Improved SARS-CoV-2 M^pro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies

2.1. Structure guided development of M^pro inhibitors using independent variations in P2 and P3

Development of improved inhibitors began with sampling variations in the P2 and P3 sites of GC376 in order to assess which types of combinations would facilitate the greatest increase in binding to M^pro (Fig. 1 ). The cyclic glutamine analog in the P1 site of the inhibitor was maintained due to the numerous favorable interactions of this residue observed with the S1 pocket of M^pro, as determined from our previous study [6]. The aldehyde warhead was also conserved due to the ability to easily and favorably form the bisulfite prodrug in a single step, combined with the proven efficacy of this group, as was shown with GC376 to treat FIP in felines [14].

Based on our previous crystal structures, the S2 pocket of SARS-CoV-2 M^pro responsible for binding of the leucine moiety in GC376 was largely hydrophobic [6], and so lipophilic variants of the leucine isopropyl group in the P2 site of the inhibitor were chosen such as cyclopropyl (1a), cyclohexyl (1b), and phenyl (1c) (Table 1 ). Replacement with a phenyl group (1c) yielded only moderate improvements in IC₅₀ values using a FRET substrate-enzyme kinetic assay, while the greatest improvement was found when a cyclopropyl group (1a) was utilized. Replacement with a cyclohexyl group (1b) led to a decrease in performance of the resultant inhibitor, as indicated by increased IC₅₀ values (Table 1). These conclusions were further verified via co-crystallization of these inhibitors with M^pro and the observation that the cyclopropyl group of 1a was able to insert deeper into the S2 site of the protease, which may explain the increase in binding affinity (Fig. 2 and Fig. 3 A).

Table 1.

Singly modified aldehyde derivatives of GC373. GC373 and aldehyde derivative structures containing single modifications at either the P2 (1a—1c) or P3 (1d—1h) positions are shown, along with SARS-CoV-2 M^pro IC₅₀ and SARS-CoV-2 antiviral EC₅₀ values, both with and without co-administration of efflux inhibitor CP-100356 (+CP). Cytotoxicity assays showed CC₅₀ values of >200 μM for all compounds shown. ND indicates that value was not determined.

Entry	Structure	IC50 (μM)	EC50 (μM)	EC50 (μM) +CP
GC373 [6]		0.40 ± 0.05	1.5 ± 0.3	0.32 ± 0.05
1a (PDB 7LCS)		0.05 ± 0.01	1.1 ± 0.1	0.32 ± 0.09
1b		0.61 ± 0.15	1.2 ± 0.4	ND
1c		0.23 ± 0.10	>10	ND
1d		0.13 ± 0.04	1.4 ± 0.5	ND
1e		0.15 ± 0.05	1.47 ± 0.8	ND
1f (PDB 7LDL)		0.06 ± 0.01	0.9 ± 0.1	0.25 ± 0.01
1g (PDB 7LCS)		0.27 ± 0.09	1.1 ± 0.5	ND
1h		0.35 ± 0.11	1.4 ± 0.7	ND

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Variations in the P3 site of the inhibitor were also made based on our previous work on co-crystallization of M^pro with the parent aldehyde GC373 [6]. We noticed that the location in M^pro at which the carboxybenzyl (Cbz) group binds (designated as S3) may not be the optimal position. A deeper pocket (designated as S4) can be observed in the vicinity of the Cbz group that may better accommodate the P3 position of the inhibitor, contrasting S3 which appears as a small concave depression pointing towards the surface of M^pro (Fig. 2). Therefore, we based further modifications of the P3 position of the inhibitor on two factors: enhancing favorable dipole interactions and inducing strategic conformations of the bound inhibitor.

