Computational analysis of human gut microbial prolyl oligopeptidases (POPs) reveal candidate genes as therapeutics for celiac disease.

Celiac disease (CD) is a common autoimmune disorder in which the patients are unable to digest gluten, which is present in foods made up of wheat, barley and rye. Whilst diagnosis happens late in 80% of the cases, avoidance of such foods appears to be the common solution. Alternative management strategies are required for the patients and their families since CD is also genetically carried over. Probiotic therapeutics and the consumption of appropriate enzymes, such as prolyloligopeptidases (POPs), from gut-friendly bacteria could reduce the disease burden and provide a better lifestyle for CD patients. We have examined around 5000 gut bacterial genomes and identified nearly 4000 non-redundant putative POPs. A select set of 10 gut bacterial POP sequences were subject to three-dimensional modelling, ligand docking and molecular dynamics simulations where stable interactions were observed between the POPs and gluten peptides. Our study provides sequence and structural analysis of potential POP enzymes in gut bacterial genomes, which form a strong basis to offer probiotic solutions to CD patients. In particular, these enzymes could be lead future therapeutics for this disease.

Predicting repurposed drugs targeting the NS3 protease of dengue virus using machine learning-based QSAR, molecular docking, and molecular dynamics simulations.

Dengue fever, prevalent in Southeast Asian countries, currently lacks effective pharmaceutical interventions for virus replication control. This study employs a strategy that combines machine learning (ML)-based quantitative-structure-activity relationship (QSAR), molecular docking, and molecular dynamics simulations to discover potential inhibitors of the NS3 protease of the dengue virus. We used nine molecular fingerprints from PaDEL to extract features from the NS3 protease dataset of dengue virus type 2 in the ChEMBL database. Feature selection was achieved through the low variance threshold, F-Score, and recursive feature elimination (RFE) methods. Our investigation employed three ML models - support vector machine (SVM), random forest (RF), and extreme gradient boosting (XGBoost) - for classifier development. Our SVM model, combined with SVM-RFE, had the best accuracy (0.866) and ROC_AUC (0.964) in the testing set. We identified potent inhibitors on the basis of the optimal classifier probabilities and docking binding affinities. SHAP and LIME analyses highlighted the significant molecular fingerprints (e.g. ExtFP69, ExtFP362, ExtFP576) involved in NS3 protease inhibitory activity. Molecular dynamics simulations indicated that amphotericin B exhibited the highest binding energy of -212 kJ/mol and formed a hydrogen bond with the critical residue Ser196. This approach enhances NS3 protease inhibitor identification and expedites the discovery of dengue therapeutics.

Elucidating the inhibitory mechanism of Zika virus NS2B-NS3 protease with dipeptide inhibitors: Insights from molecular docking and molecular dynamics simulations.

Microcephaly, Guillain-Barré syndrome, and potential sexual transmission stand as prominent complications associated with Zika virus (ZIKV) infection. The absence of FDA-approved drugs or vaccines presents a substantial obstacle in combatting the virus. Furthermore, the inclusion of pregnancy in the pharmacological screening process complicates and extends the endeavor to ensure molecular safety and minimal toxicity. Given its pivotal role in viral assembly and maturation, the NS2B-NS3 viral protease emerges as a promising therapeutic target against ZIKV. In this context, a dipeptide inhibitor was specifically chosen as a control against 200 compounds for docking analysis. Subsequent molecular dynamics simulations extending over 200 ns were conducted to ascertain the stability of the docked complex and confirm the binding of the inhibitor at the protein's active site. The simulation outcomes exhibited conformity to acceptable thresholds, encompassing parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), ligand-protein interaction analysis, ligand characterization, and surface area analysis. Notably, analysis of ligand angles bolstered the identification of prospective ligands capable of inhibiting viral protein activity and impeding virus dissemination. In this study, the integration of molecular docking and dynamics simulations has pinpointed the dipeptide inhibitor as a potential candidate ligand against ZIKV protease, thereby offering promise for therapeutic intervention against the virus.

