Computational Design of Miniproteins as SARS-CoV-2 Therapeutic Inhibitors

A rational therapeutic strategy is urgently needed for combating SARS-CoV-2 infection. Viral infection initiates when the SARS-CoV-2 receptor-binding domain (RBD) binds to the ACE2 receptor, and thus, inhibiting RBD is a promising therapeutic for blocking viral entry. In this study, the structure of lead antiviral candidate binder (LCB1), which has three alpha-helices (H1, H2, and H3), is used as a template to design and simulate several miniprotein RBD inhibitors. LCB1 undergoes two modifications: structural modification by truncation of the H3 to reduce its size, followed by single and double amino acid substitutions to enhance its binding with RBD. We use molecular dynamics (MD) simulations supported by ab initio density functional theory (DFT) calculations. Complete binding profiles of all miniproteins with RBD have been determined. The MD investigations reveal that the H3 truncation results in a small inhibitor with a −1.5 kcal/mol tighter binding to RBD than original LCB1, while the best miniprotein with higher binding affinity involves D17R or E11V + D17R mutation. DFT calculations provide atomic-scale details on the role of hydrogen bonding and partial charge distribution in stabilizing the minibinder:RBD complex. This study provides insights into general principles for designing potential therapeutics for SARS-CoV-2.

Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993

Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from Streptomyces griseoflavus (SgfSL). In this work, we have focused on optimizing the substrate-binding pocket (SBP) of SgfSL while simultaneously adjusting the oxygen reduction process. SgfSL variants with three single (Met199Ala, Met199Gly, and Tyr230Ala) and three double amino acid residues substitutions (Met199Gly/His165Ala, His165Ala/Arg240His, Met199Gly/Arg240His) were constructed, purified, and investigated. Combination of substitutions in the SBP and in the tunnel leading to Cu2 ion (Met199Gly/Arg240His) increased SgfSL catalytic activity towards ABTS by 5-fold, and towards 2.6-DMP by 16-fold. The high activity of the Met199Gly/Arg240His variant can be explained by the combined effect of the SBP geometry optimization (Met199Gly) and increased proton flux via the tunnel leading to Cu2 ion (Arg240His). Moreover, the variant with Met199Gly and His165Ala mutations did not significantly increase SgfSL’s activity, but led to a drastic shift in the optimal pH of 2.6-DMP oxidation. These results indicate that His 165 not only regulates oxygen access, but it also participates in proton transport in 2D laccases.

Impaired Ca2+ Sensitivity of a Novel GCAP1 Variant Causes Cone Dystrophy and Leads to Abnormal Synaptic Transmission Between Photoreceptors and Bipolar Cells

Guanylate cyclase-activating protein 1 (GCAP1) is involved in the shutdown of the phototransduction cascade by regulating the enzymatic activity of retinal guanylate cyclase via a Ca2+/cGMP negative feedback. While the phototransduction-associated role of GCAP1 in the photoreceptor outer segment is widely established, its implication in synaptic transmission to downstream neurons remains to be clarified. Here, we present clinical and biochemical data on a novel isolate GCAP1 variant leading to a double amino acid substitution (p.N104K and p.G105R) and associated with cone dystrophy (COD) with an unusual phenotype. Severe alterations of the electroretinogram were observed under both scotopic and photopic conditions, with a negative pattern and abnormally attenuated b-wave component. The biochemical and biophysical analysis of the heterologously expressed N104K-G105R variant corroborated by molecular dynamics simulations highlighted a severely compromised Ca2+-sensitivity, accompanied by minor structural and stability alterations. Such differences reflected on the dysregulation of both guanylate cyclase isoforms (RetGC1 and RetGC2), resulting in the constitutive activation of both enzymes at physiological levels of Ca2+. As observed with other GCAP1-associated COD, perturbation of the homeostasis of Ca2+ and cGMP may lead to the toxic accumulation of second messengers, ultimately triggering cell death. However, the abnormal electroretinogram recorded in this patient also suggested that the dysregulation of the GCAP1–cyclase complex further propagates to the synaptic terminal, thereby altering the ON-pathway related to the b-wave generation. In conclusion, the pathological phenotype may rise from a combination of second messengers’ accumulation and dysfunctional synaptic communication with bipolar cells, whose molecular mechanisms remain to be clarified.

