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. 2024 Jan 10;15(2):250–257. doi: 10.1021/acsmedchemlett.3c00498

Development of a GC-376 Based Peptidomimetic PROTAC as a Degrader of 3-Chymotrypsin-like Protease of SARS-CoV-2

Deborah Grifagni †,‡, Elena Lenci ‡, Alessia De Santis †,‡, Andrea Orsetti †,‡, Carlo Giorgio Barracchia §, Filomena Tedesco ‡, Raffaele Bellini Puglielli ‡, Francesca Lucarelli †,‡, Angela Lauriola §, Michael Assfalg §, Francesca Cantini †,‡, Vito Calderone †,‡, Daniele Guardavaccaro §, Andrea Trabocchi ‡,*, Mariapina D’Onofrio §,*, Simone Ciofi-Baffoni †,‡,∥,*
PMCID: PMC10860180  PMID: 38352832

Abstract

graphic file with name ml3c00498_0006.jpg

We have applied a proteolysis targeting chimera (PROTAC) technology to obtain a peptidomimetic molecule able to trigger the degradation of SARS-CoV-2 3-chymotrypsin-like protease (3CLPro). The PROTAC molecule was designed by conjugating a GC-376 based dipeptidyl 3CLPro ligand to a pomalidomide moiety through a piperazine–piperidine linker. NMR and crystallographic data complemented with enzymatic and cellular studies showed that (i) the dipeptidyl moiety of PROTAC binds to the active site of the dimeric state of SARS-CoV-2 3CLPro forming a reversible covalent bond with the sulfur atom of catalytic Cys145, (ii) the linker and the pomalidomide cereblon-ligand of PROTAC protrude from the protein, displaying a high degree of flexibility and no interactions with other regions of the protein, and (iii) PROTAC reduces the protein levels of SARS-CoV-2 3CLPro in cultured cells. This study paves the way for the future applicability of peptidomimetic PROTACs to tackle 3CLPro-dependent viral infections.

Keywords: Peptidomimetics, Bioconjugate chemistry, COVID-19, 3-Chymotrypsin-like protease, PROTAC, Infectious diseases

Proteolysis targeting chimera (PROTAC) technology provides an attractive approach to modulate protein levels of therapeutic target by hijacking the cellular machinery responsible for the physiological elimination of endogenous proteins.1−3 This strategy has been widely established to be successful in targeting cancer-related proteins,4 with more than a dozen drug candidates entering clinical investigation.5−7 On the contrary, the applicability of PROTAC technology in the field of antivirals remains marginal.8−12 Only a few studies of PROTAC molecules targeting viral proteins have been reported in the case of hepatitis and influenza viruses,13−15 and just a preliminary attempt investigating the applicability of PROTAC technology against coronaviruses (CoV) has been documented.16 The latter study targets the viral spike protein receptor binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). PROTAC degraders acting against the major viral protease (3-chymotrypsin-like protease, 3CLPro hereafter), one of the best characterized viral drug targets to date,17−19 have been only hypothesized20,21 or computationally predicted.22 3CLPro turns out to be an ideal viral target for several reasons: (i) it is shared by all Coronavirus genera as well as by members of the large genus Enterovirus in the picornavirus family,23 (ii) it is essential for viral replication,24,25 (iii) it has unique features of its cleavage site recognition that are absent in closely related human homologues,26 and (iv) it has a high degree of structural similarity among 3CLPro proteins of all Coronaviruses genera and 3C proteases of enteroviruses.23

In this study, we envisaged selecting 3CLPro of SARS-CoV-2 as a target for degradation activated via PROTAC technology, and we provide a PROTAC molecule specifically targeting and degrading the dimeric SARS-CoV-2 3CLPro protein. This PROTAC molecule has been designed exploiting a structural biology approach to target the active site of SARS-CoV-2 3CLPro by a peptidomimetic27 ligand on one side, and to target cereblon (CRBN) by the immunomodulatory drug pomalidomide on the other side. CRBN is one of the substrate receptors of the CRL4 E3 ubiquitin ligase complex and is responsible for the polyubiquitination of the substrate (i.e., SARS-CoV-2 3CLPro in this approach), which is the step required to trigger substrate degradation by the proteasome.28,29 The use of CRBN in the design of PROTAC recruiters has been prompted by the discovery and the structural characterization of the interaction between CRBN and immunomodulatory imide drugs.30−32 In the last years, many CRBN-based PROTACs have been designed to target a variety of substrate proteins for proteasomal degradation.33 The immunomodulatory drug pomalidomide is a frequently used CRBN ligand to design PROTAC degraders.34,35 In our designed PROTAC molecule, the pomalidomide and the peptidomimetic ligands were joined through a rigid linker previously shown to be optimal in CRBN-based PROTAC degraders.36 X-ray crystallography and solution NMR were applied to structurally characterize the interaction between PROTAC and SARS-CoV-2 3CLPro, and cellular studies were used to investigate the degradation activity of the PROTAC molecule.

