Development and characterization of pore-blocking small molecules against cholesterol-dependent cytolysins as anti-virulence strategy

Cholesterol-dependent cytolysins (CDC) are acknowledged virulence factors of a wide range of pathogenic gram-positive bacteria like Streptococci, Clostridia, and Listeria etc. CDC selectively target membranes that contain cholesterol and induce pathogenesis in the host organisms by versatile mechanisms. Bacteria release CDC during an infection, which infuriate the infection and the treatment of infection becomes challenging with the contemporary regime of anti-infectives. The noxious responses of CDC include direct toxicity via pore formation, manipulation of cellular signaling to promote bacterial survival and evasion of the immune system. Worldwide, millions of deaths are associated with CDC like PLY, PFO, LLO etc. Therefore, CDC have been considered as a potential target for drugs. In the research of the last few decades, multiple natural and synthetic small molecules have been proposed as anti-toxins, however, due to lack of specificity, none of them were contemplated for clinical testing. Thus, to date there is no specific treatment available to counter CDC manifestations and the goal of this project is to develop and validate specific small molecules against CDC. PLY was selected as a prototype CDC for testing of small molecules. In our earlier work, a model of PLY-pore was developed to virtually screen half a million molecules from online databases that could block the oligomerization and ultimately pore formation of PLY. Pore-Blocker-1 (PB-1), was the only molecule that actively inhibited PLY in a hemolysis assay and, thereon, its optimization led to the discovery of PB-2 with ten times improved activity. In this dissertation, PB-1 and 2 were validated in the BLI assay. Cryo-TEM analysis showcased that PB-2 prevented the PLY penetration in membranes of cholesterol-liposomes. By analyzing several closely related derivatives of PB-2 the pharmacophore of inhibitors was identified, which enabled the effective alteration of scaffolds to produce PB-3, a more chemically stable and potent inhibitor of PLY. PB-3 exhibited an IC50 of 3 µM in the hemolysis assay and a KD value of 256 nM against PLY in the BLI assay. Analogous potency of PB-3 was observed in the LDH assay and cellular microscopy. PB-3 was generated and quantified through the protein-catalyzed ligation of precursor fragments. The actual mechanism of inhibitors was unveiled by evaluating the activity of inhibitors against multiple PLY-mutants and in contrast to the virtually proposed mechanism of binding, a cysteine mutant of PLY suggested that PB-3 might be binding to a cysteine in the membrane-binding domain of PLY. Then, BLI and MS investigations of cysteine mutant PLY and wild type confirmed the cysteine-mediated reversible covalent interaction between PB-3 and PLY. Afterwards, PB-3 was observed barely active against other cysteine-proteases (PTP-1B and SARS-CoV2), confirming the increased affinity of PB-3 toward PLY. PB-3 blocked PLY in a bacterial infection-model assay, this experiment further affirms selectively of PB-3 to PLY because the cell culture medium contained 13000-fold more free-cysteine than PLY. Finally, PB-3 was examined against two further CDC, PFO and ILY. PFO is analogous to PLY and possesses a cysteine residue in the undecapeptide and when PB-3 was tested, it was nearly 10 times more potent against PFO than PLY. Conversely, ILY is devoid of cysteine and PB-3 was expectedly inactive against ILY. In the light of these results, we presumably consider PB-3 as an inhibitor of cysteine-containing CDC.Cholesterin-abhängige