Protein-inhibitor Complexes Research Articles

The Inhibition of Serum Amyloid A Protein Aggregation by a Five-Residue Peptidomimetic: Structural and Morphological Insights.

Serum amyloid A (SAA) is a small protein consisting of 104 residues and, under physiological conditions, exists mainly in hexameric form. It belongs to the positive acute-phase proteins, which means that its plasma concentration increases rapidly in response to injury, inflammation, and infection. The accumulation of SAA molecules promotes the formation of amyloid aggregates, which deposit extracellularly in many organs, causing their dysfunction. In our previous work, we successfully designed a peptidomimetic that inhibited the aggregation of amyloidogenic SAA fragments. In the present paper, we show how the same inhibitor, named saa3Dip, affects the oligomerization and aggregation processes of MetSAA1.1 protein. The thioflavin T assay showed that saa3Dip inhibited its fibrillization. The measurement of the internal fluorophore fluorescence (Trp) showed differences that occurred in the tertiary structure of MetSAA1.1 in the presence of the inhibitor, which was also confirmed by CD spectra in the aromatic range. FTIR results suggested that saa3Dip could stabilize some fragments of the native structure of MetSAA1.1, which was confirmed by determining the melting temperature (Tm) of the protein-inhibitor complex. AFM images demonstrated that the presence of saa3Dip prevented the formation of large SAA aggregates. Our results suggest that saa3Dip stabilizes the native conformation of MetSAA1.1.

Ligand-based pharmacophore modeling, virtual screening, and molecular dynamics simulations of Pfhsp90 fingerprints in Plasmodium malaria treatment

The World Health Organization (WHO) documents malaria as one of the leading causes of high morbidity and mortality worldwide. The disease affects millions and kills thousands of people annually. Efforts to reduce the global burden of malaria have prompted the WHO to recommend prevention strategies such as using antimalarial drugs. However, these strategies have been ineffective because of antimalarial drug resistance. The only efficacious malaria treatment is Artemisinin-based Combination Therapy (ACT). However, the extended ACT clearance times, linked to the emergence of artemisinin monotherapy resistance recorded most recently in Africa and the Great Mekong region, pose a danger to its efficacy. Therefore, better efficacious antimalarial drugs are required. Since P. falciparum heat shock protein 90 (PfHsp90) is a well-characterized malaria drug target, this study uses it to discover more efficacious antimalarial drugs. An in silico approach was used to discover PfHsp90 inhibitors with pharmacological properties against Plasmodium malaria using a molecular dynamics simulation (MDS) and hierarchical virtual screening. Geldanamycin (GDM), a well-known anti-PfHsp90 compound, was used to identify PfHsp90 inhibitors with pharmacological properties against Plasmodium malaria by screening it against the ZINC20 database via the ZINCPHARMER web server. This virtual screening process resulted in 17 hits. These ZINCPHARMER hits were subjected to drug-likeness and pharmacokinetics properties analysis in the SwissADME web server, and nine of them satisfied the requirements. The nine ZINC20 compounds were docked with PfHsp90 using the PyRx software version 0.8 to understand their interactions. From the molecular docking results, ZINC09060002 (−8.2 kcal/mol), ZINC72133064 (−7.8 kcal/mol), ZINC72163401 (−7.7 kcal/mol), ZINC72358537 (−8.1 kcal/mol), and ZINC72358557 (−7.6 kcal/mol) had better binding affinities to PfHsp90 than GDM (−7.5 kcal/mol). The stability of these molecularly docked protein–inhibitor complexes was assessed through MDS using GROMACS 2022. ZINC72163401, ZINC72358537, and ZINC72358557 formed stable complexes with PfHsp90. The lead compounds were subjected to in vitro validation for their inhibitory capability. They showed promising inhibition of parasite growth with IC50 values ranging between 200 and 400 ng/mL. In this regard, the three PfHsp90 inhibitors can be further developed as potential antimalarial drugs. However, further structural optimization studies and clinical (in vivo) tests are necessary to ascertain the antimalarial activity of these compounds in humans.

