These drugs reduce damage to mul tiple organs by inhibiting inflammation and enhancing im mune function.Catechins from green tea, cyanovirinN, several terpene compounds, and in silico propolis deriva tives are potential PLpro inhibitors (Jiang et al. 2022).
Repurposing available drugs presents a viable option for discovering potential COVID19 treatments.This ap

Prediction of PLpro Inhibitor as an Antiviral with CADD
Computeraided drug design (CADD) is a method for dis covering and developing new drugs using computational tools and techniques (Coumar 2021).The emergence of vast databases containing genomic, chemical, and phar macological information presents novel drug discovery and repurposing opportunities.In the preliminary phases of drug development, computational prediction is a wise option to procure valuable initial data while economiz ing costs and time relative to conventional experimental methodologies (Luo et al. 2017).Targetbased virtual lig and screening is an effective initial method for searching for drug candidates.This method predicted the potential use of Remdesivir as an RdRp inhibitor in less than two months since SARSCoV2 was first identified, using a drug database and a selfbuilt database of natural products (Wu et al. 2020).Erlina et al. (2022) reported that through virtual screening, several compounds such as 8methylthiooctyl glucosinolate, sinigrin, and glucoputranjivin contained in Indonesian herbal plants have the potential to become an tiviral effects through the PLpro inhibition mechanism.The validation of these results can be confirmed further through experimental tests with enzymatic assays.

PLpro Inhibitor Activity Properties for AntiCOVID-19
Several studies have shown that small molecules have the potential as antivirals that inhibit PLpro.Compound GRL0617 is proven to inhibit PLpro with IC 50 of 2.21 μM (Armstrong et al. 2021).Zhao et al. (2021) found that cryptotanshinone and tanshinone I, active components derived from Salvia miltiorrhiza and commonly used in traditional Chinese medicine (TCM), could inhibit PLpro with IC 50 values of 5.63 μM and 2.21 μM, respectively.Dihydrotanshione I from Salvia miltiorrhiza is also very potent, with an IC 50 of 0.59 µM (Park et al. 2012; Lim et al. 2021).Potential PLpro inhibitor, shown in Table 2.In silico and in vitro studies have proven that GRL 0617 and its analogs have the potential as antivirals for COVID19.GRL0617 works as a PLpro inhibitor by forming hydrogen bonds with two amino acids, Asp164 and Gln269, as evidenced by the PLpro molecule's inter action, corresponding to the PDB ID 7CMD (Figure 4).However, this compound is metabolically labile because it contains the naphthalene ring.So, it is difficult to pro ceed to the next steps in developing new drugs (Gao et al. 2021; Tan et al. 2022).

Conclusions
COVID19 has emerged as the most significant life threatening disease caused by the highly pathogenic SARSCoV2.The virus constantly evolves and has devel oped mechanisms to mutate and evade the human immune system, potentially losing efficacy in some treatments (Fan et al. 2022).Extensively used antivirals for COVID19 treatment may also lead to mutations and inevitable resis tance to the current antivirals.RdRp inhibitors showed weaker efficacy than protease inhibitors.
Paxlovid, as a 3CLpro inhibitor, is the firstline antivi ral for SARSCoV2 infection therapy (National Institute of Health (NIH) 2022).This fact proves that proteases have the potential to be effective target receptors for an tiviral discovery.PLpro plays a more critical role than 3CLpro in the virus life cycle because not just for protein cleavage and maturation, PLpro also inhibits the body's immune response.So, screening for PLpro inhibitors will lead to the discovery of novel antivirals with good effi cacy.Drug discovery from herbal compounds with PLpro as the target has not been explored, and the potential for obtaining new antivirals is still wide open.
Although it has been widely studied, the expression of PLpro in E. coli is quite challenging.Combining particu lar tag proteins makes it possible to address the issues in inclusion bodies formation and low production of soluble protein to obtain a higher yield of soluble PLpro to be used as a good target for drug discovery.

TABLE 1
Summary of recombinant PLpro production.
(Kuo et al. 2021) FIGURE 3 Strategy to produce recombinant PLpro which are constructed using certain expression vector and produced from E. coli cells system.