In order to make improvements at the P3 position based on enhancing dipole interactions, we synthesized a number of structural variants containing polar groups at the benzyl ring of the Cbz group. One variant included a 3-fluorobenzyl group (1d) to evaluate the possibility of H-bond formation. Another replaced the benzyl group with a 3-chlorophenylethyl group (1e) to evaluate the effect of increased length and flexibility via the introduction of an additional methylene. Selection of a 4-methoxyindole (1f) for evaluation was based on previously reported high affinity of this functionality in binding to SARS-CoV M^pro [28]. The second parameter, inducing advantageous conformations, was evaluated via stereospecific installation of a methyl group (1g and 1h) at the methylene position of the Cbz group in the parent compound. This methyl group may be able to induce an insertion of the phenyl group into the nearby S4 position of M^pro. FRET substrate-enzyme kinetic assays showed that all of these modifications at the P3 position lead to improved IC₅₀ values over GC373, indicating that both of the aforementioned factors played a role in the binding and interactions found at that position (Table 1). The greatest increases in binding affinity resulted from modification of the benzyl group to a 4-methoxyindole variant (1f), followed by a 3-fluorobenzyl (1d) and 3-chlorophenylethyl variant (1e). These were then followed by installation of a methyl group, with the (S) absolute configuration (1g) outperforming the (R) variant (1h). As noted in Table 1, 1h does not exhibit substantially better IC₅₀ values than the parent compound, providing evidence that the absolute stereochemistry at this position plays a modest role in the degree of binding improvement. Taken together, it appears that while both electronic and steric factors play a role, dipole-dipole interactions have a larger effect on improvements in IC₅₀ than that of initial conformation.

2.2. Combination of most potent P2/P3 modifications

After identifying a number of variations in the P2 and P3 site that lead to improved inhibitor binding to M^pro, we combined these modifications to synthesize analogs with potentially improved properties. The optimal modification for the P2 site of the inhibitor was a cyclopropyl moiety, and we therefore combined this with variations at the P3 site. Four additional analogs were obtained (1i, 1j, 1k, and 1l), as documented in Table 2 and Fig. S1. Surprisingly, it appeared that there was little or no improvement in IC₅₀ values with these analogs as compared to their singly modified variants (1d, 1e, 1f, 1g, Table 1).

Table 2.

Doubly modified aldehyde derivatives of GC373. GC373 and aldehyde derivatives structures containing modifications at both the P2 and P3 positions are shown, along with SARS-CoV-2 M^pro IC₅₀ and SARS-CoV-2 antiviral EC₅₀ values, both with and without co-administration of efflux inhibitor CP-100356 (+CP). Cytotoxicity assays showed CC₅₀ values of >200 μM for all compounds shown.

Entry	Structure	IC50 (μM)	EC50 (μM)	EC50 (μM) +CP
1i (PDB 7LCO)		0.14 ± 0.04	1.1 ± 0.2	0.25 ± 0.1
1j		0.24 ± 0.09	0.8 ± 0.2	0.28 ± 0.02
1k		0.05 ± 0.01	0.7 ± 0.1	0.27 ± 0.4
1l		0.43 ± 0.03	2.0 ± 0.5	1.2 ± 0.8

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In order to improve selectivity, we further transformed a number of these analogs into the bisulfite adduct prodrug versions. In our previous work we found that transformation of the parent aldehyde to the bisulfite adduct resulted in improved IC₅₀ and EC₅₀ values [6], likely a result of increased hydrophilicity and generally improved solubility. The most potent analog from the singular P2 and P3 rounds of modification (1a and 1f) as well as the four combination analogs (1i—1l) were transformed to the sodium bisulfite adducts in order to see whether this improved binding would take place in this instance as well (Table 3 ). Interestingly, singularly modified compounds 1a and 1f displayed slightly increased IC₅₀ values upon transformation to bisulfite adducts 2a and 2b. However, doubly modified compounds 2c, 2d, 2e, and 2f (Table 3, Fig. S2) showed the expected decreases in IC₅₀ values as compared to the parent aldehydes (1i, 1j, 1k, and 1l, Table 2). As such, it appears that in many cases, bisulfite adducts of the parent aldehydes indeed lead to improved IC₅₀ values. Of note is that each combination analog as a bisulfite adduct displayed a higher binding affinity for M^pro compared to GC376, ranging from a 2.5–5.0-fold improvement. Testing of variants of GC376 in which the Na⁺ cation was replaced with a K⁺ cation (3a) or choline (3b) was also conducted, showing very similar IC₅₀ values to that of the parent Na ⁺ form (Table 3, Fig. S3), and demonstrating retained effectiveness combined with increased aqueous solubility (discussed below).