Jack bean urease inhibition by different root fractions of Cleome gynandra L – Kinetic mechanism and computational molecular modelling

Natural products with urease inhibiting potentialities would be valuable for enhancing nitrogen fertilizer formulations and developing new therapeutics against infectious diseases. Main focus of this research was to assess the inhibitory impact of various parts of Cleome gynandra (Cleomaceae) on jack bean urease activity, identify the responsible compounds, and ascertain their mode of enzyme inhibition and interactions with urease enzyme. Urease inhibitory potentiality of different plant parts of C. gynandra was determined using the phenol-hypochlorite method and mode of enzyme inhibition by Lineweaver-Burk kinetic analysis. LC-HRMS was carried out to identify the compounds of the active fractions. Molecular docking and simulations were executed to find out the binding affinity and interaction pattern of the isolated compounds at the active site of urease enzyme and to verify stability of protein-inhibitor complexes. The root of C. gynandra exhibited highest urease inhibitory potentiality with IC50 value of 1.529 mg/ml compared with leaf and stem. Among the different root fractions, water fraction revealed maximum anti-urease activity with IC50 values of 1.477 mg/ml followed by methanol (1.655 mg/ml) and acetone fractions (1.955 mg/ml). Inhibition kinetics indicated that these fractions were strong inhibitors of urease and exhibited a non-competitive mode of inhibition with Ki values of 184.911 µg/ml, 147.34 µg/ml, and 233.75 µg/ml respectively at 1000 mg/L concentration. LC-HRMS analysis confirmed the occurrence of glucocapparin, fluticasone propionate and lauryl hydrogen sulfate etc. in water fraction. Molecular modelling and simulation study suggested strong and stable interactions between the specified compounds of water fraction and active site of urease. Hence, the water fraction of C. gynandra roots represent a valuable source of natural urease inhibitors, for possible therapeutic applications and sustainable agricultural practices.

Prediction of the binding mechanism of a selective DNA methyltransferase 3A inhibitor by molecular simulation

DNA methylation is an epigenetic mechanism that introduces a methyl group at the C5 position of cytosine. This reaction is catalyzed by DNA methyltransferases (DNMTs) and is essential for the regulation of gene transcription. The DNMT1 and DNMT3A or -3B family proteins are known targets for the inhibition of DNA hypermethylation in cancer cells. A selective non-nucleoside DNMT3A inhibitor was developed that mimics S-adenosyl-l-methionine and deoxycytidine; however, the mechanism of selectivity is unclear because the inhibitor–protein complex structure determination is absent. Therefore, we performed docking and molecular dynamics simulations to predict the structure of the complex formed by the association between DNMT3A and the selective inhibitor. Our simulations, binding free energy decomposition analysis, structural isoform comparison, and residue scanning showed that Arg688 of DNMT3A is involved in the interaction with this inhibitor, as evidenced by its significant contribution to the binding free energy. The presence of Asn1192 at the corresponding residues in DNMT1 results in a loss of affinity for the inhibitor, suggesting that the interactions mediated by Arg688 in DNMT3A are essential for selectivity. Our findings can be applied in the design of DNMT-selective inhibitors and methylation-specific drug optimization procedures.

Identification of new pharmacophore against SARS-CoV-2 spike protein by multi-fold computational and biochemical techniques

