However, enteroviruses are very different from coronaviruses. While both of them have a single-stranded RNA genome of positive polarity, that of enteroviruses is very small just 7—9 kb , whereas coronaviruses feature the largest RNA genome known to date 27—34 kb. Enteroviruses are small, naked particles, whereas coronaviruses are much larger and enveloped. Nevertheless, a related feature shared by these two groups of viruses is their type of major protease, 21 which in the enteroviruses is encoded by the 3C region of the genome hence the protease is designated 3C pro.
In coronaviruses, nonstructural protein 5 Nsp5 is the main protease M pro. This dimerization is essential for the catalytic activity of the CoV M pro , whereas the enteroviral 3C pro Figure 1 B functions as a monomer. The fact that there is no known human protease with a specificity for Gln at the cleavage site of the substrate increases the attractiveness of this viral target, as there is hope that the inhibitors to be developed will not show toxicity versus the host cell.
Indeed, neither the enterovirus 3C pro inhibitor rupintrivir, which was developed as a treatment of the common cold caused by HRV, nor the peptide aldehyde inhibitor of the coronavirus M pro that was recently demonstrated to lead to complete recovery of cats from the normally fatal infection with Feline Infectious Peritonitis Virus FIPV showed any toxic effects on humans or cats, respectively.
High Resolution Image. Here we describe the structure-based design, synthesis, and evaluation of the inhibitory activity of a series of compounds with broad-spectrum activities afforded by studying the structure—activity relationships mainly with respect to the P2 position of the peptidomimetics. Notably, unexpected differences between alpha- and betacoronavirus M pro were found in this study. The structural foundation of these was elucidated in detail in a subproject involving the M pro of HCoV NL63; because of its volume, this work will be published separately Zhang et al.
This moiety has been found to be a good mimic of glutamine and enhance the power of the inhibitors by up to fold, most probably because, compared to the flexible glutamine side chain, the more rigid lactam leads to a reduction of the loss of entropy upon binding to the target protease. Scheme 1. Synthesis Scheme 1 started with the dianionic alkylation of N -Boc glutamic acid dimethyl ester with bromoacetonitrile. As expected, this alkylation occurred in a highly stereoselective manner, giving 1 as the exclusive product.
In the following step, the cyano group of 1 was subjected to hydrogenation. The in situ cyclization of the resulting intermediate afforded the lactam 2. The lactam derivative 3 was generated by removal of the protecting group of 2. The key intermediates 6 were obtained via the condensation of the lactam derivative 3 and the N-capped amino acids 5.
The ester group of compounds 6 was then reduced to the corresponding alcohol. Oxidation of the alcohol products 7 by Dess—Martin periodinane generated the aldehydes 8 , followed by nucleophilic addition with isocyanides, gave rise to compounds 9 under acidic conditions.
Table 1. To enable the rapid and biosafe screening of antivirals against corona- and enteroviruses, a noninfectious, but replication-competent SARS-CoV replicon was used 33 along with subgenomic replicons of CVB3 34 and EV-A71 a kind gift from B. Zhang, Wuhan, China. The easily detectable reporter activity firefly or Renilla luciferase of these replicons has previously been shown to reflect viral RNA synthesis.
Table 3. Apparently, the compound is able to bind to this M pro in the absence of the peptide substrate, but cannot compete with the substrate for the binding site due to low affinity. A similar observation has been made in one of our previous studies, where we were able to determine the crystal structure of a complex between the inactive Michael-acceptor compound SG74 and the EV-D68 3C pro ref 29 ; PDB entry 3ZV9.
Compounds 11b — 11e and 11g — 11l were synthesized in an attempt to do so; however, none of them showed better inhibitory activity against the majority of the recombinant proteases, compared to the parent compound, 11a see Supplementary Results. Residues from the lid, in particular Met49 in the case of SARS-CoV M pro , can thus make hydrophobic interactions with the P2 substituent of the inhibitor, whereas such interaction is missing in the enterovirus 3C pro s.
In addition, the S2 pocket is of different sizes in the various proteases. In contrast, the crystal structure of the complex between 11a and HCoV-NL63 M pro , against which the compound is inactive, demonstrates that the P2-benzyl group cannot fully enter the S2 pocket of the enzyme because of the restricted size of this site Figure 3 b. To explore the sensitivity of the S2 pocket toward a polar substituent in the para position of the benzyl group, we synthesized compound 11m , carrying a 4-fluorobenzyl group in P2.
