3.2  Coronavirus PLpro inhibitors
Along with the Mpro, papain‐like protease (PLpro) also cleaves polyproteins which is an important process for viral replication. PLpro cleaves at the first three positions creating three nonstructural functional proteins (nsp1‐nsp3). In particular, nsp3 is central for the generation of the viral replication complex. The multifunctionality of PLpro in deubiquitinating, de‐ISGylation (ISG: interferon‐stimulated gene), 195 ,  196  and in the evasion of the innate immune response make PLpro an attractive antiviral drug target.
PLpro is a cysteine protease and its active site contains a catalytic triad composing of Cys112‐His273‐Asp287. Cys112 behaves as a nucleophile, and His273 is a general acid‐base. Asp287 helps His273 to align perfectly, thus promoting His to deprotonate Cys‐SH.
Ghosh et al. 197  contributed significantly to the development of SARS‐CoV‐1 PLpro inhibitors based on the naphthalene scaffold. Two lead compounds 157 and 158 (Figure 36) were identified by an HTS of a chemical library containing greater than 50 000 compounds. They both inhibit PLpro of SARS‐CoV‐1 at a moderate potency (IC50 20.1 and 59 µM, respectively). The (R)‐enantiomer of compound 157 was found to be a greater than twofold more potent inhibitor of PLpro when compared with its racemic mixture (157). Subsequent SAR studies highlighted the 2‐naphthyl substitution as an important structural requirement rather than at the position 1 of the naphthyl ring in addition to the presence of o‐methyl and m‐amino groups, in the other phenyl ring. Compound 159 displayed the best inhibitory activity of PLpro (IC50, 0.6 µM) and acts in a noncovalent reversible manner with a K i value of 0.49 µM. 198  Compound 159 also showed moderate antiviral activity in Vero cells with an EC50 value of 14.5 µM.
Figure 36  SARS‐CoV‐1 PLpro inhibitors based on naphthalene scaffold. PLpro, papain‐like protease; SARS‐CoV, severe acute respiratory syndrome coronavirus Compound 159 was further scrutinized by investigating the importance of the amide NH, and the effect of the substituent on the benzamide ring (160–164). Among them, compounds 163 and 164 exhibited the most potent enzymatic (163: IC50, 0.46 µM; 164: IC50, 1.23 µM) and cell‐based antiviral (163: EC50, 6.0 µM; 164: EC50, 5.2 µM) activities.
Next, the same group studied the SARs for compound 158 further. This led to the discovery of compound 165 with high PLpro inhibitory activity of SARS‐CoV‐1 (IC50, 0.32 µM) and antiviral activity (EC50, 9.1 µM) in Vero cells. 199  The mode of action of 165 was found to be a noncovalent, competitive inhibition of PLpro. Unlike the previous series, the stereochemistry at the α‐methyl group did not make a significant difference in inhibition of PLpro. For example, both (S)‐ and (R)‐methyl inhibitors, 165 (IC50, 0.32 µM; EC50, 9.1 µM and 166 (IC50, 0.56 µM; EC50, 9.1 µM), respectively, shared equipotent inhibitory activity in enzymatic and cell‐based assays.
Further SARs of 159 and 165 were investigated to improve the activity. However, no significant improvement in the activity was observed for the prepared compounds either in the enzymatic or cell‐based bioassay. Compounds 167–169 (Figure 36) displayed the best inhibitory activities. Especially, the m‐fluoro‐substituted benzamide derivative 168 (IC50, 0.15 µM; EC50, 5.4 µM) showed the best inhibition activity against PLpro. It also inhibited SARS‐CoV‐1 in the cell‐based bioassay. Both compounds 168 and 169 were metabolically more stable when compared to 167.
HTS of a chemical library of 25000 molecules identified 170 (Figure 37) as a dual SARS‐CoV‐1 PLpro (IC50, 10.9 µM) and MERS‐CoV PLpro (IC50, 6.2 µM) inhibitor. 200  This compound acts via competitive inhibition against MERS‐CoV PLpro, yet via allosteric inhibition against SARS‐CoV‐1 PLpro. This compound also exhibited a preference for SARS‐CoV‐1 PLpro and MERS‐CoV PLpro versus two human homologs of the PLpro, ubiquitin C‐terminal hydrolase, (hUCH‐L1) and (hUCH‐L3).
Figure 37  Broad spectral PLpro inhibitors from different sources. PLpro, papain‐like protease; SARS‐CoV, severe acute respiratory syndrome coronavirus Chou et al. 201  identified thiopurine (171) and 6‐thioguanine (172) as SARS‐CoV‐1 PLpro inhibitors by the screening of a library containing 160 compounds. The thiocarbonyl group was important for PLpro inhibition. However, the toxicity of these anticancer agents limits their therapeutic utility as anti‐SARS agents.
In 2012, Park et al. 202  reported a tanshinone derivative 173 as a SARS‐CoV‐1 PLpro inhibitor with an IC50 value of 0.8 µM. The same research group also described diarylheptanoids blocking SARS‐CoV‐1 PLpro. In particular compound 174 performed as the best inhibitor of SARS‐CoV PLpro with an IC50 value of 4.1 µM. An α,β‐unsaturated carbonyl functionality was crucial for effective inhibition. The geranylated flavonoid 175 was another plant‐derived natural product, which displayed SARS‐CoV‐1 PLpro inhibition with an IC50 value of 5.0 µM. 203
In 2017, Park et al. 204  assessed the inhibitory activity of polyphenols isolated from B. Papyrifera against SARS‐CoV PLpro and MERS‐CoV PLpro. Two of them (176 and 177 Figure 37) displayed moderate inhibition at both SARS‐CoV‐1 PLpro and MERS‐CoV PLpro with a noncompetitive mechanism of action.
Disulfiram (151; Figure 35) was also reported as a SARS‐CoV‐1 PLpro inhibitor (IC50, 24.1 µM), 205  probably by reacting with the active site cysteine, thereby covalently modifying the enzyme target, as was reported for other targets.