A structure-based pharmacophore modelling approach to identify and design new neprilysin (NEP) inhibitors: An in silico-based investigation.

Neutral endopeptidase or neprilysin (NEP) cleaves the natriuretic peptides, bradykinin, endothelin, angiotensin II, amyloid ß protein, substance P, etc., thus modulating their effects on heart, kidney, and other organs. NEP has a proven role in hypertension, heart disease, renal disease, Alzheimer's, diabetes, and some cancers. NEP inhibitor development has been in focus since the US FDA approved a combination therapy of angiotensin II type 1 receptor inhibitor (valsartan) and NEP inhibitor (sacubitril) for use in heart failure. Considering the importance of NEP inhibitors the present work focuses on the designing of a potential lead for NEP inhibition. A structure-based pharmacophore modelling approach was employed to identify NEP inhibitors from the pool of 1140 chemical entities obtained from the ZINC database. Based on the docking score and pivotal interactions, ten molecules were selected and subjected to binding free energy calculations and ADMET predictions. The top two compounds were studied further by molecular dynamics simulations to determine the stability of the ligand-receptor complex. ZINC0000004684268, a phenylalanine derivative, showed affinity and complex stability comparable to sacubitril. However, in silico studies indicated that it may have poor pharmacokinetic parameters. Therefore, the molecule was optimized using bioisosteric replacements, keeping the phenylalanine moiety intact, to obtain five potential lead molecules with an acceptable pharmacokinetic profile. The works thus open up the scope to further corroborate the present in silico findings with the biological analysis.

Discovery of blood-brain barrier permeable and orally bioavailable caffeine-based amide derivatives as acetylcholinesterase inhibitors.

Caffeine is one of the privileged natural products that shows numerous effects on the central nervous system. Herein, thirty-one caffeine-based amide derivatives were synthesized and evaluated in vitro for their anticholinesterase activity. The introduction of the amide group to the caffeine core augmented its anticholinesterase activity from an IC50 value of 128 to 1.32 µM (derivative, 6i). The SAR study revealed that N7 substitution on caffeine core is favorable over N1, and the presence of amide 'carbonyl' as a part of the linker contributes to the biological activity. The caffeine core of 6i exhibits interactions with the peripheral anionic site, whereas the N-benzyl ring fits nicely inside the catalytic anionic site. Analog 6i inhibits AChE in a mixed-type mode (Ki 4.58 µM) and crosses the BBB in an in-vitro PAMPA assay. Compound 6i has a descent metabolic stability in MLM (>70% remaining after 30 min) and favorable oral pharmacokinetics in Swiss albino mice.

Thermostable Xanthine Oxidase Activity from Bacillus pumilus RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization.

Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051 U/mg dcw (dry cell weight) to 0.209 U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t1/2 at 70 and 80 °C were 5 and 1 h, respectively. Activity of this enzyme was strongly inhibited by Hg(2+), Ag(+) and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.

Bioactivity predictions and virtual screening using machine learning predictive model.

Recently, there has been significant attention on machine learning algorithms for predictive modeling. Prediction models for enzyme inhibitors are limited, and it is essential to account for chemical biases while developing them. The lack of repeatability in available models and chemical bias issues constrain drug discovery and development. A new prediction model for enzyme inhibitors has been developed, and the model efficacy was checked using Dipeptidyl peptidase 4 (DPP-4) inhibitors. A Python script was prepared and can be provided for personal use upon request. Among various machine learning algorithms, it was found that Random Forest offers the best accuracy. Two models were compared, one with diverse training and test data and the other with a random split. It was concluded that machine learning predictive models based on the Murcko scaffold can address chemical bias concerns. In-silico screening of the Drug Bank database identified two molecules against DPP-4, which are previously proven hit molecules. The approach was further validated through molecular docking studies and molecular dynamics simulations, demonstrating the credibility and relevance of the developed model for future investigations and potential translation into clinical applications.Communicated by Ramaswamy H. Sarma.

A perspective on the development of small molecular neprilysin inhibitors (NEPi) with emphasis on cardiorenal disease.