Table 3.

Bisulfite adduct derivatives of GC373. GC376 and bisulfite adduct derivatives singly (2a, 2b) or doubly (2c—2f) modified at the P2 or P3 positions, or with variations in counter ion (3a, 3b) are shown, along with SARS-CoV-2 M^pro IC₅₀ and SARS CoV-2 antiviral EC₅₀ values, both with and without co-administration of efflux inhibitor CP-100356 (+CP). Cytotoxicity assays showed CC₅₀ values of >200 μM for all compounds shown. ND indicates that value was not determined.

Entry	Structure	IC50 (μM)	EC50 (μM)	EC50 (μM) +CP
GC376 [6]		0.19 ± 0.04	0.9 ± 0.2	0.25 ± 0.02
2a		0.08 ± 0.03	0.9 ± 0.3	0.27 ± 0.2
2b		0.07 ± 0.02	1.7 ± 0.3	0.32 ± 0.04
2c		0.07 ± 0.01	0.57 ± 0.07	0.19 ± 0.06
2d		0.08 ± 0.02	0.7 ± 0.2	0.18 ± 0.04
2e		0.04 ± 0.01	1.8 ± 0.4	0.29 ± 0.03
2f		0.05 ± 0.03	1.2 ± 0.2	0.5 ± 0.1
3a		0.20 ± 0.04	0.8 ± 0.3	ND
3b		0.18 ± 0.04	1.2 ± 0.2	ND

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2.3. Structural analysis by X-Ray crystallography

In order to better understand interactions of our new inhibitors with the M^pro active site, crystal structures were concurrently obtained throughout the inhibitor development process (Table S1). As previously demonstrated [6], these new structures confirm the formation of a covalent bond between the aldehyde warhead and the active site cysteine of M^pro (Cys145, C–S bond lengths 1.8–2.0 Å). The carbinol hydroxyl maintains favorable hydrogen bonds with the amide NH from both Cys145 and Gly143 of the protease with (1a, 1f, 1g) or without (1i) the coordination of an additional water molecule (Fig. 3A—3D). In accordance with the parent GC373, the lactam carbonyl of the Gln surrogate forms a strong hydrogen bond with the side chain of His163 in the S1 site in all four structures (Fig. 3A—3D). Meanwhile, the lactam NH is also engaged in hydrogen bonds with the side chain oxygen of Glu166 and the backbone oxygen of Phe140, as well as interactions with the N-terminal serine from the other M^pro protomer [26]. Similarly to the Leu-containing analogs 1f and 1g, the cyclopropylalanine-bearing compounds 1a and 1i (Fig. 3A and B) maintain close C–H ^… π contacts between His41 and the cyclopropyl group, filling the S2 pocket in a more compact fashion than the parent GC373-M^pro structure (PDB 6WTM) where the P2 position does not extend as deeply into the S2 binding pocket [6]. An interesting feature was observed upon examination of the P3 substituent. In the case of a bare Cbz group (i.e. GC373 and 1a), the P3 residue assumes a tilted conformation pointing towards S3 and the solvent exposed surface of the protease (Fig. 3A). In contrast, substitution of Cbz by a 3-fluorobenzyl group (1i, Fig. 3B) or a 4-methoxyindole group (1f, Fig. 3C), or by installation of an (S)-methyl group at the benzyl methylene (1g, Fig. 3D), forces a relocation of the P3 substituent into the S4 pocket, allowing for additional interactions with that side of the protease. Consequently, in the crystal structure of 1g, the S–Me group points towards S3 (protease surface), thus defining the attached methylene group as a bifurcation point in inhibitor design (Fig. 3D). Such a space-filling expansion towards both S3 (protease surface) and S4 (binding pocket) has so far not been reported and suggests a promising approach to seal the entrance of the catalytic cleft with a hydrophobic lid. Simultaneously, the N-terminal head group is shown to maintain interactions with Ala188 and Pro168. Of additional note, substitution of the Cbz group with the 4-methoxyindole amide moiety in 1f (Fig. 3C) leads to a tighter fit in the S4 pocket of M^pro and does not require the coordination of any additional solvent molecules, contrasting a related SARS-CoV-2 M^pro inhibitor structure wherein an indole required the presence of DMSO (PDB 6M0K) [29]. In addition to the NH^…O hydrogen bond between the backbone of Glu166 and the terminal inhibitor amide carbonyl O atom, another hydrogen bond between the backbone carbonyl O atom of Glu166 and the indole NH stabilizes the inhibitor in its position.