Cytolysine (CDC) sind anerkannte Virulenzfaktoren einer breiten Palette pathogener grampositiver Bakterien wie Streptokokken, Clostridien und Listerien usw. CDC zielen selektiv auf Membranen ab, die Cholesterin enthalten, und induzieren die Pathogenese in den Wirtsorganismen durch vielseitige Mechanismen. Bakterien setzen während einer Infektion CDCs frei, die die Invasivität der Infektion erhöhen, und die Behandlung von Infektionen wird mit der derzeitigen Therapie mit Antiinfektiva zu einer Herausforderung. Zu den schädlichen Reaktionen von CDC gehören die direkte Toxizität über die Porenbildung, die Manipulation der zellulären Signalübertragung zur Förderung des bakteriellen Überlebens und die Umgehung des Immunsystems. Millionen von Todesfällen weltweit sind mit CDC wie PLY, PFO, LLO usw. verbunden. Daher wurden CDC als potenzielles Arzneimittelziel angesehen. Während der Forschung der letzten Jahrzehnte wurden mehrere natürliche und synthetische kleine Moleküle als Antitoxine vorgeschlagen, aufgrund mangelnder Spezifität wurde jedoch keines von ihnen für klinische Tests in Betracht gezogen. In unserer früheren Forschung wurde ein Modell von PLY-Poren entwickelt, um virtuell eine halbe Million Moleküle aus Online-Datenbanken zu Screening, die die Oligomerisierung und letztendlich die Porenbildung von PLY blockieren könnten. Pore Blocker-1 (PB-1) war das einzige Molekül, das PLY in einem Hämolyse-Assay aktiv hemmte, und seine Optimierung führte anschließend zur Entdeckung von PB-2 mit zehnfach verbesserter Aktivität. In dieser Dissertation wurden PB-1 und 2 mit dem BLI-Test validiert. Die Cryo-TEM-Analyse zeigte, dass PB-2 das Eindringen von PLY in die Membranen von Cholesterin-Liposomen verhinderte. Das Pharmakophor der Inhibitoren wurde durch die Analyse mehrerer eng verwandter Derivate von PB-2 identifiziert, was eine effektive Veränderung der Gerüste ermöglichte, um PB-3, einen chemisch stabileren und stärkeren Inhibitor von PLY, zu entwickeln. PB-3 hatte eine IC50 von 3 µM im Hämolyse-Assay und einen KD-Wert von 256 nM für PLY im BLI-Assay. Eine ähnliche Wirksamkeit von PB-3 wurde im LDH-Assay und in der Zellmikroskopie nachgewiesen. PB-3 wurde durch die protein-katalysierte Ligation von Precursor-Fragmenten erzeugt und quantifiziert. PB-3 wurde durch die Proteintemplat-unterstützte Ligation von Precursor-Fragmenten erzeugt und quantifiziert. Der wahre Mechanismus der Inhibitoren wurde durch die Auswertung der Aktivität der Inhibitoren gegen mehrere PLY-Mutanten entdeckt. Im Kontrast zu dem virtuell vorgeschlagenen Bindungsmechanismus deutete eine Cystein-Mutante von PLY darauf hin, dass PB-3 an ein Cystein in der Membran-Bindungsdomäne von PLY binden könnte. Anschließend bestätigten BLI und MS Untersuchungen der Cystein-Mutante PLY und des Wildtyps die Cystein-vermittelte reversible kovalente Interaktion zwischen PB-3 und PLY. Im Anschluss daran wurde PB-3 als kaum aktiv gegen andere Cystein-Proteasen (PTP-1B und SARS-CoV2) identifiziert, was die erhöhte Affinität von PB-3 gegenüber PLY bestätigt. PB-3 blockierte PLY in einem bakteriellen Infektionsmodell-Assay. Dieses Experiment bestätigt außerdem die Selektivität von PB-3 für PLY, da das Zellkulturmedium 13000-mal mehr freies Cystein als PLY enthielt. Zum Schluss wurde PB-3 gegen zwei weitere CDC, PFO und ILY, getestet. PFO ist analog zu PLY und enthält Cystein, und bei der Prüfung von PB-3 wurde festgestellt, dass es fast zehnmal stärker gegen PFO wirkt als PLY. Umgekehrt ist ILY frei von Cystein und PB-3 war erwartungsgemäß inaktiv gegenüber ILY. In Anbetracht dieser Ergebnisse halten wir PB-3 vermutlich für einen Hemmstoff gegen Cystein-haltiges CDC

