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Biology subjects

Guzzo, C. R.

Publications and source records attributed to Guzzo, C. R..

10 recordsLinked to original sources

Pharmacokinetic-Pharmacodynamic Trade-offs in SARS-CoV-2 Main Protease Inhibitors Unveiled through Machine Learning and Molecular Dynamics Simulations

The SARS-CoV-2 main protease (Mpro) is a validated therapeutic target for inhibiting viral replication. Despite the screening of over 55,000 compounds, few candidates have advanced clinically, underscoring the difficulty in optimizing both target affinity and drug-like properties. Thus, developing effective Mpro inhibitors requires balancing high-affinity binding with favorable pharmacokinetic (PK) properties, such as solubility and permeability. To address this challenge, we integrated machine learning (ML) and molecular dynamics (MD) simulations to investigate the trade-offs between pharmacodynamic (PD) and PK properties in Mpro inhibitor design. We developed ML models to classify Mpro inhibitors based on experimental IC50 data, combining molecular descriptors with structural insights from MD simulations. Our Support Vector Machine (SVM) model achieved strong performance (training accuracy = 0.84, ROC AUC = 0.91; test accuracy = 0.79, ROC AUC = 0.86), while our logistic regression model (training accuracy = 0.78, ROC AUC = 0.85; test accuracy = 0.76, ROC AUC = 0.83) identified key molecular features influencing activity, including quantitative estimation of drug-likeness (QED), Log P and molecular weight (ExactMolWt). Notably, PK descriptors often exhibited opposing trends to binding affinity: hydrophilic features enhanced binding affinity but compromised PK properties, whereas hydrogen bonding, hydrophobic and{pi} -{pi} interactions in subsites S2 and S3/S4 are fundamental for binding affinity. Our findings highlight the need for a balanced approach in Mpro inhibitor design, strategically targeting these subsites may balance PD and PK properties. This study provides a computational framework for rational Mpro inhibitor discovery, combining ML and MD to investigate the complex interplay between enzyme inhibition and drug-likeness. These insights may guide in hit-to-lead optimization of the novel next-generation Mpro inhibitors of the SARS-CoV-2 with preclinical and clinical potential.

pharmacology and toxicology↗

The protective function of an immunity protein against the cis-toxic effects of a Xanthomonas Type IV Secretion System Effector

Many bacterial species use specialized secretion systems to translocate proteinaceous toxic effectors into target bacterial cells. In most cases, effectors are encoded in bicistronic operons with their cognate immunity proteins. The current model is that immunity proteins could, in principle, provide protection in two different ways: i) by avoiding self-intoxication (suicide or cis-intoxication) or ii) by inhibiting intoxication due to "friendly-fire" translocation from neighboring sister cells (fratricide or trans-intoxication). Here, we set out to distinguish between these two protection mechanisms in the case of the bactericidal Xanthomonas citri Type IV Secretion System (X-T4SS), where killing is due to the action of a cocktail of secreted effectors (X-Tfes) that are inhibited by their cognate immunity proteins (X-Tfis). We use a set of X. citri mutants lacking multiple X-Tfe/X-Tfi pairs to show that X-Tfis are not absolutely required to protect against trans-intoxication. Our investigation then focused on the in vivo function of the lysozyme-like effector X-TfeXAC2609 and its cognate immunity protein X-TfiXAC2610. We observe the accumulation of damage in the X. citri cell envelope and inhibition of biofilm formation due to the action of X-TfeXAC2609 in the absence of X-TfiXAC2610. We show that X-TfeXAC2609 toxicity is independent of an active X-T4SS and that X-TfiXAC2610 protects the cell colony against X-TfeXAC2609-induced cis-intoxication via autolysis. In vitro assays employing X-TfiXAC2610 mutants were used to test and validate an AlphaFold2-derived model of the X-TfeXAC2609-X-TfiXAC2610 complex which presents topological similarities with the distantly related Tse1/Tsi1 complex from P. aeruginosa and the the i-type lysozyme from Meretrix lusoria (MI-iLys) in complex with PliI-Ah from Aeromonas hydrophila. While immunity proteins in other systems have been shown to protect against attacks by sister cells (trans-intoxication), this is the first description of an antibacterial secretion system in which the immunity proteins are dedicated to protecting cells against cis-intoxication.

biochemistry↗

Cooperative and structural relationships of the trimeric Spike with infectivity and antibody escape of the strains Delta (B.1.617.2) and Omicron (BA.2, BA.5, and BQ.1)

