Design, Synthesis, and Biological Evaluation of Trisubstituted Piperazine Derivatives as Noncovalent Severe Acute Respiratory Syndrome Coronavirus 2 Main Protease Inhibitors with Improved Antiviral Activity and Favorable Druggability.

The ongoing transmission of SARS-CoV-2 necessitates the development of additional potent antiviral agents capable of combating the current highly infectious variants and future coronaviruses. Here, we present the discovery of potent nonpeptide main protease (Mpro) inhibitors with prominent antiviral activity and improved pharmacokinetic properties. Three series of 1,2,4-trisubstituted piperazine derivatives were designed and synthesized, and the optimal GC-78-HCl demonstrated high enzyme-inhibitory potency (IC50 = 0.19 µM) and exhibited excellent antiviral activity (EC50 = 0.40 µM), reaching the same level as Nirmatrelvir (EC50 = 0.38 µM). Additionally, GC-78-HCl displayed potent antiviral activities against various SARS-CoV-2 variants as well as HCoV-OC43 and HCoV-229E, indicating its potential broad-spectrum anticoronaviral activity. Notably, the pharmacokinetic properties of GC-78-HCl were somewhat enhanced compared to those of the lead compound. Furthermore, the cocrystal and molecular docking elucidated the mechanism of action. In conclusion, we discovered a novel nonpeptidic Mpro inhibitor with promising antiviral activity and a favorable pharmacokinetic profile.

Discovery of novel sulfonamide substituted indolylarylsulfones as potent HIV-1 inhibitors with better safety profiles.

Indolylarylsulfones (IASs) are classical HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a unique scaffold and possess potent antiviral activity. To address the high cytotoxicity and improve safety profiles of IASs, we introduced various sulfonamide groups linked by alkyl diamine chain to explore the entrance channel of non-nucleoside inhibitors binding pocket. 48 compounds were designed and synthesized to evaluate their anti-HIV-1 activities and reverse transcriptase inhibition activities. Especially, compound R10L4 was endowed with significant inhibitory activity towards wild-type HIV-1 (EC50(WT) = 0.007 µmol/L, SI = 30,930) as well as a panel of single-mutant strains exemplified by L100I (EC50 = 0.017 µmol/L, SI = 13,055), E138K (EC50 = 0.017 µmol/L, SI = 13,123) and Y181C (EC50 = 0.045 µmol/L, SI = 4753) which were superior to Nevirapine and Etravirine. Notably, R10L4 was characterized with significantly reduced cytotoxicity (CC50 = 216.51 µmol/L) and showed no remarkable in vivo toxic effects (acute and subacute toxicity). Moreover, the computer-based docking study was also employed to characterize the binding mode between R10L4 and HIV-1 RT. Additionally, R10L4 presented an acceptable pharmacokinetic profile. Collectively, these results deliver precious insights for next optimization and indicate that the sulfonamide IAS derivatives are promising NNRTIs for further development.

Discovery and Crystallographic Studies of Nonpeptidic Piperazine Derivatives as Covalent SARS-CoV-2 Main Protease Inhibitors.

The spread of SARS-CoV-2 keeps threatening human life and health, and small-molecule antivirals are in demand. The main protease (Mpro) is an effective and highly conserved target for anti-SARS-CoV-2 drug design. Herein, we report the discovery of potent covalent non-peptide-derived Mpro inhibitors. A series of covalent compounds with a piperazine scaffold containing different warheads were designed and synthesized. Among them, GD-9 was identified as the most potent compound with a significant enzymatic inhibition of Mpro (IC50 = 0.18 µM) and good antiviral potency against SARS-CoV-2 (EC50 = 2.64 µM), similar to that of remdesivir (EC50 = 2.27 µM). Additionally, GD-9 presented favorable target selectivity for SARS-CoV-2 Mpro versus human cysteine proteases. The X-ray co-crystal structure confirmed our original design concept showing that GD-9 covalently binds to the active site of Mpro. Our nonpeptidic covalent inhibitors provide a basis for the future development of more efficient COVID-19 therapeutics.

Discovery and Crystallographic Studies of Trisubstituted Piperazine Derivatives as Non-Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity and Low Toxicity.

The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (Mpro) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide Mpro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits Mpro with high potency (IC50 = 0.40 µM) and displays excellent antiviral activity (EC50 = 1.1 µM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC50 > 100 µM) and excellent target selectivity for SARS-CoV-2 Mpro (IC50 > 50 µM for cathepsins B, F, K, L, and caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.