Prying the lid open: Atomic-level insights on sialoglycan-TMPRSS2 coordination in HKU1 entry.

The pre-fusion coronavirus HKU1 spike binds host sialoglycans and proteinaceous receptor TMPRSS2 for cell entry. In this issue of Cell, three papers by Fernández et al., McCallum et al., and Wang et al. provide structural information on HKU1 spike interactions with host receptors, providing insights into its multi-step opening.

Gene cloning, IPTG-independent auto-induction and characterization of a novel hyperstable S9 prolyl oligopeptidase having lipolytic activity from Thermotoga naphthophila RKU-10T with applications.

A hyperstable lipase from Thermotoga naphthophila (TnLip) was cloned and overexpressed as a soluble and active monomeric protein in an effectual mesophilic host system. Sequence study revealed that TnLip is a peptidase S9 prolyl oligopeptidase domain (acetyl esterase/lipase-like protein), belongs to alpha/beta (α/ß)-hydrolase superfamily containing a well-conserved α/ß-hydrolase fold and penta-peptide (GLSAG) motif. Various cultivation and induction strategies were applied to improve the heterologous expression and bacterial biomass, but TnLip intracellular activity was enhanced by 14.25- fold with IPTG-independent auto-induction approach after 16 h (26 °C, 150 rev min-1) incubation. Purified TnLip (35 kDa) showed peak activity at 85 °C in McIlvaine buffer (pH 7.0-8.0), and has great stability over a broad range of pH (5.0-10.0), and temperature (40-85 °C) for 8 h. TnLip exhibited prodigious resistance toward various commercial detergents, chemical additives, and salt. TnLip activity was improved by 170.51 %, 130.67 %, 127.42 %, 126.54 %, 126.61 %, 120.32 %, and 116.31 % with 50 % (v/v) of methanol, ethanol, n-butanol, isopropanol, acetone, glycerol, and acetic acid, respectively. Moreover, with 3.0 M of NaCl, and 10 mM of Ca2+, Mn2+, and Mg2+ TnLip activity was augmented by 210 %, 185.64 %, 152.03 %, and 116.26 %, respectively. TnLip has an affinity with various substrates (p-nitrophenyl ester and natural oils) but maximal hydrolytic activity was perceived with p-nitrophenyl palmitate (pNPP, 3600 U mg-1) and olive oil (1182.05 U mg-1). The values of Km (0.576 mM), Vmax (4216 µmol mg-1 min-1), VmaxKm-1 (7319.44 min-1), kcat (1106.74 s-1), and kcatKm-1 (1921.42 mM-1 s-1) were calculated using pNPP substrate. Additionally, TnLip degraded animals' fats and removed oil stains within 3 h and 5 min, respectively. All these features make halo-alkali-thermophilic TnLip as an auspicious contender for laundry detergents (cleaning bio-additive), fat degradation, wastewater treatment and endorse eco-friendly stewardship along with various other biotechnological applications.

Comparative analysis of thermal adaptations of extremophilic prolyl oligopeptidases.

Prolyl oligopeptidases from psychrophilic, mesophilic, and thermophilic organisms found in a range of natural environments are studied using a combination of protein structure prediction, atomistic molecular dynamics, and trajectory analysis to determine how the S9 protease family adapts to extreme thermal conditions. We compare our results with hypotheses from the literature regarding structural adaptations that allow proteins to maintain structure and function at extreme temperatures, and we find that, in the case of prolyl oligopeptidases, only a subset of proposed adaptations are employed for maintaining stability. The catalytic and propeller domains are highly structured, limiting the range of mutations that can be made to enhance hydrophobicity or form disulfide bonds without disrupting the formation of necessary secondary structure. Rather, we observe a pattern in which overall prevalence of bound interactions (salt bridges and hydrogen bonds) is conserved by using increasing numbers of increasingly short-lived interactions as temperature increases. This suggests a role for an entropic rather than energetic strategy for thermal adaptation in this protein family.