Mass Spectrometry Guided Discovery and Design of Novel Asperphenamate Analogs From Penicillium astrolabium Reveals an Extraordinary NRPS Flexibility

Asperphenamate is a small peptide natural product that has gained much interest due to its antitumor activity. In the recent years numerous bioactive synthetic asperphenamate analogs have been reported, whereas only a handful of natural analogs either of microbial or plant origin has been discovered. Herein we describe a UHPLC-HRMS/MS and amino acid supplement approach for discovery and design of novel asperphenamate analogs. Chemical analysis of Penicillium astrolabium, a prolific producer of asperphenamate, revealed three previously described and two novel asperphenamate analogs produced in significant amounts, suggesting a potential for biosynthesis of further asperphenamate analogs by varying the amino acid availability. Subsequent growth on proteogenic and non-proteogenic amino acid enriched media, revealed a series of novel asperphenamate analogs, including single or double amino acid exchange, as well as benzoic acid exchange for nicotinic acid, with the latter observed from a natural source for the first time. In total, 22 new asperphenamate analogs were characterized by HRMS/MS, with one additionally confirmed by isolation and NMR structure elucidation. This study indicates an extraordinary nonribosomal peptide synthetase (NRPS) flexibility based on substrate availability, and therefore the potential for manipulating and designing novel peptide natural products in filamentous fungi.

Unravelling ceftazidime/avibactam resistance of KPC-28, a KPC-2 variant lacking carbapenemase activity

Background KPC-like carbapenemases have spread worldwide with more than 30 variants identified that differ by single or double amino-acid substitutions. Objectives To describe the steady-state kinetic parameters of KPC-28, which differs from KPC-2 by a H274Y substitution and the deletion of two amino acids (Δ242-GT-243). Methods The blaKPC-2, blaKPC-3, blaKPC-14 and blaKPC-28 genes were cloned into a pTOPO vector for susceptibility testing or into pET41b for overexpression, purification and subsequent kinetic parameter (Km, kcat) determination. Molecular docking experiments were performed to explore the role of the amino-acid changes in the carbapenemase activity. Results Susceptibility testing revealed that Escherichia coli producing KPC-28 displayed MICs that were lower for carbapenems and higher for ceftazidime and ceftazidime/avibactam as compared with KPC-2. The catalytic efficiencies of KPC-28 and KPC-14 for imipenem were 700-fold and 200-fold lower, respectively, than those of KPC-2, suggesting that Δ242-GT-243 in KPC-28 and KPC-14 is responsible for reduced carbapenem hydrolysis. Similarly, the H274Y substitution resulted in KPC-28 in a 50-fold increase in ceftazidime hydrolysis that was strongly reversed by clavulanate. Conclusions We have shown that KPC-28 lacks carbapenemase activity, has increased ceftazidime hydrolytic activity and is strongly inhibited by clavulanate. KPC-28-producing E. coli isolates display an avibactam-resistant ESBL profile, which may be wrongly identified by molecular and immunochromatographic assays as the presence of a carbapenemase. Accordingly, confirmation of carbapenem hydrolysis will be mandatory with assays based solely on blaKPC gene or gene product detection.

The Evolution of Azole Resistance inCandida albicansSterol 14α-Demethylase (CYP51) through Incremental Amino Acid Substitutions

ABSTRACTRecombinantCandida albicansCYP51 (CaCYP51) proteins containing 23 single and 5 double amino acid substitutions found in clinical strains and the wild-type enzyme were expressed inEscherichia coliand purified by Ni2+-nitrilotriacetic acid agarose chromatography. Catalytic tolerance to azole antifungals was assessed by determination of the concentration causing 50% enzyme inhibition (IC50) using CYP51 reconstitution assays. The greatest increase in the IC50compared to that of the wild-type enzyme was observed with the five double substitutions Y132F+K143R (15.3-fold), Y132H+K143R (22.1-fold), Y132F+F145L (10.1-fold), G307S+G450E (13-fold), and D278N+G464S (3.3-fold). The single substitutions K143R, D278N, S279F, S405F, G448E, and G450E conferred at least 2-fold increases in the fluconazole IC50, and the Y132F, F145L, Y257H, Y447H, V456I, G464S, R467K, and I471T substitutions conferred increased residual CYP51 activity at high fluconazole concentrations.In vitrotesting of select CaCYP51 mutations inC. albicansshowed that the Y132F, Y132H, K143R, F145L, S405F, G448E, G450E, G464S, Y132F+K143R, Y132F+F145L, and D278N+G464S substitutions conferred at least a 2-fold increase in the fluconazole MIC. The catalytic tolerance of the purified proteins to voriconazole, itraconazole, and posaconazole was far lower and limited to increased residual activities at high triazole concentrations for certain mutations rather than large increases in IC50values. Itraconazole was the most effective at inhibiting CaCYP51. However, when tested against CaCYP51 mutant strains, posaconazole seemed to be the most resistant to changes in MIC as a result of CYP51 mutation compared to itraconazole, voriconazole, or fluconazole.