COVID-19 appeared as a highly contagious disease after its outbreak in December 2019 by the virus, named SARS-CoV-2. The threat, which originated in Wuhan, China, swiftly became an international emergency. Among different genomic products, spike protein of virus plays a crucial role in the initiation of the infection by binding to the human lung cells, therefore, SARS-CoV-2’s spike protein is a promising therapeutic target. Using a combination of a structure-based virtual screening and biochemical assay, this study seeks possible therapeutic candidates that specifically target the viral spike protein. A database of ~ 850 naturally derived compounds was screened against SARS-CoV-2 spike protein to find natural inhibitors. Using virtual screening and inhibitory experiments, we identified acetyl 11-keto-boswellic acid (AKBA) as a promising molecule for spike protein, which encouraged us to scan the rest of AKBA derivatives in our in-house database via 2D-similarity searching. Later 19 compounds with > 85% similarity with AKBA were selected and docked with receptor binding domain (RBD) of spike protein. Those hits declared significant interactions at the RBD interface, best possess and excellent drug-likeness and pharmacokinetics properties with high gastrointestinal absorption (GIA) without toxicity and allergenicity. Our in-silico observations were eventually validated by in vitro bioassay, interestingly, 10 compounds (A3, A4, C3, C6A, C6B, C6C, C6E, C6H, C6I, and C6J) displayed significant inhibitory ability with good percent inhibition (range: > 72–90). The compounds C3 (90.00%), C6E (91.00%), C6C (87.20%), and C6D (86.23%) demonstrated excellent anti-SARS CoV-2 spike protein activities. The docking interaction of high percent inhibition of inhibitor compounds C3 and C6E was confirmed by MD Simulation. In the molecular dynamics simulation, we observed the stable dynamics of spike protein inhibitor complexes and the influence of inhibitor binding on the protein’s conformational arrangements. The binding free energy ΔGTOTAL of C3 (−38.0 ± 0.08 kcal/mol) and C6E (−41.98 ± 0.08 kcal/mol) respectively indicate a strong binding affinity to Spike protein active pocket. These findings demonstrate that these molecules particularly inhibit the function of spike protein and, therefore have the potential to be evaluated as drug candidates against SARS-CoV-2.

Rational design for novel heterocyclic based Donepezil analogs for Alzheimer’s disease: an in silico approach

Alzheimer’s disease (AD) is a progressive neurodegenerative disease and has devastating impacts on the elderly population. During the last two decades, there has been a significant focus on developing effective and safe treatments for AD. Acetylcholinesterase (AChE) has been identified as one of the primary therapeutic targets for developing drug candidates for AD. However, there is still a need for more efficient therapies. In this study, our aim is to design a new series of heterocyclic-based AChE inhibitors inspired by a standard drug. Here, we carried out molecular docking, drug-likeliness characteristics, and molecular dynamics (MD) to predict important pharmacophore features and understand the inhibitory mechanism of the designed inhibitors towards the AChE. We have designed 112 new derivatives by replacing the piperidine moiety of Donepezil with the different five and six-membered heterocyclic rings and selected 15 compounds that show higher or comparable docking scores as compared to standard Donepezil and pose no risk for carcinogenicity. Furthermore, MD results imply the structural stability of the selected docked complexes and seven exhibit a stronger binding affinity towards the AChE than Donepezil. Thus, heterocyclic-based derivatives based on oxazole, pyrazole, and tetrahydropyran may be potential therapeutic candidates for AD. Our structure-based drug design approach allows us to identify and gain insight into the structural stability of the inhibitor-protein complex and the inhibition mechanism of the newly designed inhibitors. The present finding might be an initial selection for developing a new inhibitor for AD and provide a direction for further experiments on its biological activities. Communicated by Ramaswamy H. Sarma

Discovery of a Hidden Pocket beneath the NES Groove by Novel Noncovalent CRM1 Inhibitors.

Protein localization is frequently manipulated to favor tumor initiation and progression. In cancer cells, the nuclear export factor CRM1 is often overexpressed and aberrantly localizes many tumor suppressors via protein-protein interactions. Although targeting protein-protein interactions is usually challenging, covalent inhibitors, including the FDA-approved drug KPT-330 (selinexor), were successfully developed. The development of noncovalent CRM1 inhibitors remains scarce. Here, by shifting the side chain of two methionine residues and virtually screening against a large compound library, we successfully identified a series of noncovalent CRM1 inhibitors with a stable scaffold. Crystal structures of inhibitor-protein complexes revealed that one of the compounds, B28, utilized a deeply hidden protein interior cavity for binding. SAR analysis guided the development of several B28 derivatives with enhanced inhibition on nuclear export and growth of multiple cancer cell lines. This work may benefit the development of new CRM1-targeted therapies.