Exploring the antiviral inhibitory activity of Niloticin against the NS2B/NS3 protease of Dengue virus (DENV2).

Dengue virus (DENV2) is the cause of dengue disease and a worldwide health problem. DENV2 replicates in the host cell using polyproteins such as NS3 protease in conjugation with NS2B cofactor, making NS3 protease a promising antiviral drug-target. This study investigated the efficacy of 'Niloticin' against NS2B/NS3-protease. In silico and in vitro analyses were performed which included interaction of niloticin with NS2B/NS3-protease, protein stability and flexibility, mutation effect, betweenness centrality of residues and analysis of cytotoxicity, protein expression and WNV NS3-protease activity. Similar like acyclovir, niloticin forms strong H-bonds and hydrophobic interactions with residues LEU149, ASN152, LYS74, GLY148 and ALA164. The stability of the niloticin-NS2B/NS3-protease complex was found to be stable compared to the apo NS2B/NS3-protease in structural deviation, PCA, compactness and FEL analysis. The IC50 value of niloticin was 0.14 µM in BHK cells based on in vitro cytotoxicity analysis and showed significant activity at 2.5 µM in a concentration-dependent manner. Western blotting and qRT-PCR analyses showed that niloticin reduced DENV2 protein transcription in a dose-dependent manner. Besides, niloticin confirmed the inhibition of NS3-protease by the SensoLyte 440 WNV protease detection kit. These promising results suggest that niloticin could be an effective antiviral drug against DENV2 and other flaviviruses.

Substitutions in the nonactive site of the passenger domain on the activity of Haemophilus influenzae immunoglobulin A1 protease.

Immunoglobulin A1 (IgA1) protease is a critical virulence factor of Haemophilus influenzae that facilitates bacterial mucosal infection. This study investigates the effect of iga gene polymorphism on the enzymatic activity of H. influenzae IgA1 protease. The IgA1 protease activity was examined in the H. influenzae Rd KW20 strain and 51 isolates. Genetic variations in iga and deduced amino acid substitutions affecting IgA1 protease activity were assessed. Machine learning tools and functional complementation assays were used to analyze the effects of identified substitutions on the stability and activity of IgA1 protease, respectively. All 51 isolates exhibited similar iga expression levels. No igaB expression was detected. According to comparisons with the reference Rd KW20 strain, four substitutions in the protease domain, 26 in the nonprotease passenger domain, and two in the ß-barrel domain were associated with the change in IgA1 protease activity. No substitutions in the catalytic site of IgA1 protease were observed. Logistic regression, receiver operating characteristic curves, Venn diagrams, and protein stability analyses revealed that the substitutions Asn352Lys, Pro353Ala, Lys356Asn, Gln916Lys, and Gly917Ser, which were located in the nonactive site of the passenger domain, were associated with decreases in IgA1 protease activity and stability, whereas Asn914Lys was associated with an increase in these events. Functional complementation assays revealed that the Asn914Lys substitution increased IgA1 protease activity in the Rd KW20 strain. This study identified substitutions in the nonactive site of the passenger domain that affect both the activity and stability of H. influenzae IgA1 protease.

Synthesis, biological evaluation, and molecular modelling of substituted thiazolyl thiourea derivatives: A new class of prolyl oligopeptidase inhibitors.