GC-376 is a typical 3CLPro inhibitor that has shown antiviral activity against feline infectious peritonitis CoV in experimentally infected cats and with broad-spectrum activity against coronavirus.37,38 More recently, the antiviral activity of GC-376 against SARS-CoV-2 was demonstrated in vitro,39,40 and in vivo studies supported that GC-376 could represent a promising lead candidate for therapeutic treatment of SARS-CoV-2 infections.41 Accordingly, we designed two PROTAC molecules based on the structural information available for the SARS-CoV-2 3CLPro-GC-376 interaction. Both PROTAC molecules contain a SARS-CoV-2 3CLPro peptidomimetic ligand that conserves the key interacting elements of GC-376 and of its aldehyde derivative GC-373.42 This ligand contains an (S)-γ-lactam ring occupying the S1 subsite of SARS-CoV-2 3CLPro enzyme and a hydrophobic side chain of an amino acid, such as leucine or phenylalanine, occupying the S2 subsite (Figure 1, left). As the electrophilic warhead of both SARS-CoV-2 3CLPro ligands, we reasoned to replace the aldehyde functionality of GC-373 with a α,β-unsaturated methyl ester to achieve reversible ligands (Figure 1, left). On this structural basis, PROTACs 1 and 2 were designed and synthesized in a complementary approach, exploiting the C-terminus of the GC-376-derived dipeptidyl ligand for PROTAC 1 and its N-terminus for PROTAC 2. For the synthesis of PROTAC 1, the C-terminus of a dipeptidyl SARS-CoV-2 3CLPro ligand (named ligand 1′ hereafter) was conjugated to the CRBN ligand pomalidomide through a piperazine–piperidine linker, the latter chosen following the success of Arvinas company in delivering the first two PROTACs as clinical candidates (Figure 1, right).43 Briefly, PROTAC 1 was achieved following ester hydrolysis of 1′ and subsequent amide coupling with the amino group of piperazine–piperidine-functionalized pomalidomide in 65% yield. PROTAC 2 was achieved in 13% yield by installing the pomalidomide CRBN ligand at the N-terminus position of a tripeptidyl SARS-CoV-2 3CLPro ligand through the piperazine–piperidine linker as for PROTAC 1. The tripeptidyl ligand was synthesized from 1′ through Cbz removal and subsequent couplings at the N-terminus of valine and of succinic acid as a linker for the bioconjugation to the piperazine–piperidine-functionalized pomalidomide unit (Figure 1, right; details of the synthetic routes for PROTACs 1 and 2 are reported in Supporting Information (SI), Schemes S1 and S2, respectively).

Figure 1.

Figure 1

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Schematic representation of synthesized PROTACs 1 and 2. The synthesis is based on a core structure, shown on the left, containing common functional groups in specific positions, which are typically recognized by S1 and S2 subsites of 3CLPro proteins.

We evaluated ligand 1′ and PROTACs 1 and 2 for their ability to inhibit SARS-CoV-2 3CLPro (at 60 nM) through a fluorimetric enzyme inhibition kinetics assay using Hilyte Fluor-488-ESATLQSGLRKAK-(QXL-520)-NH2 as substrate. Ligand 1′ and PROTAC 1 showed inhibition activity with a IC50 of 1.35 μM and 21.2 μM, respectively (Table 1, and SI, Figure S1), whereas PROTAC 2 did not display any significant activity, showing only 64% inhibition at 100 μM and 0% inhibition at 10 μM. Therefore, it was not further considered in subsequent structural and cellular studies.