De novo drug designing coupled with brute force screening and structure guided lead optimization gives highly specific inhibitor of METTL3: a potential cure for Acute Myeloid Leukaemia

Expression of METTL3, a SAM dependent methyltransferase, which deposits m6A on mRNA is linked to poor prognosis in Acute Myeloid Leukaemia and other type of cancers. Down regulation of this epitranscriptomic regulator has been found to inhibit cancer progression. Silencing the methyltransferase activity of METTL3 is a lucrative strategy to design anticancer drugs. In this study 3600 commercially available molecules were screened against METTL3 using brute force screening approach. However, none of these compounds take advantage of the unique Y-shaped binding cavity of the protein, raising the need for de novo drug designing strategies. As such, 125 branched, Y-shaped molecules were designed by “stitching” together the chemical fragments of the best inhibitors that interact strongly with the METTL3 binding pocket. This results in molecules that have the three-dimensional structure and functional groups which enable it to fit in the METTL3 cavity like fingers in a glove, having unprecedented selectivity and binding affinities. The designed compounds were further refined based on Lipinski’s rule, docking score and synthetic accessibility. The molecules faring well in these criteria were simulated for 100 ns to check the stability of the protein inhibitor complex followed by binding free energy calculation.Communicated by Ramaswamy H. Sarma

Early Blockage of Mycobacterium Tuberculosis Cell-wall Synthesis via EchA\6 Inhibition to Overcome Resistance Strain: Insights from Umbrella Sampling Simulations

Background: Tuberculosis (TB) has long been the major infectious cause of mortality, ranking higher than HIV/AIDS as the most common cause of death from a single infectious agent worldwide. The EchA6 target of mycobacteria plays a vital role in synthesizing an important component of the mycobacterial outer membrane. The failure of TB treatment has prompted the investigation of novel anti-tubercular drugs. Objective: This study was aimed at blockage of Mycobacterium tuberculosis cell-wall synthesis via EchA6 inhibition to overcome resistance strain. Methods: Over 3,000,000 compounds and GSK951A (positive control) were investigated as the inhibitors in this study. The GROMACS molecular dynamic package was used to analyze the protein- inhibitor complex's conformational changes under constant temperature and pressure. Also, umbrella sampling (US) was used for free binding energy (ΔG) calculation. Results: Four compounds were chosen for the docking investigation. According to the MD analysis, the studied inhibitors demonstrated good stability and flexibility. According to ∆G obtained from US, the ∆G of GSK951A, ZINC11815220, ZINC67770050, ZINC55048326, and ZINC89700914 were -6.14 kcal mol-1, -5.25 kcal mol-1, -10.19 kcal mol-1, -8.55 kcal mol-1, and -8.37 kcal mol-1, respectively. Conclusion: In conclusion, ZINC67770050 is recommended for further study in the laboratory. This investigation is an important starting point for discovering anti-tubercular drugs using EchA6 inhibition.

In-silico analysis of potent Mosquirix vaccine adjuvant leads

BackgroundWorld Health Organization recommend the use of malaria vaccine, Mosquirix, as a malaria prevention strategy. However, Mosquirix has failed to reduce the global burden of malaria because of its inefficacy. The Mosquirix vaccine’s modest effectiveness against malaria, 36% among kids aged 5 to 17 months who need at least four doses, fails to aid malaria eradication. Therefore, highly effective and efficacious malaria vaccines are required. The well-characterized P. falciparum circumsporozoite surface protein can be used to discover adjuvants that can increase the efficacy of Mosquirix. Therefore, the study sought to undertake an in-silico discovery of Plasmodium falciparum circumsporozoite surface protein inhibitors with pharmacological properties on Mosquirix using hierarchical virtual screening and molecular dynamics simulation. ResultsMonoclonal antibody L9, an anti-Plasmodium falciparum circumsporozoite surface protein molecule, was used to identify Plasmodium falciparum circumsporozoite surface protein inhibitors with pharmacological properties on Mosquirix during a virtual screening process in ZINCPHARMER that yielded 23 hits. After drug-likeness and absorption, distribution, metabolism, excretion, and toxicity property analysis in the SwissADME web server, only 9 of the 23 hits satisfied the requirements. The 9 compounds were docked with Plasmodium falciparum circumsporozoite surface protein using the PyRx software to understand their interactions. ZINC25374360 (−8.1 kcal/mol), ZINC40144754 (−8.3 kcal/mol), and ZINC71996727 (−8.9 kcal/mol) bound strongly to Plasmodium falciparum circumsporozoite surface protein with binding affinities of less than −8.0 kcal/mol. The stability of these molecularly docked Plasmodium falciparum circumsporozoite surface protein-inhibitor complexes were assessed through molecular dynamics simulation using GROMACS 2022. ZINC25374360 and ZINC71996727 formed stable complexes with Plasmodium falciparum circumsporozoite surface protein. They were subjected to in vitro validation for their inhibitory potential. The IC50 values ranging between 250 and 350 ng/ml suggest inhibition of parasite development. ConclusionTherefore, the two Plasmodium falciparum circumsporozoite surface protein inhibitors can be used as vaccine adjuvants to increase the efficacy of the existing Mosquirix vaccine. Nevertheless, additional in vivo tests, structural optimization studies, and homogenization analysis are essential to determine the anti-plasmodial action of these adjuvants in humans.