Chemical space exploration around indolylarylsulfone scaffold led to a novel class of highly active HIV-1 NNRTIs with spiro structural features.

To thoroughly investigate the uncharted chemical space around the entrance channel of HIV-1 reverse transcriptase (RT) and to improve the physicochemical properties, we introduced different spiro ring structures with high Fsp3 values as linkers at indole-2-carboxamide, attaching to various terminal substituents to enhance the interactions with the entrance channel. All the newly designed and synthesized indolylarylsulfone (IAS) derivatives exhibited moderate to excellent potency against wild-type HIV-1 with EC50 values ranging from 0.0053 to 0.19 µM. Among them, compounds SO-7g (EC50 = 0.0053 µM) and SO-7h (EC50 = 0.009 µM, SI > 21552) were identified as the most two potent compounds, which displayed 30- and 16-fold improvement than nevirapine and zidovudine and comparable potency to efavirenz and etravirine. Moreover, SO-7g maintained the promising activity against a variety of mutant strains, especially for L100I (EC50 = 0.047 µM), K103 N (EC50 = 0.056 µM), and E138K (EC50 = 0.040 µM). Notably, the introduction of spiro rings could effectively reduce the cytotoxicity (CC50) and greatly improve the selectivity index compared to lead compound, exemplified by SO-7h (CC50 > 214.4 µM, SI > 21552) and SO-7a (CC50 > 233.2 µM, SI > 20933). Additionally, the preliminary SARs based on antiviral activity and molecular simulation perspective were analyzed with a detailed description, which could point out the direction for further structural optimization.

Medicinal chemistry strategies towards the development of effective SARS-CoV-2 inhibitors.

Novel therapies are urgently needed to improve global treatment of SARS-CoV-2 infection. Herein, we briefly provide a concise report on the medicinal chemistry strategies towards the development of effective SARS-CoV-2 inhibitors with representative examples in different strategies from the medicinal chemistry perspective.

Identification of Polyphenol Derivatives as Novel SARS-CoV-2 and DENV Non-Nucleoside RdRp Inhibitors.

The Coronavirus Disease 2019 (COVID-19) and dengue fever (DF) pandemics both remain to be significant public health concerns in the foreseeable future. Anti-SARS-CoV-2 drugs and vaccines are both indispensable to eliminate the epidemic situation. Here, two piperazine-based polyphenol derivatives DF-47 and DF-51 were identified as potential inhibitors directly blocking the active site of SARS-CoV-2 and DENV RdRp. Data through RdRp inhibition screening of an in-house library and in vitro antiviral study selected DF-47 and DF-51 as effective inhibitors of SARS-CoV-2/DENV polymerase. Moreover, in silico simulation revealed stable binding modes between the DF-47/DF-51 and SARS-CoV-2/DENV RdRp, respectively, including chelating with Mg2+ near polymerase active site. This work discovered the inhibitory effect of two polyphenols on distinct viral RdRp, which are expected to be developed into broad-spectrum, non-nucleoside RdRp inhibitors with new scaffold.

Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger.

As a powerful tool for fast and precise genome editing, the CRISPR/Cas9 system has been applied in filamentous fungi to improve the efficiency of genome alteration. However, the method of delivering guide RNA (gRNA) remains a bottleneck in performing CRISPR mutagenesis in Aspergillus species. Here we report a gRNA transcription driven by endogenous tRNA promoters which include a tRNA gene plus 100 base pairs of upstream sequence. Co-transformation of a cas9-expressing plasmid with a linear DNA coding for gRNA demonstrated that 36 of the 37 tRNA promoters tested were able to generate the intended mutation in A. niger. When gRNA and cas9 were expressed in a single extra-chromosomal plasmid, the efficiency of gene mutation was as high as 97%. Co-transformation with DNA template for homologous recombination, the CRISPR/Cas9 system resulted ~42% efficiency of gene replacement in a strain with a functioning non-homologous end joining machinery (kusA+), and an efficiency of >90% gene replacement in a kusA- background. Our results demonstrate that tRNA promoter-mediated gRNA expressions are reliable and efficient in genome editing in A. niger.

Phylogenetic Analysis of Protein Family.

With the number of sequenced genomes increasing rapidly, it is impractical to perform functional and structural analyses on all individual proteins. Phylogenetic analysis employs a combination of molecular and statistical approaches to infer or estimate relationships among individuals. It provides a credible method to explore the relationship between sequence similarity and function of proteins belonging to the same family. This chapter describes a standardized framework of phylogenetic analysis to study large protein families. Bioinformatic approaches and online tools used in phylogenetic analyses are presented.