Herein, we simulated the trimeric Spike of the variants B.1.617.2, BA.2, BA.5 and BQ.1 for 300 ns. We derived mechanisms by which the substitutions K417N, L452R, N444T and N460K may favor resistance to neutralizing antibodies. The K417N and L452R contribute to the expansion of the networks of hydrogen bonding interactions with neighboring residues, decreasing their capacity to interact with neutralizing antibodies. The SpikeBQ.1 possesses two unique K444T and N460K mutations that expand the network of hydrogen bonding interactions. This lysine also contributes one novel strong saline interaction and both substitutions may favor resistance to neutralizing antibodies. We also investigated how the substitutions D614G, P681R, and P681H impact Spike structural conformations and discuss the impact of these changes to infectivity and lethality. The prevalent D614G substitution plays a key role in the communication between the glycine and the residues of a {beta}-strand located between the NTD and the RBD, impacting the transition between up- and down-RBD states. The P681R mutation, found in the Delta variant, favors intra- and inter-protomer correlations between the subunits S1 and S2. Conversely, in Omicron sub-variants, P681H decreases the intra- and inter-protomer long-range interactions within the trimeric Spike, providing an explanation for the reduced fusogenicity of this variant. Taken together, our results enhance the knowledge on how novel mutations lead to changes in infectivity and reveal mechanisms by which SARS-CoV-2 may evade the immune system.

biophysics↗

Molecular dynamics simulations of the Spike trimeric ectodomain of the SARS-CoV-2 Omicron variant: structural relationships with infectivity, evasion to immune system and transmissibility

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is replacing Delta, the most prevalent variant worldwide from the beginning of 2021 until early 2022. The Omicron variant is highly transmissible and responsible for a new worldwide COVID-19 wave. Herein, we calculated molecular dynamics simulations of the SARS-CoV-2 trimeric spike protein of Wuhan-Hu-1 strain (wild type, WT) and the Omicron variant of concern. Structural analyses reveal that the SpikeOmicron presents more conformational flexibility than SpikeWT, mainly in the N-terminal domain (NTD) and receptor-binding domain (RBD). Such flexibility results in a broader spectrum of different conformations for SpikeOmicron, whereby the RBD can more easily visit an up-conformational state. We reported how the mutations in this variant may influence the intra- and inter-protomer contacts caused by conformational flexibility of the NTD. Based on our analysis, we suggest that the differences in conformational flexibility between SpikeOmicron and SpikeWT may explain the observed gains in infectivity, immune system evasion and transmissibility in this novel variant. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

Immunization with SARS-CoV-2 nucleocapsid protein triggers a pulmonary immune response in rats

The SARS-CoV-2 pandemic have been affecting millions of people worldwide, since the beginning of 2020. COVID-19 can cause a wide range of clinical symptoms, which varies from asymptomatic presentation to severe respiratory insufficiency, exacerbation of immune response, disseminated microthrombosis and multiple organ failure, which may lead to dead. Due to the rapid spread of SARS-CoV-2, the development of vaccines to minimize COVID-19 severity in the world population is imperious. One of the employed techniques to produce vaccines against emerging viruses is the synthesis of recombinant proteins, which can be used as immunizing agents. Based on the exposed, the aim of the present study was to verify the systemic and immunological effects of IM administration of recombinant Nucleocapsid protein (NP), derived from SARS-CoV-2 and produced by this research group, in 2 different strains of rats (Rattus norvegicus); Wistar and Lewis. For this purpose, experimental animals received 4 injections of NP, once a week, and were submitted to biochemical and histological analysis. Our results showed that NP inoculations were safe for the animals, which presented no clinical symptoms of worrying side effects, nor laboratorial alterations in the main biochemical and histological parameters, suggesting the absence of toxicity induced by NP. Moreover, NP injections successfully triggered the production of specific anti-SARS-CoV-2 IgG antibodies by both Wistar and Lewis rats, showing the sensitization to have been well sufficient for the immunization of these strains of rats. Additionally, we observed the local lung activation of the Bronchus-Associated Lymphoid Tissue (BALT) of rats in the NP groups, suggesting that NP elicits specific lung immune response. Although pre-clinical and clinical studies are still required, our data support the recombinant NP produced by this research group as a potential immunizing agent for massive vaccination, and may represent advantages upon other recombinant proteins, since it seems to induce specific pulmonary protection.

immunology↗

Molecular dynamics analysis of fast-spreading severe acute respiratory syndrome coronavirus 2 variants and their effects in the interaction with human angiotensin-converting enzyme 2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is evolving with mutations in the Spike protein, especially in the receptor-binding domain (RBD). The failure of public health measures to contain the spread of the disease in many countries has given rise to novel viral variants with increased transmissibility. However, key questions about how quickly the variants can spread and whether they can cause a more severe disease remain unclear. Herein, we performed a structural investigation using molecular dynamics simulations and determined dissociation constant (KD) values using surface plasmon resonance (SPR) assays of three fastspreading SARS-CoV-2 variants, Alpha, Beta and Gamma ones, as well as genetic factors in the host cells that may be related to the viral infection. Our results suggest that the SARS-CoV-2 variants facilitate their entry into the host cell by moderately increased binding affinities to the human ACE2 receptor, different torsions in hACE2 mediated by RBD variants, and an increased Spike exposure time to proteolytic enzymes. We also found that other host cell aspects, such as gene and isoform expression of key genes for the infection (ACE2, FURIN and TMPRSS2), may have few contributions to the SARS-CoV-2 variants infectivity. In summary, we concluded that a combination of viral and host cell factors allows SARS-CoV-2 variants to increase their abilities to spread faster than wild-type. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