Kinetics, Thermodynamics, and Structural Effects of Quinoline-2-Carboxylates, Zinc-Binding Inhibitors of New Delhi Metallo-β-lactamase-1 Re-sensitizing Multidrug-Resistant Bacteria for Carbapenems.

Carbapenem resistance mediated by metallo-β-lactamases (MBL) such as New Delhi metallo-β-lactamase-1 (NDM-1) has become a major factor threatening the efficacy of essential β-lactam antibiotics. Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy- and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors of NDM-1 with highly improved inhibitory activity and binding affinity. The most active compounds formed reversibly inactive ternary protein-inhibitor complexes with two zinc ions as proven by native protein mass spectrometry and bio-layer interferometry. Modification of the NDM-1 structure with remarkable entropic gain was shown by isothermal titration calorimetry and NMR spectroscopy of isotopically labeled protein. The best compounds were potent inhibitors of NDM-1 and other representative MBL with no or little inhibition of human zinc-binding enzymes. These inhibitors significantly reduced the minimum inhibitory concentrations (MIC) of meropenem for multidrug-resistant bacteria recombinantly expressing blaNDM-1 as well as for several multidrug-resistant clinical strains at concentrations non-toxic to human cells.

Preclinical studies of a PARP targeted, Meitner-Auger emitting, theranostic radiopharmaceutical for metastatic ovarian cancer

Advanced ovarian cancer currently has few therapeutic options. Poly(ADP-ribose) polymerase (PARP) inhibitors bind to nuclear PARP and trap the protein-inhibitor complex to DNA. This work investigates a theranostic PARP inhibitor for targeted radiopharmaceutical therapy of ovarian cancer in vitro and PET imaging of healthy mice in vivo. Methods[77Br]RD1 was synthesized and assessed for pharmacokinetics and cytotoxicity in human and murine ovarian cancer cell lines. [76Br]RD1 biodistribution and organ uptake in healthy mice were quantified through longitudinal PET/CT imaging and ex vivo radioactivity measurements. Organ-level dosimetry following [76/77Br]RD1 administration was calculated using RAPID, an in-house platform for absorbed dose in mice, and OLINDA for equivalent and effective dose in human. ResultsThe maximum specific binding (Bmax), equilibrium dissociation constant (Kd), and nonspecific binding slope (NS) were calculated for each cell line. These values were used to calculate the cell specific activity uptake for cell viability studies. The half maximal effective concentration (EC50) was measured as 0.17 (95 % CI: 0.13–0.24) nM and 0.46 (0.13–0.24) nM for PARP(+) and PARP(−) expressing cell lines, respectively. The EC50 was 0.27 (0.21–0.36) nM and 0.30 (0.22–0.41) nM for BRCA1(−) and BRCA1(+) expressing cell lines, respectively. When measuring the EC50 as a function of cellular activity uptake and nuclear dose, the EC50 ranges from 0.020 to 0.039 Bq/cell and 3.3–9.2 Gy, respectively. Excretion through the hepatobiliary and renal pathways were observed in mice, with liver uptake of 2.3 ± 0.4 %ID/g after 48 h, contributing to estimated absorbed dose values in mice of 19.3 ± 0.3 mGy/MBq and 290 ± 10 mGy/MBq for [77Br]RD1 and [76Br]RD1, respectively. Conclusion[77Br]RD1 cytotoxicity was dependent on PARP expression and independent of BRCA1 status. The in vitro results suggest that [77Br]RD1 cytotoxicity is driven by the targeted Meitner-Auger electron (MAe) radiotherapeutic effect of the agent. Further studies investigating the theranostic potential, organ dose, and tumor uptake of [76/77Br]RD1 are warranted.

