Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses

BACKGROUND: Coronaviruses (CoVs) can be classified into alphacoronavirus (group 1), betacoronavirus (group 2), and gammacoronavirus (group 3) based on diversity of the protein sequences. Their 3C-like protease (3CL(pro)), which catalyzes the proteolytic processing of the polyproteins for viral replication, is a potential target for anti-coronaviral infection. METHODOLOGY/PRINCIPAL FINDINGS: Here, we profiled the substrate specificities of 3CL(pro) from human CoV NL63 (group 1), human CoV OC43 (group 2a), severe acute respiratory syndrome coronavirus (SARS-CoV) (group 2b) and infectious bronchitis virus (IBV) (group 3), by measuring their activity against a substrate library of 19 × 8 of variants with single substitutions at P5 to P3' positions. The results were correlated with structural properties like side chain volume, hydrophobicity, and secondary structure propensities of substituting residues. All 3CL(pro) prefer Gln at P1 position, Leu at P2 position, basic residues at P3 position, small hydrophobic residues at P4 position, and small residues at P1' and P2' positions. Despite 3CL(pro) from different groups of CoVs share many similarities in substrate specificities, differences in substrate specificities were observed at P4 positions, with IBV 3CL(pro) prefers P4-Pro and SARS-CoV 3CL(pro) prefers P4-Val. By combining the most favorable residues at P3 to P5 positions, we identified super-active substrate sequences 'VARLQ↓SGF' that can be cleaved efficiently by all 3CL(pro) with relative activity of 1.7 to 3.2, and 'VPRLQ↓SGF' that can be cleaved specifically by IBV 3CL(pro) with relative activity of 4.3. CONCLUSIONS/SIGNIFICANCE: The comprehensive substrate specificities of 3CL(pro) from each of the group 1, 2a, 2b, and 3 CoVs have been profiled in this study, which may provide insights into a rational design of broad-spectrum peptidomimetic inhibitors targeting the proteases

Profiling of Substrate Specificity of SARS-CoV 3CLpro

BACKGROUND: The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome-coronavirus is required for autoprocessing of the polyprotein, and is a potential target for treating coronaviral infection. METHODOLOGY/PRINCIPAL FINDINGS: To obtain a thorough understanding of substrate specificity of the protease, a substrate library of 198 variants was created by performing saturation mutagenesis on the autocleavage sequence at P5 to P3' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer. The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high β-sheet propensity; P4 prefers small hydrophobic residues; P2 prefers hydrophobic residues without β-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1' position prefers small residues, while P2' and P3' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated a strong structure-activity relationship between the 3CL(pro) and its substrate. The substrate specificity profiled in this study may provide insights into a rational design of peptidomimetic inhibitors

Expanding the gelation properties of valine-based 3,5-diaminobenzoate organogelators with N-alkylurea functionalities

A new family of valine-containing 3,5-diaminobenzoate derivatives 2 with N-alkylurea moieties attached to the valine moieties was prepared. By appending these two new N-alkylurea chains to the molecular structure, their organogelating properties were extended from only aromatic solvents, to a wide range of other types of solvents such as alicyclic hydrocarbons, alcohols and polar solvents such as DMSO and DMF. It was also found that a longer N-alkylurea chain conferred improved gelation power and higher thermal stability as compared to those of the shorter ones

A Polycyclic Aromatic Hydrocarbon Bearing an All-cis Tetrabenzo-[5.5.5.5]fenestrane (Fenestrindane) Core Merged with Two Hexa-peri-hexabenzocoronene Units

An P, Chow H-F, Kuck D. A Polycyclic Aromatic Hydrocarbon Bearing an All-cis Tetrabenzo-[5.5.5.5]fenestrane (Fenestrindane) Core Merged with Two Hexa-peri-hexabenzocoronene Units. SYNLETT. 2016;27(8):1255-1261.The successful merging of two multiply tert-butylated hexa-peri-hexabenzocoronene (HBC) units with all-cis-tetrabenzo[5.5.5.5]fenestrane (fenestrindane) at two opposite aromatic rings was described. Due to steric inhibition of the tert-butyl substituents near the merging region with the fenestrindane core, attempts to construct a highly warped but fully conjugated polycyclic aromatic hydrocarbon that contains two cycloheptatriene rings failed to materialize

Electronic and steric effects on the three-fold Scholl-type cycloheptatriene ring formation around a tribenzotriquinacene core

Ip H-W, Chow H-F, Kuck D. Electronic and steric effects on the three-fold Scholl-type cycloheptatriene ring formation around a tribenzotriquinacene core. Organic Chemistry Frontiers. 2017;4(5):817-822.Systematic studies on the role of substituents in the bay-bridging cycloheptatriene ring formation around the tribenzotriquinacene (TBTQ) core via the non-typical Scholl reaction were carried out. The electronic effect of the substituents was found to be the predominant factor that controls the ease of the cyclization reaction, while the steric effect of methoxy groups in the bay regions of the TBTQ core appears to be also significant but less important. In several cases with insufficient electronic activation and/or unfavorable steric restriction, single bay-bridging occurred with or without concomitant bridgehead hydroxylation. Alternatively, an unprecedented ring opening/closure of the TBTQ skeleton by electrophilic ipso-attack was found to intervene in other cases. Starting from the electronically and sterically most favorable precursor, a 1,4,8-tris-(2,3,4-trimethoxyphenyl)-TBTQ derivative, a new wizard-hat-shaped, three-fold baybridged TBTQ nanographene core bearing nine methoxy groups at the molecular periphery was synthesized with high efficiency

Donor-Acceptor Tribenzotriquinacene-Based Molecular Wizard Hats Bearing Three ortho-Benzoquinone Units

Hung T-Y, Kuck D, Chow H-F. Donor-Acceptor Tribenzotriquinacene-Based Molecular Wizard Hats Bearing Three ortho-Benzoquinone Units. Chemistry - A European Journal . 2022.Two pi-extended bay-bridged tribenzotriquinacenes ("TBTQ wizard hats") 12 and 16 bearing three mutually conjugated, alternating veratrole-type and ortho-benzoquinone units were synthesized. The electronic properties of these complementarily arranged, nonplanar push-pull systems are affected by the fusion with the rigid, C3-symmetric TBTQ core to a different extent, as revealed by X-ray structural analysis, UV-vis spectroscopy and cyclovoltammetry. The combination of three quinone units within the original TBTQ core and three veratrole-type bay bridging units in 12 gives rise to a more efficiently pi-conjugated chromophore, as reflected by the shallower shape of wizard hat and its absorption in the visible up to 750 nm in comparison to 16. Congener 12 contains an aromatic 18-pi electron system in contrast to the cross-conjugated analog 16. X-ray structure analysis of the precursor dodecaether 15 revealed the formation of a cage-like supramolecular dimer, in which the peripheral dioxane-type ether groups interlace by twelve noncovalent C-H..O bonds. © 2022 Wiley-VCH GmbH