Prolyl oligopeptidase (POP) is a compelling therapeutic target associated with aging and neurodegenerative disorders due to its pivotal role in neuropeptide processing. Despite initial promise demonstrated by early-stage POP inhibitors, their progress in clinical trials has been halted at Phase I or II. This impediment has prompted the pursuit of novel inhibitors. The current study seeks to contribute to the identification of efficacious POP inhibitors through the design, synthesis, and comprehensive evaluation (both in vitro and in silico) of thiazolyl thiourea derivatives (5a-r). In vitro experimentation exhibited that the compounds displayed significant higher potency as POP inhibitors. Compound 5e demonstrated an IC50 value of 16.47 ± 0.54 µM, representing a remarkable potency. A meticulous examination of the structure-activity relationship indicated that halogen and methoxy substituents were the most efficacious. In silico investigations delved into induced fit docking, pharmacokinetics, and molecular dynamics simulations to elucidate the intricate interactions, orientation, and conformational changes of these compounds within the active site of the enzyme. Moreover, our pharmacokinetic assessments confirmed that the majority of the synthesized compounds possess attributes conducive to potential drug development.

Unraveling the mechanism of small molecule induced activation of Staphylococcus aureus signal peptidase IB.

Staphylococcus aureus signal peptidase IB (SpsB) is an essential enzyme for protein secretion. While inhibition of its activity by small molecules is a well-precedented mechanism to kill bacteria, the mode of activation is however less understood. We here investigate the activation mechanism of a recently introduced activator, the antibiotic compound PK150, and demonstrate by combined experimental and Molecular Dynamics (MD) simulation studies a unique principle of enzyme stimulation. Mass spectrometric studies with an affinity-based probe of PK150 unravel the binding site of PK150 in SpsB which is used as a starting point for MD simulations. Our model shows the localization of the molecule in an allosteric pocket next to the active site which shields the catalytic dyad from excess water that destabilizes the catalytic geometry. This mechanism is validated by the placement of mutations aligning the binding pocket of PK150. While the mutants retain turnover of the SpsB substrate, no stimulation of activity is observed upon PK150 addition. Overall, our study elucidates a previously little investigated mechanism of enzyme activation and serves as a starting point for the development of future enzyme activators.

SPRING licenses S1P-mediated cleavage of SREBP2 by displacing an inhibitory pro-domain.

Site-one protease (S1P) conducts the first of two cleavage events in the Golgi to activate Sterol regulatory element binding proteins (SREBPs) and upregulate lipogenic transcription. S1P is also required for a wide array of additional signaling pathways. A zymogen serine protease, S1P matures through autoproteolysis of two pro-domains, with one cleavage event in the endoplasmic reticulum (ER) and the other in the Golgi. We recently identified the SREBP regulating gene, (SPRING), which enhances S1P maturation and is necessary for SREBP signaling. Here, we report the cryo-EM structures of S1P and S1P-SPRING at sub-2.5 Å resolution. SPRING activates S1P by dislodging its inhibitory pro-domain and stabilizing intra-domain contacts. Functionally, SPRING licenses S1P to cleave its cognate substrate, SREBP2. Our findings reveal an activation mechanism for S1P and provide insights into how spatial control of S1P activity underpins cholesterol homeostasis.

Computational screening combined with well-tempered metadynamics simulations identifies potential TMPRSS2 inhibitors.

Type-II transmembrane serine proteases are effective pharmacological targets for host defence against viral entry and in certain cancer cell progressions. These serine proteases cleave viral spike proteins to expose the fusion peptide for cell entry, which is essential to the life cycle of the virus. TMPRSS2 inhibitors can also fight against respiratory viruses that employ them for cell entry. Our study combining virtual screening, all-atom molecular dynamics, and well-tempered metadynamics simulation identifies vicenin-2, neohesperidin, naringin, and rhoifolin as promising TMPRSS2 antagonists. The binding energies obtained are - 16.3, - 15.4, - 13.6, and - 13.8 kcal/mol for vicenin-2, neohesperidin, naringin, and rhoifolin respectively. The RMSD, RMSF, PCA, DCCM, and binding free energy profiles also correlate with the stable binding of these ligands at the active site of TMPRSS2. The study reveals that these molecules could be promising lead molecules for combating future outbreaks of coronavirus and other respiratory viruses.

