Aloin isoforms (A and B) selectively inhibits proteolytic and deubiquitinating activity of papain like protease (PLpro) of SARS-CoV-2 in vitro.

The most common host entry point of human adapted coronaviruses (CoV) including SARS-CoV-2 is through the initial colonization in the nostril and mouth region which is responsible for spread of the infection. Most recent studies suggest that the commercially available oral and nasal rinse products are effective in inhibiting the viral replication. However, the anti-viral mechanism of the active ingredients present in the oral rinses have not been studied. In the present study, we have assessed in vitro enzymatic inhibitory activity of active ingredients in the oral mouth rinse products: aloin A and B, chlorhexidine, eucalyptol, hexetidine, menthol, triclosan, methyl salicylate, sodium fluoride and povidone, against two important proteases of SARS-CoV-2 PLpro and 3CLpro. Our results indicate only aloin A and B effectively inhibited proteolytic activity of PLpro with an IC50 of 13.16 and 16.08 µM. Interestingly, neither of the aloin isoforms inhibited 3CLpro enzymatic activity. Computational structural modelling of aloin A and B interaction with PLpro revealed that, both aloin isoforms form hydrogen bond with Tyr268 of PLpro, which is critical for their proteolytic activity. Furthermore, 100 ns molecular dynamics (MD) simulation studies predicted that both aloin isoforms have strong interaction with Glu167, which is required for PLpro deubiquitination activity. Our results from the in vitro deubiquitinase inhibition assay show that aloin A and B isomers exhibit deubiquitination inhibitory activity with an IC50 value of 15.68 and 17.51 µM, respectively. In conclusion, the isoforms of aloin inhibit both proteolytic and the deubiquitinating activity of SARS-CoV-2 PLpro, suggesting potential in inhibiting the replication of SARS-CoV-2 virus.

Monitoring protein conformational changes using fluorescent nanoantennas.

Understanding the relationship between protein structural dynamics and function is crucial for both basic research and biotechnology. However, methods for studying the fast dynamics of structural changes are limited. Here, we introduce fluorescent nanoantennas as a spectroscopic technique to sense and report protein conformational changes through noncovalent dye-protein interactions. Using experiments and molecular simulations, we detect and characterize five distinct conformational states of intestinal alkaline phosphatase, including the transient enzyme-substrate complex. We also explored the universality of the nanoantenna strategy with another model protein, Protein G and its interaction with antibodies, and demonstrated a rapid screening strategy to identify efficient nanoantennas. These versatile nanoantennas can be used with diverse dyes to monitor small and large conformational changes, suggesting that they could be used to characterize diverse protein movements or in high-throughput screening applications.

Functional antibody characterization via direct structural analysis and information-driven protein-protein docking.

Detailed description of the mechanism of action of the therapeutic antibodies is essential for the functional characterization and future optimization of potential clinical agents. We recently developed KD035, a fully human antibody targeting vascular endothelial growth factor receptor 2 (VEGFR2). KD035 blocked VEGF-A, and VEGF-C-mediated VEGFR2 activation, as demonstrated by the in vitro binding and competition assays and functional cellular assays. Here, we report a computational model of the complex between the variable fragment of KD035 (KD035(Fv)) and the domains 2 and 3 of the extracellular portion of VEGFR2 (VEGFR2(D2-3)). Our modeling was guided by a priori experimental information including the X-ray structures of KD035 and related antibodies, binding assays, target domain mapping and comparison of KD035 affinity for VEGFR2 from different species. The accuracy of the model was assessed by molecular dynamics simulations, and subsequently validated by mutagenesis and binding analysis. Importantly, the steps followed during the generation of this model can set a precedent for future in silico efforts aimed at the accurate description of the antibody-antigen and more broadly protein-protein complexes.

Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents.

Emerging outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is a major threat to public health. The morbidity is increasing due to lack of SARS-CoV-2 specific drugs. Herein, we have identified potential drugs that target the 3-chymotrypsin like protease (3CLpro), the main protease that is pivotal for the replication of SARS-CoV-2. Computational molecular modeling was used to screen 3987 FDA approved drugs, and 47 drugs were selected to study their inhibitory effects on SARS-CoV-2 specific 3CLpro enzyme in vitro. Our results indicate that boceprevir, ombitasvir, paritaprevir, tipranavir, ivermectin, and micafungin exhibited inhibitory effect towards 3CLpro enzymatic activity. The 100 ns molecular dynamics simulation studies showed that ivermectin may require homodimeric form of 3CLpro enzyme for its inhibitory activity. In summary, these molecules could be useful to develop highly specific therapeutically viable drugs to inhibit the SARS-CoV-2 replication either alone or in combination with drugs specific for other SARS-CoV-2 viral targets.