Neprilysin is a cell surface metallo-endopeptidase, commonly identified as neutral endopeptidase (NEP), that plays a crucial role in the cleavage of peptides, for example, natriuretic peptides, angiotensin II, enkephalins, endothelin, bradykinin, substance P, glucagon-like peptide and amyloid beta. In the case of heart failure, a significant upsurge in NEP activity and expression enhances the degradation of natriuretic peptides. Therefore, NEP inhibitors have gained attention in the field of cardiology. NEP has been studied for over 40 years; however, it has recently gained attention with the US FDA approval of a fixed dose combination of sacubitril (NEP inhibitor) and valsartan (AT-1 inhibitor) for chronic heart failure treatment. The present review elucidates the role of neprilysin in cardiorenal disease, its pathophysiology, and how NEP inhibition benefits. It also summarizes the research advances in NEP inhibitors (NEPi) and their structure-activity relationships. Moreover, the review provides insight into NEPi effectiveness - alone or combined with other cardiorenal protective agents. It is expected to help medicinal chemists synthesize and develop novel NEPi.

FDA approved fused-pyrimidines as potential PI3K inhibitors: a computational repurposing approach.

Fused pyrimidine scaffold is present in several US FDA-approved drugs for various therapeutic indications. Drug repurposing (or drug repositioning) involves the analysis of existing clinically approved drugs for new therapeutic indications. Phosphoinositide-3-kinase (PI3K), via the regulatory PI3K pathway, is involved in cell growth, proliferation, differentiation, survival, and angiogenesis. It is also considered a target in anticancer drug development as it promotes the growth of cancerous cells and increases resistance to anticancer therapy. The present work employed computational techniques like molecular docking, MMGBSA analysis, and molecular dynamics simulations to explore the PI3K inhibition by FDA-approved drugs with fused pyrimidine scaffold. The work identifies Lapatinib as a pan-class I PI3K inhibitor and Dipyridamole as an γ isoform-specific PI3K inhibitor and is reported here.Communicated by Ramaswamy H. Sarma.

Methoxy-naphthyl-Linked N-Benzyl Pyridinium Styryls as Dual Cholinesterase Inhibitors: Design, Synthesis, Biological Evaluation, and Structure-Activity Relationship.

The multifaceted nature of Alzheimer's disease (AD) indicates the need for multitargeted agents as potential therapeutics. Both cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), play a vital role in disease progression. Thus, inhibiting both ChEs is more beneficial than only one for effectively managing AD. The present study provides a detailed lead optimization of the e-pharmacophore-generated pyridinium styryl scaffold to discover a dual ChE inhibitor. A structure-activity relationship analysis indicated the importance of three structural fragments, methoxy-naphthyl, vinyl-pyridinium, and substituted-benzyl, in a dual ChE inhibitor pharmacophore. The optimized 6-methoxy-naphthyl derivative, 7av (SB-1436), inhibits EeAChE and eqBChE with IC50 values of 176 and 370 nM, respectively. The kinetic study has shown that 7av inhibits AChE and BChE in a non-competitive manner with ki values of 46 and 115 nM, respectively. The docking and molecular dynamics simulation demonstrated that 7av binds with the catalytic and peripheral anionic sites of AChE and BChE. Compound 7av also significantly stops the self-aggregation of Aß. The data presented herein indicate the potential of 7av for further investigation in preclinical models of AD.

Design, Synthesis, and Pharmacological Evaluation of Embelin-Aryl/alkyl Amine Hybrids as Orally Bioavailable Blood-Brain Barrier Permeable Multitargeted Agents with Therapeutic Potential in Alzheimer's Disease: Discovery of SB-1448.