Impact of Perceived Competence and Academic Self efficacy on the Academic Major Satisfaction among University Students

The study aims to explore the impact of Perceived Competence and Academic Self efficacy on the Academic Major Satisfaction of university students. The data was collected from university students in Multan using a simple random sampling approach with a sample size of 110 individuals. The data was collected through survey. Three scales were used: Perceived competence Scale (subscale of the BMPN (Sheldon & Hilpert, 2012), Academic Self efficacy Scale (Larson, et al., 1994) and Academic major Satisfaction Scale (AMSS; Nauta, 2007) to find the impact of Perceived competence and Academic Self efficacy on the Academic major satisfaction. Statistical analysis was conducted through SPSS and the results indicated that Perceived competence has positive correlation with academic self- efficacy on academic major satisfaction and a significant impact of Perceived Competence and Academic Self efficacy on the Academic Major Satisfaction of university student’s area of this study was restricted to Multan. There was a time limitation. Future researches can include a large number of participants and also cover large research area for generalize ability. This study is significant in determining the Academic Performance of university students in the social framework of Pakistani society

Diagnostic Reliability of Paediatric Appendicitis Score & Ultrasound Scan in Children with Suspected Acute Appendicitis

Objective:  To evaluate the efficacy of Paediatric Appendicitis Score and ultrasound scan in the paediatric population. This study evaluates the best predictive measure for diagnosing acute appendicitis.Methodology: We conducted a prospective observational study on 120 children (02-13 years) in the department of Neonatal and Paediatric Surgery at The Children’s Hospital PIMS, Islamabad, with suspected appendicitis. Each patient was evaluated based on their Paediatric Appendicitis Score and ultrasound findings. We performed open appendectomy under general anaesthesia in patients with Paediatric Appendicitis Score ≥06 or who presented with severe tenderness on cough, TLC more than 10,000/mm3, or positive ultrasound findings.Results: In our study, the sensitivity and specificity of the Paediatric Appendicitis Score were 92.6% and 72.0%, respectively. Its sensitivity and specificity were 84.09% and 85.71%, respectively, with cutoff ≥6 combined with ultrasound findings. The combined sensitivity and specificity of Paediatric Appendicitis Score <6 and ultrasound were 85.71% and 94.44%, respectively.Conclusion: Paediatric Appendicitis Score is a useful diagnostic tool for suspected appendicitis in pediatric population. Its efficacy is significantly increased when combined with ultrasound. Combined application of the Paediatric Appendicitis Score with ultrasonography helps to rule out negative appendectomies and reduces complications associated with delayed diagnosis

Narrative Review: Use of Competent Stimulating Peptide in Gene Transfer Via Suicide Plasmid in Streptococcus pneumoniae

Natural competency for genetic transformation of Streptococcus pneumoniae causes the emergence of novel or non-vaccine preventable pneumococcal serotypes. This phenomenon has become a global concern as it can spread quickly in the population through inhalation and close contact. The colonisation of S. pneumoniae at the upper respiratory tract can either become commensal or pathogenic. Once the bacterium invades into the body system, it will secrete its toxin and virulence protein to facilitate the invasion. Besides, S. pneumoniae can undergo natural biological transformation via uptake of exogenous DNA by horizontal gene transfer for integration and recombination of the genome. S. pneumoniae natural transformation is aided by competence-stimulating peptide (CSP) that induces the competence of bacteria. Natural transformation cascade of S. pneumoniae via CSP is triggered in the presence of conserved 17-amino acids peptide which is regulated and encoded by comC, comD and comE operon, where comC is responsible in secreting precursor CSP. Nowadays, researchers transforming S. pneumoniae by inserting the mutated S. pneumoniae gene through a vector, suicide plasmid. Suicide plasmids such as pID701, pAUL-A and pVA891 can be transferred but cannot replicate in the bacteria. Homologous recombination process occurs once the mutated gene of suicide plasmid is integrated with wild-type S. pneumoniae. Previous studies had used the transformation of suicide plasmid into S. pneumoniae as it can integrate with host DNA at specific insert for gene transfer. But there is no evidence on the role of CSP in horizontal/gene replacement via suicide plasmid in Streptococcus pneumoniae. This narrative review's scope as per defined purpose statement is to relate and recommend the use of competent stimulating peptide in efficient horizontal gene transfer via suicide plasmids in Streptococcus pneumoniae.Keywords: Streptococcus pneumoniae transformation; CSP-based transformation; Gene transfer via suicide plasmid; Genetic exchange; Competence-stimulating peptid

A Critical Study on Acylating and Covalent Reversible Fragment Inhibitors of SARS-CoV-2 Main Protease Targeting the S1 Site with Pyridine