Use of protease substrate specificity screening in the rational design of selective protease inhibitors with unnatural amino acids: Application to HGFA, matriptase, and hepsin.

Inhibition of the proteolytic processing of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis. Here, we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries (PS-SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF/MSP activating serine proteases, HGFA, matriptase, and hepsin. These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease. We demonstrate that by varying the P2, P3, and P4 positions of the inhibitor with unnatural amino acids based on the protease substrate preferences learned from HyCoSuL, we can predictably modify the potency and selectivity of the inhibitor. We identified the tetrapeptide JH-1144 (8) as a single digit nM inhibitor of HGFA, matriptase and hepsin with excellent selectivity over Factor Xa and thrombin. These unnatural peptides have increased metabolic stability relative to natural peptides of similar structure. The tripeptide inhibitor PK-1-89 (2) has excellent pharmacokinetics in mice with good compound exposure out to 24 h. In addition, we obtained an X-ray structure of the inhibitor MM1132 (15) bound to matriptase revealing an interesting binding conformation useful for future inhibitor design.

Structural basis of TMPRSS2 zymogen activation and recognition by the HKU1 seasonal coronavirus.

The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.

Human coronavirus HKU1 recognition of the TMPRSS2 host receptor.

The human coronavirus HKU1 spike (S) glycoprotein engages host cell surface sialoglycans and transmembrane protease serine 2 (TMPRSS2) to initiate infection. The molecular basis of HKU1 binding to TMPRSS2 and determinants of host receptor tropism remain elusive. We designed an active human TMPRSS2 construct enabling high-yield recombinant production in human cells of this key therapeutic target. We determined a cryo-electron microscopy structure of the HKU1 RBD bound to human TMPRSS2, providing a blueprint of the interactions supporting viral entry and explaining the specificity for TMPRSS2 among orthologous proteases. We identified TMPRSS2 orthologs from five mammalian orders promoting HKU1 S-mediated entry into cells along with key residues governing host receptor usage. Our data show that the TMPRSS2 binding motif is a site of vulnerability to neutralizing antibodies and suggest that HKU1 uses S conformational masking and glycan shielding to balance immune evasion and receptor engagement.

In silico screening of potential plant peptides against the non-structural proteins of dengue virus.

BACKGROUND OBJECTIVES: Peptides isolated from different sources of plants have the advantages of specificity, lower toxicity, and increased therapeutic effects; hence, it is necessary to search for newer antivirals from plant sources for the treatment of dengue viral infections. METHODS: In silico screening of selected plant peptides against the non-structural protein 1, NS3 protease domain (NS2B-NS3Pro) with the cofactor and ATPase/helicase domain (NS3 helicase domain/NS3hel) of dengue virus was performed. The physicochemical characteristics of the peptides were calculated using Protparam tools, and the allergenicity and toxicity profiles were assessed using allergenFP and ToxinPred, respectively. RESULTS: Among the tested compounds, Ginkbilobin demonstrated higher binding energy against three tested nonstructural protein targets. Kalata B8 demonstrated maximum binding energy against NSP-1 and NSP-2, whereas Circulin A acted against the NSP3 protein of dengue virus. INTERPRETATION CONCLUSION: The three compounds identified by in silico screening can be tested in vitro, which could act as potential leads as they are involved in hampering the replication of the dengue virus by interacting with the three prime non-structural proteins.

Identification of novel and potential inhibitors against the dengue virus NS2B/NS3 protease using virtual screening and biomolecular simulations.