Table 1. Inhibitory Activity of Ligand 1′, PROTACs 1 and 2,a and of Representative SARS-CoV-2 Inhibitors against 3CLPro.

compd IC50 (μM)
Ligand 1′ 1.35 ± 0.34
PROTAC 1 21.2 ± 5.8
PROTAC 2 N/A
representative 3CLProinhibitors reported IC50 (μM)
GC-37641,44,45 0.19 ± 0.04
GC-37345 0.40 ± 0.05
Nirmatrelvir46 0.051 ± 0.01
Boceprevir47,48 5.40 ± 1.53
Dalcetrapib-thiol49 14.4 ± 3.3
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a

Mean from three different assays, errors were in the range of 5–10% of the reported values; IC50 values were retrieved from dose–response assays as the concentration of compound required for 50% inhibition, as estimated by nonlinear correlation using GraphPad Prism software.

In order to elucidate the binding mode of PROTAC 1, solution NMR was applied to screen the interaction50 between PROTAC 1 and dimeric SARS-CoV-2 3CLPro. Specifically, NMR titration experiments were performed by stepwise addition of PROTAC 1 to 15N-labeled dimeric SARS-CoV-2 3CLPro. Upon subsequent additions of PROTAC 1, chemical shift changes were observed in the 1H–15N heteronuclear single quantum coherence (HSQC) maps of dimeric SARS-CoV-2 3CLPro, corresponding to a slow exchange regime on the NMR time scale (Figure 2A). This means that signals of dimeric SARS-CoV-2 3CLPro decreased in intensity, and those of a new species, assigned to the adduct formed between dimeric SARS-CoV-2 3CLPro and PROTAC 1, appeared and increased in intensity during the titration (Figure 2A, insets). On reaching the 1:1 SARS-CoV-2 3CLPro-PROTAC 1 ratio, the signals of SARS-CoV-2 3CLPro completely disappeared and the signals of the PROTAC 1–protein complex reached their maximal intensity, indicating that the complex was fully formed at the 1:1 molar ratio. These data strongly support that a tight binding of PROTAC 1 to the protein occurred. Most of the affected residues were found close to the Cys-His catalytic dyad of SARS-CoV-2 3CLPro (Figure 2B), thus revealing that PROTAC 1 specifically binds to the active site of the enzyme. The NMR data were confirmed by a fluorescence quench on the spectra recorded with or without PROTAC 1 addition after excitation of the tryptophan residues (SI, Figure S2A).

Figure 2.

Figure 2

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Mapping the interaction between PROTAC 1 and dimeric SARS-CoV-2 3CLPro. (A) Overlay of 1H–15N HSQC maps of 15N-labeled SARS-CoV-2 3CLPro (red) and a 1:1 15N-labeled SARS-CoV-2 3CLPro-PROTAC 1 mixture (black). Some residues affected by the interaction are highlighted. In the insets, 1H–15N HSQC maps at 1:0 (red), 1:0.5 (green), and 1:1 (black) SARS-CoV-2 3CLPro-PROTAC 1 ratios are shown for some residues that are in slow exchange regime on the NMR time scale upon complex formation. (B) The backbone NH protons of the residues affected by the interaction between PROTAC 1 and dimeric SARS-CoV-2 3CLPro are shown as pink and orange spheres on the two subunits (depicted in orange and pink) of the dimeric structure of SARS-CoV-2 3CLPro (PDB 8OKC). The side chains of the Cys-His catalytic dyad are shown in yellow on the pink subunit.

To further characterize the protein-PROTAC 1 interaction, NMR experiments were performed titrating 15N-labeled dimeric SARS-CoV-2 3CLPro with ligand 1′. We observed chemical shift changes in a slow exchange on the NMR time scale (SI, Figure S3), which reproduced those observed in the titration between PROTAC 1 and SARS-CoV-2 3CLPro. This result indicates that the dipeptidyl moiety of PROTAC 1 is actively involved in binding to the active site of the enzyme, while the linker and the pomalidomide moieties of PROTAC 1 do not significantly interact with SARS-CoV-2 3CLPro. To provide information on the structural organization of the protein in the presence of PROTAC 1 and ligand 1′, circular dichroism (CD) spectra were recorded on the dimeric SARS-Cov2 3CLPro in the presence and absence of PROTAC 1 or ligand 1′ (SI, Figure S4). The recorded spectra are fully superimposable, thus showing that no significant changes of the secondary structure of the dimeric SARS-Cov2 3CLPro occurred upon the PROTAC 1 interaction. Finally, to ascertain whether PROTAC 1 could recognize monomeric SARS-CoV-2 3CLPro, NMR titration experiment were carried out by adding PROTAC 1 to a monomeric form of SARS-CoV-2 3CLPro (SI, Figures S5 and S6). No significant chemical shift changes were observed for the monomeric form, indicating no binding of PROTAC 1 to the protein (SI, Figure S6). Thus, we conclude that the dimeric state of SARS-CoV-2 3CLPro is specifically targeted by PROTAC 1 and only the dimeric state of SARS-CoV-2 3CLPro can be subjected to protein degradation.