2.4. Assessment of inhibitory activity and cytotoxicity: plaque reduction and viral assays

While IC₅₀ values provide a drug potency in vitro and aid in inhibitor design, EC₅₀ values provide information regarding the drug-potential of the molecule in a living system. We therefore tested our compounds in a viral plaque reduction assay using Vero E6 mammalian cells. In order to assess possible cytotoxic effects, the compounds were also tested using a CellTiter-Glo assay in the same cell line and were found to display therapeutic indices of >200. All inhibitor variants containing lone P2 site modifications (1a—1c), with the exception of 1c, exhibited lower EC₅₀ values as compared to the parent GC373, with the cyclopropyl modification (1a) appearing to perform the best. Replicating this, lone P3 site modifications also led to a number of compounds with universally improved EC₅₀ values, with the best performers being 1f and 1g (Table 1). Combining the best-performing modifications at each site in the molecule then furnished a number of doubly modified inhibitors (1i—1l), with two (1j and 1k) demonstrating a further increase in potency and analogous decrease in EC₅₀ values (Table 2). The corresponding sodium bisulfite adducts were derived from the most potent compound from each singly modified group (2a and 2b from 1a and 1f, respectively). While 2a exhibited comparable EC₅₀ values to GC376, indicating similar potential as a drug candidate, 2b displayed a distinctly higher EC₅₀ value, hinting at possible efflux effects (Table 3). Sodium bisulfite adducts that combined a cyclopropyl group at the P2 position along with P3 substitutions (2c—2f), had lower EC₅₀ values in two out of four cases, 2c and 2d. Based on a recent report in the literature, it was noted that related compounds may be susceptible to cellular efflux, leading to increased EC₅₀ values [30]. In order to determine if this was the case, the EC₅₀ values for a number of our candidates (1a, 1f, 1i—1l, and 2a—2f) were reevaluated in the presence of CP-100356, a P-gp efflux inhibitor (Table 1, Table 2, and Table 3). When administered together with CP-100356, all but three (1l, 2b, and 2f) of the tested derivatives performed on par or better than the analogous parent GC373 or GC376. Furthermore, the presence of an indole moiety may contribute to efflux, as noted by the comparatively high initial EC₅₀ values of 2b and 2e coupled with a significant decrease upon administration of CP-100356, though not to the same extent as reported for related substances [30]. This drop was less significant for the remaining analogs, especially for 2c and 2d (Table 3), indicating that while there are some cellular efflux effects present, these new compounds possess the advantage of not requiring the co-administration of efflux inhibitors to be effective, thereby potentially simplifying pharmaceutical development. The results indicate 2c and 2d are the most promising inhibitors reported herein (Fig. 4 ), exhibiting both IC₅₀ and EC₅₀ values that are superior to those of GC376, even in the absence of efflux inhibitors. Replacement of the indole with a 3-fluorobenzyl or 3-chlorophenylethyl group provides the added benefit of increased synthetic yields, contrasting previously described indole-containing inhibitors [2,30], which may facilitate reaction scale-up during process development.