The complex and multifaceted nature of Alzheimer's disease has brought about a pressing demand to develop ligands targeting multiple pathways to combat its outrageous prevalence. Embelin is a major secondary metabolite of Embelia ribes Burm f., one of the oldest herbs in Indian traditional medicine. It is a micromolar inhibitor of cholinesterases (ChEs) and ß-site amyloid precursor protein cleaving enzyme 1 (BACE-1) with poor absorption, distribution, metabolism, and excretion (ADME) properties. Herein, we synthesize a series of embelin-aryl/alkyl amine hybrids to improve its physicochemical properties and therapeutic potency against targeted enzymes. The most active derivative, 9j (SB-1448), inhibits human acetylcholinesterase (hAChE), human butyrylcholinesterase (hBChE), and human BACE-1 (hBACE-1) with IC50 values of 0.15, 1.6, and 0.6 µM, respectively. It inhibits both ChEs noncompetitively with ki values of 0.21 and 1.3 µM, respectively. It is orally bioavailable, crosses blood-brain barrier (BBB), inhibits Aß self-aggregation, possesses good ADME properties, and protects neuronal cells from scopolamine-induced cell death. The oral administration of 9j at 30 mg/kg attenuates the scopolamine-induced cognitive impairments in C57BL/6J mice.

A tale of two waves: Delineating diverse genomic and transmission landscapes driving the COVID-19 pandemic in Pune, India.

BACKGROUND: Modern response to pandemics, critical for effective public health measures, is shaped by the availability and integration of diverse epidemiological outbreak data. Tracking variants of concern (VOC) is integral to understanding the evolution of SARS-CoV-2 in space and time, both at the local level and global context. This potentially generates actionable information when integrated with epidemiological outbreak data. METHODS: A city-wide network of researchers, clinicians, and pathology diagnostic laboratories was formed for genome surveillance of COVID-19 in Pune, India. The genomic landscapes of 10,496 sequenced samples of SARS-CoV-2 driving peaks of infection in Pune between December-2020 to March-2022, were determined. As a modern response to the pandemic, a "band of five" outbreak data analytics approach was used. This integrated the genomic data (Band 1) of the virus through molecular phylogenetics with key outbreak data including sample collection dates and case numbers (Band 2), demographics like age and gender (Band 3-4), and geospatial mapping (Band 5). RESULTS: The transmission dynamics of VOCs in 10,496 sequenced samples identified B.1.617.2 (Delta) and BA(x) (Omicron formerly known as B.1.1.529) variants as drivers of the second and third peaks of infection in Pune. Spike Protein mutational profiling during pre and post-Omicron VOCs indicated differential rank ordering of high-frequency mutations in specific domains that increased the charge and binding properties of the protein. Time-resolved phylogenetic analysis of Omicron sub-lineages identified a highly divergent BA.1 from Pune in addition to recombinant X lineages, XZ, XQ, and XM. CONCLUSIONS: The band of five outbreak data analytics approach, which integrates five different types of data, highlights the importance of a strong surveillance system with high-quality meta-data for understanding the spatiotemporal evolution of the SARS-CoV-2 genome in Pune. These findings have important implications for pandemic preparedness and could be critical tools for understanding and responding to future outbreaks.

A perspective on oligonucleotide therapy: Approaches to patient customization.

It is estimated that the human genome encodes 15% of proteins that are considered to be disease-modifying. Only 2% of these proteins possess a druggable site that the approved clinical candidates target. Due to this disparity, there is an immense need to develop therapeutics that may better mitigate the disease or disorders aroused by non-druggable and druggable proteins or enzymes. The recent surge in approved oligonucleotide therapeutics (OT) indicates the imminent potential of these therapies. Oligonucleotide-based therapeutics are of intermediate size with much-improved selectivity towards the target and fewer off-target effects than small molecules. The OTs include Antisense RNAs, MicroRNA (MIR), small interfering RNA (siRNA), and aptamers, which are currently being explored for their use in neurodegenerative disorders, cancer, and even orphan diseases. The present review is a congregated effort to present the past and present of OTs and the current efforts to make OTs for plausible future therapeutics. The review provides updated literature on the challenges and bottlenecks of OT and recent advancements in OT drug delivery. Further, this review deliberates on a newly emerging approach to personalized treatment for patients with rare and fatal diseases with OT.