To gain further insight into the interaction mode of PROTAC 1 with SARS-CoV-2 3CLPro, the crystal structures of SARS-CoV-2 3CLPro in complex with ligand 1′ and PROTAC 1 were obtained (see SI, Table S1). The polypeptide structure of both adducts is well superimposable with previously reported structures of SARS-CoV-2 3CLPro.51,52 By comparing the structure of the apo form of SARS-CoV-2 3CLPro with those of the two adducts with ligand 1′ and PROTAC 1, only loop regions showed local backbone RMSD values higher than average, indicating that the binding of either ligand 1′ or PROTAC 1 to the protein does not require significant structural rearrangement of the protein active site. Concerning the occupancy of PROTAC 1 and ligand 1′, we found that the electron density is quite well-defined for most atoms of ligand 1′ (SI, Figure S7A) and, in the case of PROTAC 1, the electron density is better defined for the chemical moiety corresponding to ligand 1′ (SI, Figure S7B). For the remaining PROTAC 1 structure, the electron density is virtually absent, indicating a high degree of mobility of the linker and of the pomalidomide moiety as well as the absence of interactions with other regions of the protein. This suggests that this molecular fragment protrudes out of the protein and is completely solvent exposed (Figure 3), in agreement with solution NMR data and in support of the fruitful involvement of PROTAC 1 in recruiting CRL4 E3 ubiquitin ligase. The binding of ligand 1′ and PROTAC 1 to the SARS-CoV-2 3CLPro protein is quite similar for the structural part that is shared by the two compounds, highlighting their similar positioning in the protein active site (Figure 4A). The most relevant feature in common between the two compounds is their binding mode to the catalytic Cys145. Accordingly, the crystallographic structures of both adducts revealed that the distance between the sulfur atom of Cys145 and the Cβ carbon of the α,β-unsaturated amide moiety of ligand 1′ and PROTAC 1 spontaneously refines to a value around 1.7 Å, which is typical of a C–S covalent bond. From a chemical point of view, it is known that the formation of a covalent bond between the unsaturated β-carbon and sulfur atoms has been already observed to occur between analogous peptidomimetic inhibitors and SARS-CoV 3CLPro.53

Figure 3.

Figure 3

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Close-up of the binding of PROTAC 1 to the SARS-CoV-2 3CLPro active site. The dipeptidyl moiety of PROTAC 1 with a well-defined electron density is in red. The piperazine–piperidine linker and the pomalidomide moiety with absent electron density are shown in cyan.

Figure 4.

Figure 4

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Binding mode of PROTAC 1 and ligand 1′ to SARS-CoV-2 3CLPro by X-ray crystallography. (A) Superimposition of PROTAC 1 (green) and ligand 1′ (cyan) in the respective structures showing a similar binding mode to that of the protein. (B) Ligplot schematic view of the binding modes of PROTAC 1 (left) and ligand 1′ (right). The residues of SARS-CoV-2 3CLPro involved in covalent, hydrophobic, and hydrogen-bonding contacts with PROTAC 1 and ligand 1′ are indicated according to the legend. The red circles and ellipses identify the residues that interact with the ligand (PROTAC 1 or ligand 1′) on both structures upon superposition of the two structures.

The formation of a covalent complex between SARS-CoV-2 3CLPro and ligand 1′ or PROTAC 1 was confirmed by matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry data. Incubation of SARS-CoV-2 3CLPro with PROTAC 1 (exact mass 885 Da) or ligand 1′ (exact mass 459 Da) yielded a peak shift from the protein mass by 881 and 458 Da, respectively (SI, Figure S2B–C). Increased mass corresponds to a single molecule of PROTAC 1 or ligand 1′ covalently captured by SARS-CoV-2 3CLPro. Besides the covalent bond, other polar and nonpolar interactions keep both ligand 1′ and PROTAC 1 in place.