Molecular and functional basis of a novel Amazonian Dark Earth Esterase 1 (Ade1) with hysteresis behavior and quorum-quenching activity

Amazon Dark Earth (ADE) soil is rich in organic compounds and its fertility has been associated with a high diversity of microorganisms. Herein, we investigate the biochemical and functional features of a novel esterase, Ade1, obtained from a metagenomic library of Amazonian Dark Earth soils of the Amazonian Rainforest, in Brazil. The esterases cleave ester bonds to form a carboxylic and an alcohol group. Esterases and lipases are enzymes found in almost all living organisms, demonstrating their biological relevance. We reported that Ade1 belongs to an /{beta}-hydrolase superfamily. We suggest that Ade1 is a moonlighting enzyme with hysteresis behavior and quorum-quenching activity, which may play a key role in the metabolism of a Gram-negative proteobacteria. In addition, molecular dynamics simulations reveal that the hysteresis behavior is directly associated with structural properties of the cap domain. Our findings reveal details of the molecular basis, catalytic and structural mechanisms of a novel /{beta}-hydrolase, which may be applied to other esterases of biotechnological, food, and/or pharmaceutical interest. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

Molecular dynamics reveals complex compensatory effects of ionic strength on the SARS-CoV-2 Spike/hACE-2 interaction

The SARS-CoV-2 pandemic has already killed more than 800,000 people worldwide. To gain entry, the virus uses its spike protein to bind to host hACE-2 receptors on the host cell surface and mediate fusion between viral and cell membranes. As initial steps leading to virus entry involves significant changes in protein conformation as well as in the electrostatic environment in the vicinity of the spike-hACE-2 complex, we explored the sensitivity of the interaction to changes in ionic strength through computational simulations and surface plasmon resonance. We identified two regions in the receptor-binding domain (RBD), E1 and E2, which interact differently with hACE-2. At high salt concentration, E2-mediated interactions are weakened but are compensated by strengthening E1-mediated hydrophobic interactions. These results provide a detailed molecular understanding of spike RBD/hACE-2 complex formation and stability under a wide range of ionic strengths. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/267351v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1a7dc02org.highwire.dtl.DTLVardef@15d3c78org.highwire.dtl.DTLVardef@2d09c1org.highwire.dtl.DTLVardef@db78a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

Hypertonic saline solution inhibits SARS-CoV-2 in vitro assay

We are facing an unprecedented global health crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At this date more than 680 thousand people have died due to coronavirus disease 2019 (COVID-19). Unfortunately, until now no effective treatment to combat the virus and vaccine are available. We performed experiments to test if hypertonic saline solution is able to inhibit virus replication in vitro. Our data shows that 260 mM NaCl (1.5%) inhibits 100% SARS-CoV-2 replication in Vero cells. Furthermore, our results suggest that the virus replication inhibition is due to an intracellular mechanism and not due to the dissociation between spike SARS-CoV-2 protein and its human receptor angiotensin-converting enzyme 2 interaction. NaCl depolarizes the plasma membrane supposedly associated with the inhibition of the SARS-CoV-2 life cycle. This observation could lead to simple, safe and low cost interventions at various stages of COVID-19 treatment, improving the prognosis of infected patients, thereby mitigating the social and economic costs of the pandemic.

microbiology↗

A Superfamily of T6SS Antibacterial Effectors Displaying L,D-carboxypeptidase Activity Towards Peptidoglycan

Type VI secretion systems (T6SSs) are contractile nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized a Salmonella SPI-6 T6SS antibacterial effector called Tae5STM (type VI amidase effector 5). Tae5STM is toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tai5STM). Microscopy analysis revealed that cells expressing the effector stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovered similarities between Tae5STM and the catalytic domain of L,D-transpeptidase. Point mutations on conserved catalytic histidine and cysteine residues abrogated toxicity. Biochemical assays revealed that Tae5STM displays L,D-carboxypeptidase activity, cleaving peptidoglycan tetrapeptides between meso-diaminopimelic acid3 and D-alanine4. Phylogenetic analysis showed that Tae5STM homologs constitutes a new superfamily of T6SS-associated amidase effectors distributed among -, {beta}- and {gamma}-proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition.

microbiology↗