SARS coronavirus main proteases (3CL proteases) have been validated as pharmacological targets for the treatment of coronavirus infections. Current inhibitors of SARS main protease, including the clinically admitted drug nirmatrelvir are peptidomimetics with the downsides of this class of drugs including limited oral bioavailability, cellular permeability, and rapid metabolic degradation. Here, we investigate covalent fragment inhibitors of SARS Mpro as potential alternatives to peptidomimetic inhibitors in use today. Starting from inhibitors acylating the enzyme's active site, a set of reactive fragments was synthesized, and the inhibitory potency was correlated with the chemical stability of the inhibitors and the kinetic stability of the covalent enzyme-inhibitor complex. We found that all tested acylating carboxylates, several of them published prominently, were hydrolyzed in assay buffer and the inhibitory acyl-enzyme complexes were rapidly degraded leading to the irreversible inactivation of these drugs. Acylating carbonates were found to be more stable than acylating carboxylates, however, were inactive in infected cells. Finally, reversibly covalent fragments were investigated as chemically stable SARS CoV-2 inhibitors. Best was a pyridine-aldehyde fragment with an IC50 of 1.8 μM at a molecular weight of 211 g/mol, showing that pyridine fragments indeed are able to block the active site of SARS-CoV-2 main protease

Commands of Synthetic Biology to Modernize and Re-design the Biological Systems

The scope of synthetic biology continues to expand and has encompassed a huge number of biological features. Its scope starts from scratch, enabling the de novo synthesis of biological systems. It has re-designed the biological systems and empowered the production of synthetic genes, RNA, DNA and proteins by undertaking the control of pathways involved in genetic regulation. It has increased the production of nano-scale RNA architectures and synthetic biological circuits which either have therapeutic or other productive uses. Furthermore, advancements in synthetic biology have enabled the generation of diversity through methods such as epPCR and site-directed mutagenesis, allowing for the creation of complex genetic variations. Additionally, synthetic biology intersects with computer engineering to design functional biological devices and circuits, utilizing computational analysis to guide the design process. Moreover, ethical and regulatory considerations are paramount in synthetic biology, with careful examination required to address dual-use concerns, environmental impacts, and issues of social justice and equitable access to benefits. As synthetic biology continues to advance, it presents opportunities to address pressing challenges in fields ranging from medicine and agriculture to environmental conservation and beyond. Thus, the fusion of synthetic biology with other scientific disciplines holds promise for transformative innovation and societal benefit. The present discussion enlightened the core of generating complex biological systems and has given a brief overview on the fusion of synthetic biology with other fields of science.Keywords: Biological Systems; Genetic Regulation; Synthetic biology circuits 

Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins

Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well

Vitamin D Detection Using Electrochemical Biosensors: A Comprehensive Overview

Vitamin D plays a vital role in health; therefore, there is a need for a sensitive, selective, quick, and easy technique for its determination. Previous research has proposed electrochemical biosensors based on different carbon materials that are functionalized with various electrochemical biosensors. However, the existing problems and future opportunities for these sensors need further research. The practical use of electrochemical biosensors for vitamin D detection is attributed to their ability to detect vitamin D from diverse samples, including vitamin D production, in nature. This chapter provides recent investigations on the utilization of electrochemical biosensors for vitamin D detection such as Ab-25OHD/SPE/FMTAD, CYP27B1/GCE, SiO2/GO/Ni(OH)2/GCE, BSA/Ab-VD2/CD-CH/ITO, BSA/Anti VD/Fe3O4 PANnFs/ITO, BSA/Ab-VD/Asp-Gd2O3NRs/ITO, 25OHD, 25OHD Antibody, IoT-Enabled Enzyme Embossed Biosensor, Au-Pt NPs/APTES/FTO, and GCN-β-CD/Au nanocomposite. The chapter aims to provide a comprehensive overview of the recent developments in electrochemical biosensors for accurate and efficient vitamin D detection

Chemical Evolution of Antivirals Against Enterovirus D68 through Protein-Templated Knoevenagel Reactions

The generation of bioactive molecules from inactive precursors is a crucial step in the chemical evolution of life, however, mechanistic insights into this aspect of abiogenesis are scarce. Here, we investigate the protein-catalyzed formation of antivirals by the 3C-protease of enterovirus D68. The enzyme induces aldol condensations yielding inhibitors with antiviral activity in cells. Kinetic and thermodynamic analyses reveal that the bioactivity emerges from a dynamic reaction system including inhibitor formation, alkylation of the protein target by the inhibitors, and competitive addition of non-protein nucleophiles to the inhibitors. The most active antivirals are slowly reversible inhibitors with elongated target residence times. The study reveals first examples for the chemical evolution of bio-actives through protein-catalyzed, non-enzymatic C−C couplings. The discovered mechanism works under physiological conditions and might constitute a native process of drug development