In the case of PROTAC 1, the side chain of His41, and the backbone of Phe140 and Glu166 were found as the protein residues involved in direct hydrogen bonding interactions (Figure 4B, left). In addition, a water molecule bridges the nitrogen atom of Gln189 via hydrogen bonding. Several residues are involved in hydrophobic interactions (Figure 4B, left), in agreement with most of the molecule atoms being nonpolar. In the case of the ligand 1′-protein structure, protein–ligand interactions are similar but not identical to those found in the PROTAC 1–protein structure (Figure 4B, right), as the hydrogen bonding network partially differs. In particular, the hydrogen bond involving the His41 side chain is absent, the one involving the Phe140 backbone is looser, and those involving Glu166 and the water molecule bridging Gln189 are strictly maintained (Figure 4B, right). Two additional hydrogen bonds are present involving the backbone amides of Cys145 and of Gly143 and the same oxygen of the ester group of the ligand. The absence of such hydrogen bonds in the PROTAC 1–protein structure is due to the substitution of the methyl ester of ligand 1′ with the bulky piperazine–piperidine linker (Figure 4B, left). Although the occurrence of these slightly different hydrogen bond networks, all the residues involved in the molecular recognition of SARS-CoV-2 3CLPro with both PROTAC 1 and ligand 1′ are conserved among human coronavirus main proteases, with the only exception of Asn 142 (SI, Figure S8). This finding supports the idea that the active sites of other viral proteases might also be targeted by PROTAC 1.

Considering the covalent C–S bond formation detected in the crystal structure between Cys145 and the Cβ carbon of the α,β-unsaturated amide moiety of PROTAC 1, we investigated whether PROTAC 1 is a reversible covalent ligand for SARS-CoV-2 3CLPro by applying an assay protocol already available in the literature.54 Thus, the enzyme was incubated with or without a large excess of PROTAC 1 for 30 min. Then, such mixtures were diluted 100-fold into a reaction buffer containing a fluorogenic substrate to initiate the enzymatic reaction of SARS-CoV-2 3CLPro. The progress curves for these samples were obtained, and the corresponding initial velocities were measured and compared to those of the enzyme incubated and diluted in the absence of PROTAC 1. This experiment revealed the complete recovery of the enzyme activity (SI, Figure S9), thus showing the reversibility of the covalent C–S bond formed between SARS-CoV-2 3CLPro and PROTAC 1. As a general rule for a PROTAC molecule, combining covalent bond formation with its reversibility is expected to represent the best condition to efficiently degrade target proteins in a cellular context.55 Indeed, the reversibility ensures dissociation and recycling of the PROTAC molecule, whereas the covalency confers higher selectivity.56 Overall, these effects favor low doses usage of PROTAC molecules.

Finally, we investigated the degradation efficiency in the cellular context by assaying whether PROTAC 1 affects SARS-CoV-2 3CLPro protein levels in cultured cells. We first generated HeLa cells stably expressing hemagglutinin (HA)-epitope C-terminally tagged SARS-CoV-2 3CLPro by lentiviral transduction (Figure 5A). These cells were then treated with increasing concentrations of PROTAC 1 and the expression of SARS-CoV-2 3CLPro was examined by immunoblotting. We observed a pronounced decrease in SARS-CoV-2 3CLPro levels in the presence of PROTAC 1 at concentrations that did not affect the cell viability (Figure 5B–D).

Figure 5.

Figure 5

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Effects of PROTAC 1 on SARS-CoV-2 3CLPro protein levels and cell viability in HeLa cells. (A) HeLa cells transduced with lentiviruses expressing C-terminally tagged SARS-CoV-2 3CLPro were analyzed by immunoblotting with antibodies specific for the indicated proteins before (preselection expressing HA-epitope) and after (postselection) incubation with puromycin. (B) HeLa cells expressing HA-epitope C-terminally tagged SARS-CoV-2 3CLPro were treated with PROTAC 1 at the indicated concentrations for 24 h. Cells were harvested and lysed. Whole cell extracts were analyzed by immunoblotting with antibodies specific to the indicated proteins. The graph shows the quantification of SARS-CoV-2 3CLPro levels normalized to actin levels. (C) HeLa cells expressing HA-epitope C-terminally tagged SARS-CoV-2 3CLPro were treated with 80 μM PROTAC 1 for the indicated times. Cells were analyzed by immunoblotting as in (B). The graph shows the quantification of SARS-CoV-2 3CLPro levels normalized to actin levels. (D) Viability of HeLa cells treated with PROTAC 1 for 72 h at the indicated concentration was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are reported as mean ± SEM; n = 3.