2.5. NMR studies with ¹³C-labelled GC373 and GC376: formation of a dynamic equilibrium of stereoisomeric mixtures in aqueous media

As the mechanism of GC373 and GC376 involves binding of a defined stereoisomer [6], we investigated the properties of GC373 and isomer distribution of both GC373 and GC376 in aqueous media. Versions of GC373 and GC376 having a¹³C-label at the carbonyl carbon (Fig. S4) were synthesized. An initial HSQC NMR experiment of GC373 in CDCl₃ showed a single prominent signal in the aldehyde region corresponding to the ¹³C-label (Fig. S5A). A second HSQC NMR experiment wherein GC373 was dissolved in aqueous solution (0.5% DMSO‑d ₆ in D₂O) showed that the single aldehyde peak disappeared. It was replaced by 2 signals at ppm values corresponding to formation of two hydrate diastereomers (Fig. S5B) [31], indicating that GC373 epimerizes rapidly and exists almost entirely as the hydrated form in aqueous media [6]. Loss of stereochemical integrity at the α-carbon adjacent to the ¹³C-label in aqueous media is facile for aldehydes of amino acids. Comparison of peak areas reveals a hydrate diastereomer distribution of ca. 7:3. In the presence of M^pro, there is appearance of an additional peak due to a hemithioacetal formed by binding of a single stereoisomer in the active site of the enzyme. The hydrate diastereomer distribution remained the same as a result of interconversion between the remaining diastereomeric unbound species, suggesting that this equilibrium is dynamic (Fig. S5C). Analogous HSQC NMR experiments with GC376 ¹³C-labelled at the carbonyl carbon also demonstrated a distribution of diastereomers, but only 3 distinct signals were observed at a ratio of 36:46:18 (Fig. S5D). There are 4 different stereochemical permutations possible (Fig. S4), but the range over which their chemical shift values in HSQC spectra may be distributed is limited (Fig. S5E). Hence, it appears that the middle signal is likely an aggregate resulting from spectral overlap of 2 distinct stereochemical species. This study supports the proposal that in aqueous solution, diastereomers of GC373, its hydrate, and GC376, exist in a dynamic stereochemical equilibrium, with only the correct aldehyde isomer binding as a single hemithioacetal in the active site of M^pro.

2.6. Increasing solubility by counter ion variation and investigating aggregation phenomena

In order to help assess the potential of GC376 for administration to humans, a number of solubility experiments were undertaken. We sought to increase the aqueous solubility of GC376 and hypothesized that the cation counterpart to the bisulfite adduct could be replaced with alternative species that exhibited better properties. Potassium and choline were selected as possible candidates owing to larger solvation radii and the generally regarded as safe (GRAS) nature of choline [32]. The potassium and choline counterparts to GC376 (designated as GC376-K (3a) and GC376-Cho (3b)) were synthesized from GC373 using solutions of potassium or choline bisulfite. Both derivatives demonstrated better solubility in aqueous media, with increases in molar solubility of approximately 30% for GC376-K (3a) and over 90% for GC376-Cho (3b) compared to GC376. Additionally, GC376-Cho did not appear to form the same turbid solution as GC376 at 50 mM in water (see below). Furthermore, for inhibitors 3a and 3b, cellular plaque reduction assays with live virus revealed no significant difference in inhibitory activity as compared to the parent compound GC376 (Table 3), demonstrating that cation replacement is a viable strategy to significantly increase aqueous solubility while retaining activity. This approach represents an attractive general avenue for obtaining more favorable solubility profiles in ion-paired drugs and prodrugs.

It was observed that addition of H₂O or D₂O to powdered GC376 (sodium salt) initially formed clear, transparent solutions at a concentration of ca. 440 mM (Fig. S6A). Further dilution of these transparent solutions resulted in the formation of cloudy colloidal suspensions (Fig. S6B) that eventually became transparent again at sufficiently high dilutions (Fig S6C). While initially unexpected, this led us to hypothesize that GC376 was forming micelles – a possibility owing to the amphipathic nature of the compound. The bisulfite moiety may function as a polar head group while the largely non-polar peptide backbone and associated side chains may function as a hydrophobic tail, akin in structure to that of a fatty acid. To test this hypothesis, we conducted diffusion-ordered spectroscopy (DOSY) experiments [33] in D₂O at concentrations of 440 mM and 5 mM (Fig. S7), as these concentrations both resulted in the formation of clear solutions. Based on these experiments, a clear difference in diffusion coefficients could be observed, corresponding to 1.2 × 10⁻¹⁰ m²/s at 440 mM and 4.0 × 10⁻¹⁰ m²/s at 5 mM. Accordingly, these diffusion order values correspond to hydrodynamic radii of 20.6 Å and 6.2 Å (41.2 Å and 12.4 Å in diameter) respectively [34] – a distinct difference suggesting the formation of micelles at 440 mM. For comparison, the particle size of extensively studied dodecyl phosphocholine (DPC) micelles in water are noted to be on the order of 64 Å to 72 Å in diameter [35].