Enzyme - Switch sensors for therapeutic drug monitoring of immunotherapies

Therapeutic monoclonal antibodies (TmAb) have emerged as effective treatments for a number of cancers and autoimmune diseases. However, large interpatient disparities in the pharmacokinetics of TmAb treatment requires close therapeutic drug monitoring (TDM) to optimise dosage for individual patients. Here we demonstrate an approach for achieving rapid, sensitive quantification of two monoclonal antibody therapies using a previously described enzyme switch sensor platform. The enzyme switch sensor consists of a β-lactamase – β-lactamase inhibitor protein (BLA-BLIP) complex with two anti-idiotype binding proteins (Affimer proteins) as recognition elements. The BLA-BLIP sensor was engineered to detect two TmAbs (trastuzumab and ipilimumab) by developing constructs incorporating novel synthetic binding reagents to each of these mAbs. Trastuzumab and ipilimumab were successfully monitored with sub nM sensitivity in up to 1% serum, thus covering the relevant therapeutic range. Despite the modular design, the BLA-BLIP sensor was unsuccessful in detecting two further TmAbs (rituximab and adalimumab), an explanation for which was explored. In conclusion, the BLA-BLIP sensors provide a rapid biosensor for TDM of trastuzumab and ipilimumab with the potential to improve therapy. The sensitivity of this platform alongside its rapid action would be suitable for bedside monitoring in a point-of-care (PoC) setting.

Computational investigation of Moringa oleifera phytochemicals targeting EGFR: molecular docking, molecular dynamics simulation and density functional theory studies

Epidermal growth factor receptor (EGFR) is a prominent target for anticancer therapy due to its role in activating several cell signaling cascades. Clinically approved EGFR inhibitors are reported to show treatment resistance and toxicity, this study, therefore, investigates Moringa oleifera phytochemicals to find potent and safe anti-EGFR compounds. For that, phytochemicals were screened based on drug-likeness and molecular docking analysis followed by molecular dynamics simulation, density functional theory analysis and ADMET analysis to identify the effective inhibitors of EGFR tyrosine kinase (EGFR-TK) domain. Known EGFR-TK inhibitors (1-4 generations) were used as control. Among 146 phytochemicals, 136 compounds showed drug-likeness, of which Delta 7-Avenasterol was the most potential EGFR-TK inhibitor with a binding energy of −9.2 kcal/mol followed by 24-Methylenecholesterol (–9.1 kcal/mol), Campesterol (–9.0 kcal/mol) and Ellagic acid (–9.0 kcal/mol). In comparison, the highest binding affinity from control drugs was displayed by Rociletinib (–9.0 kcal/mol). The molecular dynamics simulation (100 ns) exhibited the structural stability of native EGFR-TK and protein-inhibitor complexes. Further, MM/PBSA computed the binding free energies of protein complex with Delta 7-Avenasterol, 24-Methylenecholesterol, Campesterol and Ellagic acid as −154.559 ± 18.591 kJ/mol, −139.176 ± 19.236 kJ/mol, −136.212 ± 17.598 kJ/mol and −139.513 ± 23.832 kJ/mol, respectively. Non-polar interactions were the major contributors to these energies. The density functional theory analysis also established the stability of these inhibitor compounds. ADMET analysis depicted acceptable outcomes for all top phytochemicals without displaying any toxicity. In conclusion, this report has identified promising EGFR-TK inhibitors to treat several cancers that can be further investigated through laboratory and clinical tests.

In-silico drug design for the novel Karachi-NF001 strain of brain-eating amoeba: Naegleria fowleri.