Approximately 3.9 billion individuals are vulnerable to dengue infection, a prevalent cause of tropical diseases worldwide. Currently, no drugs are available for preventing or treating Flavivirus diseases, including Dengue, West Nile, and the more recent Zika virus. The highly conserved Flavivirus NS2B-NS3 protease, crucial for viral replication, is a promising therapeutic target. This study employed in-silico methodologies to identify novel and potentially effective anti-dengue small molecules. A pharmacophore model was constructed using an experimentally validated NS2B-NS3 inhibitor, with the Gunner Henry score confirming the model's validity. The Natural Product Activity and Species Source (NPASS) database was screened using the validated pharmacophore model, yielding a total of 60 hits against the NS2B-NS3 protease. Furthermore, the docking finding reveals that our newly identified compounds from the NPASS database have enhanced binding affinities and established significant interactions with allosteric residues of the target protein. MD simulation and post-MD analysis further validated this finding. The free binding energy was computed in terms of MM-GBSA analysis, with the total binding energy for compound 1 (-57.3 ± 2.8 and - 52.9 ± 1.9 replica 1 and 2) indicating a stronger binding affinity for the target protein. Overall, this computational study identified these compounds as potential hit molecules, and these findings can open up a new avenue to explore and develop inhibitors against Dengue virus infection.

The LexA-RecA* structure reveals a cryptic lock-and-key mechanism for SOS activation.

The bacterial SOS response plays a key role in adaptation to DNA damage, including genomic stress caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein filament that forms on single-stranded DNA, binds to and stimulates autoproteolysis of the repressor LexA. Here, we present the structure of the complete Escherichia coli SOS signal complex, constituting full-length LexA bound to RecA*. We uncover an extensive interface unexpectedly including the LexA DNA-binding domain, providing a new molecular rationale for ordered SOS gene induction. We further find that the interface involves three RecA subunits, with a single residue in the central engaged subunit acting as a molecular key, inserting into an allosteric binding pocket to induce LexA cleavage. Given the pro-mutagenic nature of SOS activation, our structural and mechanistic insights provide a foundation for developing new therapeutics to slow the evolution of antibiotic resistance.

Study of the Conformational Dynamics of Prolyl Oligopeptidase by Mass Spectrometry: Lessons Learned.

Ion mobility mass spectrometry (IM-MS) can be used to analyze native proteins according to their size and shape. By sampling individual molecules, it allows us to study mixtures of conformations, as long as they have different collision cross sections and maintain their native conformation after dehydration and vaporization in the mass spectrometer. Even though conformational heterogeneity of prolyl oligopeptidase has been demonstrated in solution, it is not detectable in IM-MS. Factors that affect the conformation in solution, binding of an active site ligand, the stabilizing Ser554Ala mutation, and acidification do not qualitatively affect the collision-induced unfolding pattern. However, measuring the protection of accessible cysteines upon ligand binding provides a principle for the development of MS-based ligand screening methods.

Identification of DPP-IV inhibitory peptides derived from buffalo colostrum: Mining through bioinformatics, in silico and in vitro approaches.

Bioactive peptides derived from foods provide physiological health benefits beyond nutrition. This study focused on profiling small peptide inhibitors against two key serine proteases, dipeptidyl peptidase-IV (DPP-IV) and prolyl oligopeptidase (POP). DPP-IV is a well-known protein involved in diverse pathways regulating inflammation, renal, cardiovascular physiology, and glucose homeostasis. POP is yet another key target protein for neurodegenerative disorders. The study evaluated peptide libraries of buffalo colostrum whey and fat globule membrane proteins derived from pepsin and pepsin-pancreatin digestion through in silico web tools and structure-based analysis by molecular docking and binding free-energy estimation, followed by in vitro assay for DPP-IV inhibition for the lead peptides. The bioinformatic study indicated 49 peptides presented motifs with DPP-IV inhibition while 5 peptides with sequences for POP inhibition. In the molecular docking interactions study, 22 peptides interacted with active site residues of DPP-IV and 3 peptides with that of POP. The synthesized peptides, SFVSEVPEL and LTFQHNF inhibited DPP-IV in vitro with an IC50 of 193.5 µM and 1.782 mM, respectively. The study revealed the key residues for inhibition of DPP-IV and POP thus affirming the DPP-IV inhibitory potential of milk-derived peptides.