Studies on ligand-based pharmacophore modeling approach in identifying potent future EGFR inhibitors.

Epidermal growth factor receptor (EGFR) is a validated drug target for cancer chemotherapy. Mutations in EGFR are directly linked with the development of drug resistance and this has led for the development of newer drugs in quest for more efficacious inhibitors. The current research is focused on identifying potential and safe molecules as EGFR inhibitors by using both structure and ligand based computational approaches. In quest for finding newer moieties, we have developed a pharmacophore model utilizing drugs like lazertinib, osimertinib, nazartinib, avitinib, afatininb, and talazoparib that are known to inhibit EGFR along with their downstream signaling. Ligand-based pharmacophore model have been developed to screen the ZINC database through ZINCPharmer webserver. The server has identified 9482 best possible ligands with high pharmacophoric similarity i.e., RMSD value less than 0.2 Å. The top 10 ligands with the criteria of dock score(s) and interactions were further subjected to in silico ADMET studies giving two plausible ligands that were further subjected to Molecular Dynamics and MM/PBSA free energy calculations to ensure stability to the target site. Results deduced by in silico work in the current study may be corroborated biologically in the future. The current work, therefore, provides ample opportunity for computational and medicinal chemists to work in allied areas to facilitate the design and development of novel and more efficacious EGFR inhibitors for future experimental studies.

Exploring insights of hydroxychloroquine, a controversial drug in Covid-19: An update.

The review summarizes chloroquine (CQ) and its safer derivative hydroxychloroquine (HCQ) and its utility in Covid-19. Recently this well-established drug made its way back to the headlines during the SARS-CoV-2 pandemic. This led to an upsurge in the scientific arena with multiple research and review articles along with expert opinions and commentaries. The HCQ has received mixed judgements so far about its efficacy to be used in Covid-19 patients in a limited trial conducted all across the Globe. The purpose of our article is to put forth the history, pharmacodynamics, and pharmacokinetics, along with the existing studies favouring and disapproving the role of HCQ in the treatment of Covid-19. We grouped HCQ use at three stages, this includes HCQ for i. prophylactic use by asymptomatic health workers or peoples at higher risk; ii. patients having mild symptoms; iii. patients with extreme symptoms. The review critically discusses the underlying plausible reasons and mechanisms exploring HCQ in prophylactic management or treatment of SARS-CoV-2. Furthermore, we have critically analysed the reported pharmacokinetic parameters and compiled the proponent, opponent, or neutral opinions on the use of HCQ in Covid-19. Authors discretion is to conduct more studies considering the optimal dosing regimen and pharmacokinetics assessment.

Recent efforts for drug identification from phytochemicals against SARS-CoV-2: Exploration of the chemical space to identify druggable leads.

Nature, which remains a central drug discovery pool, is always looked upon to find a putative druggable lead. The natural products and phytochemical derived from plants are essential during a global health crisis. This class represents one of the most practical and promising approaches to decrease pandemic's intensity owing to their therapeutic potential. The present manuscript is therefore kept forth to give the researchers updated information on undergoing research in allied areas of natural product-based drug discovery, particularly for Covid-19 disease. The study briefly shreds evidence from in vitro and in silico researches done so far to find a lead molecule against Covid-19. Following this, we exhaustively explored the concept of chemical space and molecular similarity parameters for the drug discovery about the lead(s) generated from in silico-based studies. The comparison was drawn using FDA-approved anti-infective agents during 2015-2020 using key descriptors to evaluate druglike properties. The outcomes of results were further corroborated using Molecular Dynamics studies which suggested the outcomes in alignment with chemical space ranking. In a nutshell, current research work aims to provide a holistic strategic approach to drug design, keeping in view the identified phytochemicals against Covid-19.

Nanohybrid electrochemical enzyme sensor for xanthine determination in fish samples.