Naegleria fowleri (N. fowleri) is a free-living thermophilic amoeba of fresh water and soil. The amoeba primarily feeds on bacteria but can be transmitted to humans upon contact with freshwater sources. Furthermore, this brain-eating amoeba enters the human body through the nose and travels to the brain to cause primary amebic meningoencephalitis (PAM). N. fowleri has been reported globally since its discovery in 1961. Recently a new strain of N. fowleri named Karachi-NF001 was found in a patient who had traveled from Riyadh, Saudi Arabia to Karachi in 2019. There were 15 unique genes identified in the genome of the Karachi-NF001 strain compared to all the previously reported strains of N. fowleri worldwide. Six of these genes encode well-known proteins. In this study, we performed in-silico analysis on 5 of these 6 proteins, namely, Rab family small GTPase, NADH dehydrogenase subunit 11, two Glutamine-rich protein 2 proteins (locus tags: 12086 and 12110), and Tigger transposable element-derived protein 1. We conducted homology modeling of these 5 proteins followed by their active site identification. These proteins were subjected to molecular docking against 105 anti-bacterial ligand compounds as potential drugs. Subsequently, the 10 best-docked compounds were determined for each protein and ranked according to the number of interactions and their binding energies. The highest binding energy was recorded for the two Glutamine-rich protein 2 proteins with different locus tags, and results have shown that the protein-inhibitor complex was stable throughout the simulation run. Moreover, future in-vitro studies could validate the findings of our in-silico analysis and identify potential therapeutic drugs against N. fowleri infections.

Insight into designing of 2-pyridone derivatives for COVID-19 drug discovery - A computational study.

Presently, the prime global focus is on SARS-CoV-2, as no fully established, licensed medicine has been found thus far, in spite of the existence of various reports and administration of partially proven certain class of natural products. However, in case of natural products, the extraction and purification limit their application. This situation drives researchers to explore synthetically viable drugs. In the present investigation, twenty-three 2-pyridone synthetic derivatives (P1-P23) have been theoretically tested for their suitability as potential inhibitors for COVID-19 main protease through DFT, molecular docking, and molecular dynamics simulations. DFT calculations offer insights into structure–property relationships, while ADMET studies indicate the pharmacological characteristics of these molecules. Molecular docking studies ascertain the nature and mode of interactions of these entities with COVID-19 main protease. Furthermore, covalent docking has been carried out to verify the feasibility of the formation of a covalent bond with the active site. The top protein-inhibitor complexes, such as P18, P11, and P12, were identified based on their glide score. These molecules, along with the covalent docked complexes, namely P18 and P16, were selected and subjected to molecular dynamics simulations. The 100 ns simulation process exhibited that the covalent docked ones, due to their stable form could serve as lead compounds against SARS-CoV-2. Hence, this study affirms the potential candidature of 2-pyridone-based inhibitors.

Solid state synthesis of bispyridyl-ferrocene conjugates with unusual site selective 1,4-Michael addition, as potential inhibitor and electrochemical probe for fibrillation in amyloidogenic protein

Destabilization of protein's native conformation to intermediates rich in β-sheet structure may lead to amyloid fibrillation involving a complex process of aggregation that are primarily responsible for various neurodegenerative disorders. Design of flexible, functional molecules capable of disruption or inhibition of fibrillar aggregates at their initial stages can be an effective approach to restrict or prevent amyloid related diseases. In this perspective, strategic association of ferrocenyl based electro-active moiety with heterocyclic rings can generate flexible, biocompatible molecular system with significant role in protein interactions, electrochemical monitoring and improved biological activity. Therefore, synthesis of molecules containing multi-pyridine ring framework conjugated to a ferrocenyl scaffold have been explored using a unique solid state, redmud supported, solvent free reaction method which led to the formation of ferrocenyl bis-pyridylenone (2) and a rare class of ferrocene based 1,4-Michael addition compound (3). The 1,4-Michael addition attack at an olefinic carbon with bulkier, electron donating ferrocenyl substituent has been unprecedented. Inhibitory potential of di-functionalized ferrocenyl-bispyridylenone conjugate (2) against amyloid fibrillation and its ability to serve as electrochemical probe on the disaggregation process of BSA have been explored. The study revealed significant inhibition of amyloid fibrils during aggregation process and also showed disruption of pre-formed BSA fibrils. The redox active inhibitor molecule was also used as potential electrochemical probe to monitor the inhibition of the fibrillation process. Molecular dynamics simulation confirmed distinct hydrogen bond interactions between the inhibitor compound and the A chain of BSA and revealed enough conformational stability of the protein-inhibitor complex.