In this study, we designed and synthesized a peptidomimetic PROTAC molecule specifically targeting the dimeric SARS-CoV-2 3CLPro protein. We structurally characterized the interaction of the PROTAC molecule with SARS-CoV-2 3CLPro and observed the formation of a covalent bond between the Cβ carbon of the α,β-unsaturated amide moiety of the PROTAC 1 molecule and the catalytic Cys145 sulfur atom of SARS-CoV-2 3CLPro. The covalent reversibility of the PROTAC 1 inhibition was demonstrated as a key requirement for the efficacy of the PROTAC molecule that needs to be recycled at the cellular level once the degradation of the viral target has occurred. We finally provided the first evidence that PROTAC 1 is active at the cellular level drastically reducing protein levels of SARS-CoV-2 3CLPro without affecting cell viability, indicating that a peptidomimetic-based PROTAC strategy can be an efficient tool to block viral infections in Coronavirus genera also in the μM range.

Further studies will be aimed at improving the PROTAC efficacy in reaching the target in the cellular environment. We expect that the insights attained from this study can extend the applicability of peptidomimetic-based PROTACs to a more general setting to attack Coronavirus genera, as well as some members of the large genus Enterovirus.

Acknowledgments

This work was supported by “Fondo di beneficienza ed opere di carattere sociale e culturale” of Intesa Sanpaolo (project no. B/2021/0212), by “the European Union-NextGenerationEU-National Recovery and Resilience Plan, Mission 4 Component 2-Investment 1.5-THE-Tuscany Health Ecosystem-ECS00000017-CUP B83C22003920001, and by Italian Ministry of Education and Research (MUR) through Dipartimenti di Eccellenza 2023-2027 (DICUS 2.0) to the Department of Chemistry “Ugo Schiff” of the University of Florence. Moreover, Centro Piattaforme Tecnologiche of the University of Verona is acknowledged for providing access to the mass spectrometer and CD instruments.

Glossary

Abbreviations

PROTAC

proteolysis targeting chimera

CoV

coronaviruses

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

3CLPro

3-chymotrypsin-like protease

CRBN

cereblon

NMR

nucler magnetic resonance

HSQC

heteronuclear single quantum coherence

CD

circular dichroism

MALDI-TOF

matrix-assisted laser desorption/ionization time-of-flight

HA

hemagglutinin

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications Web site. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00498.

  • Experimental procedures and characterization data for the synthesis of PROTAC 1 and 2; inhibition curves for PROTAC 1 and ligand 1′; fluorescence and MALDI TOF mass spectrometry data monitoring complex formation of SARS-CoV-2 3CLPro with PROTAC 1 and ligand 1′; NMR data monitoring complex formation between ligand 1′ and dimeric SARS-CoV-2 3CLPro; far-UV circular dichroism spectroscopy monitoring structural changes upon complex formation of SARS-CoV-2 3CLPro with PROTAC 1 and ligand 1′; analytical gel filtration of monomeric and dimeric SARS-CoV-2 3CLPro; NMR data monitoring the interaction between PROTAC 1 and monomeric SARS-CoV-2 3CLPro; electron density of ligand 1′ and PROTAC 1; sequence alignment of 3CLPro proteins from different coronaviruses and mapping of the residues interacting with both PROTAC 1 and ligand 1′ on the SARS-CoV-2 3CLPro structure; recovery of the enzymatic activity in the SARS-CoV-2 3CLPro-PROTAC 1 adduct; HPLC chromatograms of PROTAC 1 and PROTAC 2; data collection and refinement statistics of SARS-CoV-2 3CLPro complexed with ligand 1′ and PROTAC 1; NMR spectra of ligands 1′, 2′, PROTAC 1, PROTAC 2, S7, and S12 (PDF)

Accession Codes

Coordinates and structure factors have been deposited at the PDB under the accession code 8OKB for SARS-CoV-2 3CLPro complexed with ligand 1′ and 8OKC for SARS-CoV-2 3CLPro complexed with PROTAC 1.

The authors declare no competing financial interest.

Supplementary Material

ml3c00498_si_001.pdf (2.9MB, pdf)

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