An amperometric biosensor for xanthine was designed, based on covalent immobilization of xanthine oxidase (XO) of Bacillus pumilus RL-2d onto a screen-printed multi-walled carbon nanotubes gold nanoparticle-based electrodes (Nano-Au/c-MWCNT). The carboxyl groups at the electrode surface were activated by the use of 1-Ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDC) and N-hydroxysuccinimide (NHS). The working electrode was then coated with 6 µL of xanthine oxidase (0.273 U/mg protein). The cyclic voltammetry (CV) study was done for the characterization of the sensor using [K3Fe(CN)6] as an artificial electron donor. The sensitivity (S) and the limit of detection (LOD) of the biosensor were 2388.88 µA/cm2/nM (2.388 µA/cm2/µM) and 1.14 nM, respectively. The developed biosensor was used for determination of fish meat freshness.

Nanotechnology Based Approaches in Phage Therapy: Overcoming the Pharmacological Barriers.

With the emergence and spread of global antibiotic resistance and the need for searching safer alternatives, there has been resurgence in exploring the use of bacteriophages in the treatment of bacterial infections referred as phage therapy. Although modern phage therapy has come a long way as demonstrated by numerous efficacy studies but the fact remains that till date, phage therapy has not received regulatory approval for human use (except for compassionate use).Thus, to hit the clinical market, the roadblocks need to be seriously addressed and gaps mended with modern solution based technologies. Nanotechnology represents one such ideal and powerful tool for overcoming the pharmacological barriers (low stability, poor in-vivo retention, targeted delivery, neutralisation by immune system etc.) of administered phage preparations.In literature, there are many review articles on nanotechnology and bacteriophages but these are primarily focussed on highlighting the use of lytic and temperate phages in different fields of nano-medicine such as nanoprobes, nanosensors, cancer diagnostics, cancer cell targeting, drug delivery through phage receptors, phage display etc. Reviews specifically focused on the use of nanotechnology driven techniques strictly to improve phage therapy are however limited. Moreover, these review if present have primarily focussed on discussing encapsulation as a primary method for improving the stability and retention of phage(s) in the body.With new advances made in the field of nanotechnology, approaches extend from mere encapsulation to recently adopted newer strategies. The present review gives a detailed insight into the more recent strategies which include 1) use of lipid based nano-carriers (liposomes, transfersomes etc.) 2) adopting microfluidic based approach, surface modification methods to further enhance the efficiency and stability of phage loaded liposomes 3) Nano- emulsification approach with integration of microfluidics for producing multiple emulsions (suitable for phage cocktails) with unique control over size, shape and drop morphology 4) Phage loaded nanofibers produced by electro-spinning and advanced core shell nanofibers for immediate, biphasic and delayed release systems and 5) Smart release drug delivery platforms that allow superior control over dosing and phage release as and when required. All these new advances are aimed at creating a suitable housing system for therapeutic bacteriophage preparations while targeting the multiple issues of phage therapy i.e., improving phage stability and titers, improving in-vivo retention times, acting as suitable delivery systems for sustained release at target site of infection, improved penetration into biofilms and protection from immune cell attack. The present review thus aims at giving a complete insight into the recent advances (2010 onwards) related to various nanotechnology based approaches to address the issues pertaining to phage therapy. This is essential for improving the overall therapeutic index and success of phage therapy for future clinical approval.

Exploring the magic bullets to identify Achilles' heel in SARS-CoV-2: Delving deeper into the sea of possible therapeutic options in Covid-19 disease: An update.

The symptoms associated with Covid-19 caused by SARS-CoV-2 in severe conditions can cause multiple organ failure and fatality via a plethora of mechanisms, and it is essential to discover the efficacious and safe drug. For this, a successful strategy is to inhibit in different stages of the SARS-CoV-2 life cycle and host cell reactions. The current review briefly put forth the summary of the SARS-CoV-2 pandemic and highlight the critical areas of understanding in genomics, proteomics, medicinal chemistry, and natural products derived drug discovery. The review further extends to briefly put forth the updates in the drug testing system, biologics, biophysics, and their advances concerning SARS-CoV-2. The salient features include information on SARS-CoV-2 morphology, genomic characterization, and pathophysiology along with important protein targets and how they influence the drug design and development against SARS-CoV-2 and a concerted and integrated approach to target these stages. The review also gives the status of drug design and discovery to identify the drugs acting on critical targets in SARS-CoV-2 and host reactions to treat Covid-19.