Further support of this phenomenon is found when examining the 1D ¹H NMR spectra for these two solutions, with the 440 mM sample producing a spectrum with a number of broad peaks, a known phenomenon in samples exhibiting aggregation (Fig. S8A). Contrasting this, much sharper peaks are observed in the 5 mM sample (Fig. S8B), hinting at dispersed molecules in solution. Expanding upon this, further DOSY experiments were conducted with serially diluted samples of GC376 in D₂O (Table S2). These experiments showed that GC376 possessed a critical micelle concentration of approximately 96.7 mM (Fig. S9A), establishing a new parameter of interest for the development of GC376 and related bisulfite adduct prodrugs. This experiment was repeated with the choline version of GC376 (GC376-Cho, 3b), and a corresponding critical micellar concentration of 91.1 mM was observed (Fig. S9B). Previously reported effective dosages of GC376 for felines are established at 5–10 mg/kg/day, twice daily [14]. In humans general volume limits of subcutaneous injection are about 1.5 mL [36]. Administration of a micellar solution of GC376 is a possible method for circumventing solubility issues and volume limits when considering GC376 for use in human trials. Use of GC376-Cho, with volume requirements as low as 0.11 mL per injection (calculated for a 62 kg human, 5.66 M concentration, 360 mg per injection, MW = 588 g/mol), twice daily, could help to increase patient acceptance. Administration of GC376 or related drugs as a concentrated micellar or colloid suspension along with the increased solubility resulting from the use of an alternative counterion may allow for significant improvements in the drug profile of these types of peptide-aldehyde inhibitors.

4.1. FRET peptide substrate synthesis

The FRET substrate Abz-SVTLQSG-Y(NO₂)-R used was synthesized according to the methods previously described in literature [6]. Characterization data was in accordance as described.

4.2. Isolation and purification of SARS-CoV-2 M^pro

Pure SARS-CoV-2 M^pro was obtained as described previously and confirmed according to established data [6].

4.3. Inhibition assay and determination of IC₅₀ values

IC50 values for each inhibitor tested was determined via the methods described previously in literature [6].

4.4. Crystallization of M^pro

The purified SARS-CoV-2 M^pro was dialyzed against buffer containing 10 mM NaCl and 5 mM Tris HCl pH 8.0 overnight at 4 °C. Protein was concentrated with a Millipore centrifugal filter (10 kDa MW cutoff) to a concentration of 8 mg/mL. Protein was incubated with 5 mM excess of inhibitor at 4 °C for 1 h prior to crystallization. The SARS-CoV-2 M^pro, the protein was subjected to the JCSG plus and PACT crystallization screen (Molecular Dimensions), with hits identified in several conditions for all inhibitors. Best crystals were observed with hanging drop trays at room temperature at a ratio of 1:1 with mother liquor 0.2 M Sodium chloride, 0.1 M HEPES pH 7.0, 20% w/v PEG 6000. Prior to freezing, crystals were incubated with 19% glycerol as a cryoprotectant. Data collection was take place at SSRL, beamline 12-2 with Blu-Ice using the Web-Ice interface and Canadian Light Source beamline 08B1-1 using MxDC.