We will see from examples to follow that this trend persists among all inhibitors with a smaller P2 substituent: Even though the SARS-CoV M pro S2 pocket has a larger volume than that of the enterovirus 3C pro , the enzyme can be efficiently inhibited by compounds carrying a small P2 residue that makes hydrophobic interactions with the lid Met49 and floor Met residues.
This is not only a consequence of the smaller size of the isobutyl group compared to the benzyl group, but also of its larger conformational flexibility, which allows a better fit to the binding site. The decreased activity in the case of SARS-CoV M pro and the total inactivity against HCoV-NL63 M pro indicate that the n -butyl chain is too long for the S2 pocket of these proteases, whereas the slight improvement against EV-A71 3C pro and CVB3 3C pro is probably a consequence of the extra space that is available to long and flexible substituents because of the lack of a lid covering the enterovirus 3C pro pocket.
As the n -butyl substituent in P2 of 11o was obviously too long, we next synthesized a derivative with the shorter propargyl ethinylmethyl as the P2 residue compound 11p. This led to very mediocre activities against all tested proteases. Using cyclopropyl as the P2 residue compound 11q , the IC 50 values were even higher against most of the proteases tested. Having realized that, in addition to size, the flexibility of the P2 substituent may be an important factor influencing inhibitory activity, we introduced flexibility into the phenyl ring of 11a by reducing it.
Thus, the replacement of the phenyl group by the cyclohexyl group led to a significant improvement of the inhibitory activity against the recombinant SARS-CoV M pro and to a dramatic improvement in the case of CVB3 3C pro. Even for the HCoV-NL63 M pro , against which 11a was completely inactive, greatly improved, albeit still weak activity was observed Table 1.
At this point, we decided to systematically vary the size of the ring system in P2. The replicon results were mirrored by the antiviral activity of 11s in enterovirus-infected cells Table 3 , which was weak or very weak. This plasticity is expressed in a conformational change of residue Gln, both in the main chain and in the side chain.
The main-chain conformational change is connected with a flip of the peptide between Gln and Thr The conformational variability of Gln has been noted before, both in molecular dynamics simulations 26 and in other crystal structures. The affinity of 11s for the S2 pocket of HCoV-NL63 M pro is good because of an almost ideal match of size and not requiring conformational changes, which this enzyme would not be able to undergo because of the replacement of the flexible Gln by the more rigid Pro.
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On the other hand, docking of the same compound into the crystal structure of the CVB3 3C pro revealed that the cyclopropylmethyl moiety was probably unable to generate sufficient free energy of binding because of the missing lid and the large size of the S2 pocket in the enterovirus 3C pro , thereby explaining the poor inhibitory activity of 11s against these targets. This turned out to be the one compound with acceptable IC 50 values against all tested enzymes: 1.
The activity against the replicons was between 3. In other words, the nearly equipotent 11u is indeed a compromise. Also, in enterovirus-infected cell cultures, the compound performed well, with EC 50 values of 0. On the other hand, only weak toxicity was detected for 11r in Vero or Huh-T7 cells. Preliminary pharmacokinetics tests with the compound in mice did not indicate a toxicity problem to be published elsewhere.
In addition, the amide oxygen of the inhibitor accepts a hydrogen bond from the catalytic His side chain. Alternatively, the thiohemiketal can interact with the catalytic His residue and the amide oxygen with the main-chain amides of the oxyanion hole. Depending on the exact interaction, the stereochemistry at the thiohemiketal C atom would be different. We have previously observed a similar difference in the case of aldehyde inhibitors, where the single interaction point, the oxyanion of the thiohemiacetal, can accept a hydrogen bond either from the oxyanion hole or from the catalytic His side chain, 37 resulting in different stereochemistry of the thiohemiacetal carbon.
The former displayed IC 50 values 1 order of magnitude lower than the latter, indicating that the S2 pocket of the norovirus 3CL protease is fairly small. Although we did not include the norovirus 3CL pro in our study, expanding the target range of our inhibitors to norovirus is probably a realistic undertaking.
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While our study was underway, Zeng et al. At the same time, Kim et al. Among a series of aldehydes, Prior et al. No optimization of this compound was performed, and no toxicity data have been reported. Lee et al. P2 was invariably isobutyl leucine in these compounds, suggesting that further improvement might be possible.
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In addition to Michael acceptors, peptide aldehydes have also been used to explore the inhibition of coronavirus M pro s as well as enterovirus 3C pro s. Kim et al. In nearly all studies aiming at discovering peptidomimetic inhibitors of coronavirus M pro s, P2 is invariably isobutyl leucine , and this residue has also been used in the efforts to design compounds that would inhibit enterovirus 3C pro s as well see above.