Evolving Experimental Techniques for Structure-Based Drug Design.

Structure-based drug design (SBDD) is a prominent method in rational drug development and has traditionally benefitted from the atomic models of protein targets obtained using X-ray crystallography at cryogenic temperatures. In this perspective, we highlight recent advances in the development of structural techniques that are capable of probing dynamic information about protein targets. First, we discuss advances in the field of X-ray crystallography including serial room-temperature crystallography as a method for obtaining high-resolution conformational dynamics of protein-inhibitor complexes. Next, we look at cryogenic electron microscopy (cryoEM), another high-resolution technique that has recently been used to study proteins and protein complexes that are too difficult to crystallize. Finally, we present small-angle X-ray scattering (SAXS) as a potential high-throughput screening tool to identify inhibitors that target protein complexes and protein oligomerization.

Catechol-containing compounds are a broad class of protein aggregation inhibitors: Redox state is a key determinant of the inhibitory activities

A range of neurodegenerative and related aging diseases, such as Alzheimer’s disease and type 2 diabetes, are linked to toxic protein aggregation. Yet the mechanisms of protein aggregation inhibition by small molecule inhibitors remain poorly understood, in part because most protein targets of aggregation assembly are partially unfolded or intrinsically disordered, which hinders detailed structural characterization of protein-inhibitor complexes and structural-based inhibitor design. Herein we employed a parallel small molecule library-screening approach to identify inhibitors against three prototype amyloidogenic proteins in neurodegeneration and related proteinopathies: amylin, Aβ and tau. One remarkable class of inhibitors identified from these screens against different amyloidogenic proteins was catechol-containing compounds and redox-related quinones/anthraquinones. Secondary assays validated most of the identified inhibitors. In vivo efficacy evaluation of a selected catechol-containing compound, rosmarinic acid, demonstrated its strong mitigating effects of amylin amyloid deposition and related diabetic pathology in transgenic HIP rats. Further systematic investigation of selected class of inhibitors under aerobic and anaerobic conditions revealed that the redox state of the broad class of catechol-containing compounds is a key determinant of the amyloid inhibitor activities. The molecular insights we gained not only explain why a large number of catechol-containing polyphenolic natural compounds, often enriched in healthy diet, have anti-neurodegeneration and anti-aging activities, but also could guide the rational design of therapeutic or nutraceutical strategies to target a broad range of neurodegenerative and related aging diseases.

Hit Expansion of a Noncovalent SARS-CoV-2 Main Protease Inhibitor

Inhibition of the SARS-CoV-2 main protease (Mpro) is a major focus of drug discovery efforts against COVID-19. Here we report a hit expansion of non-covalent inhibitors of Mpro. Starting from a recently discovered scaffold (The COVID Moonshot Consortium. Open Science Discovery of Oral Non-Covalent SARS-CoV-2 Main Protease Inhibitor Therapeutics. bioRxiv 2020.10.29.339317) represented by an isoquinoline series, we searched a database of over a billion compounds using a cheminformatics molecular fingerprinting approach. We identified and tested 48 compounds in enzyme inhibition assays, of which 21 exhibited inhibitory activity above 50% at 20 μM. Among these, four compounds with IC50 values around 1 μM were found. Interestingly, despite the large search space, the isoquinolone motif was conserved in each of these four strongest binders. Room-temperature X-ray structures of co-crystallized protein–inhibitor complexes were determined up to 1.9 Å resolution for two of these compounds as well as one of the stronger inhibitors in the original isoquinoline series, revealing essential interactions with the binding site and water molecules. Molecular dynamics simulations and quantum chemical calculations further elucidate the binding interactions as well as electrostatic effects on ligand binding. The results help explain the strength of this new non-covalent scaffold for Mpro inhibition and inform lead optimization efforts for this series, while demonstrating the effectiveness of a high-throughput computational approach to expanding a pharmacophore library.