Aliphatic amidase of Rhodococcus rhodochrous PA-34: Purification, characterization and application in synthesis of acrylic acid.

An intracellular aliphatic amide degrading inducible amidase produced by Rhodococcus rhodochrous PA-34 was characterized and acrylic acid synthesis from acrylamide was carried out using whole cell amidase. A bioprocess was developed at 50 ml fed batch reaction using 400 mM acrylamide feeding at an interval of 30 min resulted in the production of 4 g acrylic acid with volumetric and catalytic productivity of 80 g/l and 19 g/g/h respectively. The amidase of this organism had molecular weight of 40 kDa and was purified to 8.5 fold with 8% yield. This enzyme was active within the temperature range of 30 to 60 °C, with optimum temperature 45 °C and pH 7.5. The Vmax, Km, and kcat of purified amidase were calculated as 250 U/mg protein, 4.5 mM, and 166 sec-1 for acrylamide. The enzyme showed tolerance to metal chelating agent (EDTA) and was strongly inhibited by heavy metal ions Hg2+, Ag2+, Cu2+ and Co2+. R. rhodochrous PA-34 amidase preferentially hydrolyzed small aliphatic toxic amide such as acrylamide. Thus, the amidase of R. rhodochrous PA-34 is promising biocatalyst for the synthesis of industrially important acids and biodegradation of toxic amides.

Organic solvent tolerant metallo protease of novel isolate Serratia marcescens PPB-26: production and characterization.

Proteases are a class of enzymes that catalyze hydrolysis of peptide bonds of proteins. In this study, 221 proteolytic bacterial isolates were obtained by enrichment culture method from soils of various regions of Himachal Pradesh, India. From these a hyper producer of protease was screened and identified by morphological and physiological testing and by 16S rDNA sequence as Serratia marcescens PPB-26. Statistical optimization of physiochemical parameters enhanced the protease production by 75 %. Protease of S. marcescens PPB-26 was classified as a metalloprotease. It showed optimal activity at 30 °C, pH 7.5 (0.15 M Tris-HCl buffer) and with 0.8 % substrate concentration. It had K m = 0.3 %, V max = 34.5 µmol min-1 mg-1 protein and a half life of 2 days at 30 °C. The enzyme was stable in most metal ions but showed increased activity with Fe2+ and Cu2+ while strong inhibition with Co2+ and Zn2+. Further investigation showed that the enzyme could not only retain its activity in various organic solvents but also showed increased activity with methanol and ethanol. The reported metalloprotease is thus a potential candidate for carrying out industrial peptide synthesis.

Awareness of bispectral index monitoring system among the critical care nursing personnel in a tertiary care hospital of India.

BACKGROUND: Bispectral index monitoring system (BIS) is one of the several systems used to measure the effects of anaesthetic and sedative drugs on the brain and to track changes in the patient's level of sedation and hypnosis. BIS monitoring provides information clinically relevant to the adjustment of dosages of sedating medication. It can help the nursing personnel in preventing under- and over sedation among intensive care unit (ICU) patients. OBJECTIVE: The present study was conducted to assess the knowledge of nursing personnel working in the ICU regarding BIS. METHODS: Fifty-four subjects participated in the study. A structured questionnaire was developed to assess the knowledge of the nursing personnel regarding BIS. Focus group discussions were held among the nursing personnel to know their views regarding BIS. RESULTS: Mean age (years) of the subjects was 30.7±7.19 (21-47) years, with a female preponderance. Although the use of BIS in ICU is not common, majority (94.44%) were aware of BIS and its purpose. 79.62% of the subjects knew about its implication in patient care. The mean knowledge score of the subjects was 11.87±2.43 (maximum score being 15). CONCLUSION: There exists an awareness among the critical care nursing staff in our institution regarding BIS and its clinical implications. Its use in the critical care setting may benefit the patients in terms of providing optimal sedation.