4.5. Diffraction data collection, phase determination, model building, and refinement

X-ray diffraction data sets for SARS-CoV-2 M^pro and inhibitor complex were collected at 100 K in a cold nitrogen stream using beamlines at 12-2 at Stanford Synchrotron Radiation Lightsource (SSRL) California, USA, of wavelength 0.97946, equipped with Dectris PILATUS 6 M detector and Canadian light Source, CLSI BEAMLINE 08B1-1 coupled with PILUUS 6 M. More than 80 data sets were collected for all the five inhibitors, all of them processed and the best ones selected based on data statistics. XDS and Scala were used for processing of the data sets. The diffraction data set of the Lig124 (XU4) SARS-CoV-2 M^pro was processed at a resolution of 1.93 Å, in an orthorhombic space group P2₁2₁2 (Supplementary Table 1). For the complex of SARS-CoV-2 M^pro with ligand 180 (XTJ), the data set collected, was processed at a resolution of 1.90 Å in a space group C2, Ligand 182 (XV4) processed at resolution of 2.0 Å in a monoclinic space group P1. The data set collected for Lig128 (XTP) at resolution 1.85 Å, processed in a space group C2. The data set collected for Lig132 (XTM) in an orthorhombic space group P2₁2₁2₁ refined at resolution 1.95 Å. (Table S1). All the five structures were determined by molecular replacement with the crystal structure of the free enzyme of the SARS-CoV-2 M^pro (PDB entry 6WTM) [6] as a search model, using the Phaser program from Phenix, version v1.18.1–3855). Ligand was fit using Coot manually for all the inhibitors in the density of pre-calculated map from Phenix refinment. Refinement of all the structures was performed with phenix.refine in Phenix software. The structure refinement yielded final R_work and R_free of reasonable values. Statistics of diffraction, data processing and model refinement are given in the supplementary materials (Table S1). The model was inspected with Ramachandran plots and all show good stereochemistry. Final models displayed using PyMOL molecular graphics software (Version 2.0 Schrödinger, LLC).

4.6. Plaque reduction assay and EC₅₀ determination

Plaque reduction assay for EC50 determination was carried out with live virus in Vero cells using the methods that we have previously described [6].

4.7. Evaluation of inhibitor cytotoxicity

Cytotoxicity of inhibitors was determined using the methods that we have previously described [6].

4.8. Population distribution studies

¹³C-labelled substrate was prepared in accordance with procedures documented in the supplemental materials. NMR samples of GC373 were prepared by dissolving the sample in CDCl₃ or in the case of D₂O, as a solution in DMSO‑d ₆ that was subsequently diluted to a final concentration of 1% DMSO‑d ₆ in D₂O. Preparation of GC376 samples omitted the use of DMSO‑d ₆ as a co-solvent. Additional information and protocols for samples containing M^pro enzyme can be found previously documented in literature [6]. Experiments were executed on a 700 MHz Agilent VNMRS spectrometer equipped with a cryo-cooled HCN triple resonance Z-gradient probe.

4.9. Solubility studies

Solubility studies were carried out by depositing 10 mg of bisulfite prodrug (differing in cation – Na, K, or choline) in a small conical vial and sequentially adding H₂O in 0.5 μL increments at 25 °C. These mixtures were then alternately vortexed using a desktop instrument (30 s) and sonicated in a water bath (30 s) 3x each. The mixtures were then observed to see if and solids remained. Additional volumes of H₂O (in 0.5 μL increments) were added followed by vortexing and sonication steps until a clear, transparent mixture was obtained. Back calculation then allowed for determination of upper solubility limits for each variant of bisulfite salt.

4.10. DOSY experiments

Diffusion Ordered Spectroscopy (DOSY) measurements were performed on a 600 MHz four-channel Varian VNMRS spectrometer equipped with a HCN Z-gradient probe using OpenVNMRJ 2.1A as the acquisition and processing software. The Oneshot45 DOSY pulse sequence [39,40]was used for all diffusion measurements. All experiments were done at 27 °C. The 90° pulse was optimized for each sample using a simple pulse acquire NMR experiment with presaturation of the residual HOD during the relaxation delay. The diffusion delay was optimized for each sample using the Oneshot45 pulse sequence using 5 different pulsed field gradient strengths from 2.4 G/cm to 59.5 G/cm. Based on the optimized diffusion delay, for GC376 and 3b diffusion delays of 50 ms–200 ms were used, the final DOSY experiment was acquired using 15 different pulsed field gradient strengths from 2 G/cm to 59.5 G/cm with 12 scans for each value of gradient strength. The DOSY data were base line corrected and then processed using a two component fit, were corrected for non-uniform pulsed field gradient shapes and plotted using the DOSY module in OpenVNMRJ 2.1A.