Engineering a thermostable fungal GH10 xylanase, importance of N-terminal amino acids.

Xylanases are used in many industrial processes including pulp bleaching, baking, detergent, and the hydrolysis of plant cell wall in biofuels production. In this work we have evolved a single domain GH10 xylanase, Xyn10A_ASPNG, from Aspergillus niger to improve its thermostability. We introduced a rational approach involving as the first step a computational analysis to guide the design of a mutagenesis library in targeted regions which identified thermal important residues that were subsequently randomly mutagenized through rounds of iterative saturation mutagenesis (ISM). Focusing on five residues, four rounds of ISM had generated a quintuple mutant 4S1 (R25W/V29A/I31L/L43F/T58I) which exhibited thermal inactivation half-life (t1/2 ) at 60°C that was prolonged by 30 folds in comparison with wild-type enzyme. Whereas the wild-type enzyme retained 0.2% of its initial activity after a heat treatment of 10 min at 60°C and was completely inactivated after 2 min at 65°C, 4S1 mutant retained 30% of its initial activity after 15 min heating at 65°C. Furthermore, the mutant melting temperature (Tm ) increased by 17.4°C compared to the wild type. Each of the five mutations in 4S1 was found to contribute to thermoresistance, but the dramatic improvement of enzyme thermoresistance of 4S1 was attributed to the synergistic effects of the five mutations. Comparison of biochemical data and model structure between 4S1 and the wild-type enzyme suggested that the N-terminal coil of the enzyme is important in stabilizing GH10 xylanase structure. Based on model structure analyses, we propose that enforced hydrophobic interactions within N-terminal elements and between N- and C-terminal ends are responsible for the improved thermostability of Xyn10A_ASPNG.

Impact of an N-terminal extension on the stability and activity of the GH11 xylanase from Thermobacillus xylanilyticus.

To understand structure-function relationships in the N-terminal region of GH11 xylanases, the 17 N-terminal amino acids of the GH11 xylanase from Neocallimastix patriciarum (Np-Xyn) have been grafted onto the N-terminal extremity of the untypically short GH11 xylanase from Thermobacillus xylanilyticus (Tx-Xyn), creating a hybrid enzyme denoted NTfus. The hybrid xylanase displayed properties (pH and temperature optima) similar to those of the parental enzyme, although thermostability was lowered, with the Tm value, being reduced by 5°C. Kinetic assays using oNP-Xylo-oligosaccharides (DP2 and 3) indicated that the N-extension did not procure more extensive substrate binding, even when further mutagenesis was performed to promote this. However, these experiments confirmed weak subsite -3 for both NTfus and the parental enzyme. The catalytic efficiency of NTfus was shown to be 17% higher than that of the parental enzyme on low viscosity wheat arabinoxylan and trials using milled wheat straw as the substrate revealed that NTfus released more substituted oligosaccharide products (Xyl/Ara=8.97±0.13 compared to Xyl/Ara=9.70±0.21 for the parental enzyme), suggesting that the hybrid enzyme possesses wider substrate selectivity. Combining either the parental enzyme or NTfus with the cellulolytic cocktail Accellerase 1500 boosted the impact of the latter on wheat straw, procuring yields of solubilized xylose and glucose of 23 and 24% of theoretical yield, respectively, thus underlining the benefits of added xylanase activity when using this cellulase cocktail. Overall, in view of the results obtained for NTfus, we propose that the N-terminal extension leads to the modification of a putative secondary substrate binding site, a hypothesis that is highly consistent with previous data.

Engineering better biomass-degrading ability into a GH11 xylanase using a directed evolution strategy.