Dual-Target Inhibitors of the Folate Pathway Inhibit Intrinsically Trimethoprim-Resistant DfrB Dihydrofolate Reductases.

Trimethoprim (TMP) is widely used to treat infections in humans and in livestock, accelerating the incidence of TMP resistance. The emergent and largely untracked type II dihydrofolate reductases (DfrBs) are intrinsically TMP-resistant plasmid-borne Dfrs that are structurally and evolutionarily unrelated to chromosomal Dfrs. We report kinetic characterization of the known DfrB family members. Their kinetic constants are conserved and all are poorly inhibited by TMP, consistent with TMP resistance. We investigate their inhibition with known and novel bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK). Importantly, all are inhibited by the HPPK inhibitors, making these molecules dual-target inhibitors of two folate pathway enzymes that are strictly microbial.

Structure-Based Design of Dimeric Bisbenzimidazole Inhibitors to an Emergent Trimethoprim-Resistant Type II Dihydrofolate Reductase Guides the Design of Monomeric Analogues.

The worldwide use of the broad-spectrum antimicrobial trimethoprim (TMP) has induced the rise of TMP-resistant microorganisms. In addition to resistance-causing mutations of the microbial chromosomal dihydrofolate reductase (Dfr), the evolutionarily and structurally unrelated type II Dfrs (DfrBs) have been identified in TMP-resistant microorganisms. DfrBs are intrinsically TMP-resistant and allow bacterial proliferation when the microbial chromosomal Dfr is TMP-inhibited, making these enzymes important targets for inhibitor development. Furthermore, DfrBs occur in multiresistance plasmids, potentially accelerating their dissemination. We previously reported symmetrical bisbenzimidazoles that are the first selective inhibitors of the only well-characterized DfrB, DfrB1. Here, their diversification provides a new series of inhibitors (K i = 1.7-12.0 µM). Our results reveal two prominent features: terminal carboxylates and inhibitor length allow the establishment of essential interactions with DfrB1. Two crystal structures demonstrate the simultaneous binding of two inhibitor molecules in the symmetrical active site. Observations of those dimeric inhibitors inspired the design of monomeric analogues, binding in a single copy yet offering similar inhibition potency (K i = 1.1 and 7.4 µM). Inhibition of a second member of the DfrB family, DfrB4, suggests the generality of these inhibitors. These results provide key insights into inhibition of the highly TMP-resistant DfrBs, opening avenues to downstream development of antibiotics for combatting this emergent source of resistance.

Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening.

The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells.

Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions.

Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such "half-native" tetramers were shown to procure native-like catalytic activity, with similar KM values but kcat values 5- to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (Tm ∼12°C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active-site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation.

Fragment-based design of symmetrical bis-benzimidazoles as selective inhibitors of the trimethoprim-resistant, type II R67 dihydrofolate reductase.

The continuously increasing use of trimethoprim as a common antibiotic for medical use and for prophylactic application in terrestrial and aquatic animal farming has increased its prevalence in the environment. This has been accompanied by increased drug resistance, generally in the form of alterations in the drug target, dihydrofolate reductase (DHFR). The most highly resistant variants of DHFR are known as type II DHFR, among which R67 DHFR is the most broadly studied variant. We report the first attempt at designing specific inhibitors to this emerging drug target by fragment-based design. The detection of inhibition in R67 DHFR was accompanied by parallel monitoring of the human DHFR, as an assessment of compound selectivity. By those means, small aromatic molecules of 150-250 g/mol (fragments) inhibiting R67 DHFR selectively in the low millimolar range were identified. More complex, symmetrical bis-benzimidazoles and a bis-carboxyphenyl were then assayed as fragment-based leads, which procured selective inhibition of the target in the low micromolar range (K(i) = 2-4 µM). The putative mode of inhibition is discussed according to molecular modeling supported by in vitro tests.