BACKGROUND: Improving the hydrolytic performance of hemicellulases on lignocellulosic biomass is of considerable importance for second-generation biorefining. To address this problem, and also to gain greater understanding of structure-function relationships, especially related to xylanase action on complex biomass, we have implemented a combinatorial strategy to engineer the GH11 xylanase from Thermobacillus xylanilyticus (Tx-Xyn). RESULTS: Following in vitro enzyme evolution and screening on wheat straw, nine best-performing clones were identified, which display mutations at positions 3, 6, 27 and 111. All of these mutants showed increased hydrolytic activity on wheat straw, and solubilized arabinoxylans that were not modified by the parental enzyme. The most active mutants, S27T and Y111T, increased the solubilization of arabinoxylans from depleted wheat straw 2.3-fold and 2.1-fold, respectively, in comparison to the wild-type enzyme. In addition, five mutants, S27T, Y111H, Y111S, Y111T and S27T-Y111H increased total hemicellulose conversion of intact wheat straw from 16.7%tot. xyl (wild-type Tx-Xyn) to 18.6% to 20.4%tot. xyl. Also, all five mutant enzymes exhibited a better ability to act in synergy with a cellulase cocktail (Accellerase 1500), thus procuring increases in overall wheat straw hydrolysis. CONCLUSIONS: Analysis of the results allows us to hypothesize that the increased hydrolytic ability of the mutants is linked to (i) improved ligand binding in a putative secondary binding site, (ii) the diminution of surface hydrophobicity, and/or (iii) the modification of thumb flexibility, induced by mutations at position 111. Nevertheless, the relatively modest improvements that were observed also underline the fact that enzyme engineering alone cannot overcome the limits imposed by the complex organization of the plant cell wall and the lignin barrier.

Effect and limitation of excess ammonium on the release of O-glycans in reducing forms from glycoproteins under mild alkaline conditions for glycomic and functional analysis.

Ammonium-based alkali-catalyzed ß-elimination under nonreducing conditions was investigated in detail for the stability of the released mucin-type O-glycan chains with ß1,3-linked cores. In contrast to the previously studied ß1,4-linkage of the N-glycan-type, which was shown to be stable under the ammonium-based alkaline conditions, the ß1,3-linkage is labile toward alkaline treatment and considerable peeling was observed with both model heptasaccharides and standard glycoproteins. The former include eight reducing glucoheptasaccharides with different and commonly occurring linkages (α1,2-, ß1,2-, α1,3-, ß1,3-, α1,4-, ß1,4-, α1,6-, and ß1,6-linkages), and the latter include mucin-type bovine submaxillary mucin and bovine fetuin, which contains both O- and N-glycans. The results indicated that complete prevention of peeling under nonreducing alkali-catalyzed hydrolysis conditions remains difficult. The yields of released O- and N-glycans were also assessed by use of the two glycoproteins as models. Compared with conventional procedures, Carlson degradation for O-glycan release and PNGase F digestion for N-glycan release, the nonreducing ammonium-based alkaline hydrolysis gave lower yields. Great care has to be taken when employing such nonreducing alkaline conditions in glycomic analysis and in obtaining glycoprotein glycans for functional studies.

A high-throughput screening system for the evaluation of biomass-hydrolyzing glycoside hydrolases.

To implement a protein engineering strategy for the improvement of enzyme performance on biomass, a straightforward, robust high-throughput method was devised and tested with recombinant GH11 xylanase as acting on wheat straw. The method requires automated liquid handling equipment, but avoids the need for specialized milling and powder weighing devices and the use of labour intensive steps such as manual cutting of pipette tips. After expression in Escherichia coli cells grown in microtiter plates, recombinant xylanase was released into the culture medium and used directly for biomass hydrolysis. Reactions were monitored using a micro-3,5-dinitrosalicylic acid assay. The cumulative error of the method was less than 15%. To validate the method, randomly generated xylanase mutants were analyzed. This allowed the detection of one mutant, which produced a 74% increase in hydrolysis compared to the parental enzyme. Closer analysis revealed that this increase in activity was correlated with a twofold increase in xylanase expression.

[Behaviors of beta-glucan from Grifola frondosa in high performance gel permeation chromatography].

The high performance gel permeation chromatographic (HPGPC) behaviors of four beta-1, 3/1, 6-glucans (GF1, GF2, GF3, GF4) with different protein contents and one alpha-1, 4/1, 6-glucan (P100) from Grifola frondosa were examined with different concentrations of NaCl solution and pH values. The experimental results showed that the relative molecular mass (M(r)) of the beta-glucans sharply dropped as the NaCl concentration was less than 0.025 mol/L and then tended to be stable as the NaCl concentration was raised from 0.1 to 0.2 mol/L, the M(r) increased quickly from pH 3 to 6, and then maintained stable from pH 6 to 9, but slightly increased as the pH value was higher than 9. However, the M(r) of the alpha-1, 4/1, 6-glucan was slightly affected by either different concentrations of NaCl or different pH values. The beta-glucan existed in a super-helix structure in aqueous solution, which could be influenced by the NaCl concentration and pH value. These factors led to the different molecular aggregation states, and the increase or decrease of the M(r) of beta-glucans, displaying a variety of HPGPC behaviors.