Quorum sensing inhibitors as Therapeutics: Bacterial biofilm inhibition.

The overuse and inappropriate use of antibiotics to treat bacterial infections has led to the development of multiple drug resistant strains. Biofilm is a complex microorganism aggregation defined by the presence of a dynamic, sticky, and protective extracellular matrix made of polysaccharides, proteins, and nucleic acids. The infectious diseases are caused by bacteria that flourish within quorum sensing (QS) mediated biofilms. Efforts to disrupt biofilms have enabled the identification of bioactive molecules produced by prokaryotes and eukaryotes. The QS system is quenched predominantly by these molecules. The phenomenon is also termed as quorum sensing (QS). Both synthetic and natural substances have been discovered to be useful in QS. This review describes natural and synthetic quorum sensing inhibitors (QSIs) with the potential to treat bacterial infections. It includes the discussion on quorum sensing, mechanism of quorum sensing, effect of substituents on the activity. These discoveries could result in effective therapies using far lower dosages of medications, particularly antibiotics, are currently needed.

Highlighting the Potential Role of Exosomes as the Targeted Nanotherapeutic Carrier in Metastatic Breast Cancer.

Breast cancer, being the second most common type of cancer, is a leading cause of death in the female population. Of all the available treatments existing for breast cancer, exosomes appear as an important medium for the site targeted delivery of the drugs. Exosomes, unlike all the other extracellular vesicles, play a vital role in the transport of numerous biomolecules throughout the body and can easily be detected because of the presence of specific biomarkers. Apart from playing a wide variety of roles in the progression of many diseases, they are also responsible for tumor progression and metastasis in breast cancer. Exosomes and related engineering strategies are being discussed as nano-carrier for the delivery of different drugs in the case of breast cancer. Overall, we have discussed in this review the role of exosomes in breast cancer and the engineering strategies being devised for making them an efficient drug delivery system.

Role of Tau in Various Tauopathies, Treatment Approaches, and Emerging Role of Nanotechnology in Neurodegenerative Disorders.

A few protein kinases and phosphatases regulate tau protein phosphorylation and an imbalance in their enzyme activity results in tau hyper-phosphorylation. Aberrant tau phosphorylation causes tau to dissociate from the microtubules and clump together in the cytosol to form neurofibrillary tangles (NFTs), which lead to the progression of neurodegenerative disorders including Alzheimer's disease (AD) and other tauopathies. Hence, targeting hyperphosphorylated tau protein is a restorative approach for treating neurodegenerative tauopathies. The cyclin-dependent kinase (Cdk5) and the glycogen synthase kinase (GSK3ß) have both been implicated in aberrant tau hyperphosphorylation. The limited transport of drugs through the blood-brain barrier (BBB) for reaching the central nervous system (CNS) thus represents a significant problem in the development of drugs. Drug delivery systems based on nanocarriers help solve this problem. In this review, we discuss the tau protein, regulation of tau phosphorylation and abnormal hyperphosphorylation, drugs in use or under clinical trials, and treatment strategies for tauopathies based on the critical role of tau hyperphosphorylation in the pathogenesis of the disease. Pathology of neurodegenerative disease due to hyperphosphorylation and various therapeutic approaches including nanotechnology for its treatment.

Time Series Analysis of SARS-CoV-2 Genomes and Correlations among Highly Prevalent Mutations.

The efforts of the scientific community to tame the recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seem to have been diluted by the emergence of new viral strains. Therefore, it is imperative to understand the effect of mutations on viral evolution. We performed a time series analysis on 59,541 SARS-CoV-2 genomic sequences from around the world to gain insights into the kinetics of the mutations arising in the viral genomes. These 59,541 genomes were grouped according to month (January 2020 to March 2021) based on the collection date. Meta-analysis of these data led us to identify significant mutations in viral genomes. Pearson correlation of these mutations led us to the identification of 16 comutations. Among these comutations, some of the individual mutations have been shown to contribute to viral replication and fitness, suggesting a possible role of other unexplored mutations in viral evolution. We observed that the mutations 241C>T in the 5' untranslated region (UTR), 3037C>T in nsp3, 14408C>T in the RNA-dependent RNA polymerase (RdRp), and 23403A>G in spike are correlated with each other and were grouped in a single cluster by hierarchical clustering. These mutations have replaced the wild-type nucleotides in SARS-CoV-2 sequences. Additionally, we employed a suite of computational tools to investigate the effects of T85I (1059C>T), P323L (14408C>T), and Q57H (25563G>T) mutations in nsp2, RdRp, and the ORF3a protein of SARS-CoV-2, respectively. We observed that the mutations T85I and Q57H tend to be deleterious and destabilize the respective wild-type protein, whereas P323L in RdRp tends to be neutral and has a stabilizing effect. IMPORTANCE We performed a meta-analysis on SARS-CoV-2 genomes categorized by collection month and identified several significant mutations. Pearson correlation analysis of these significant mutations identified 16 comutations having absolute correlation coefficients of >0.4 and a frequency of >30% in the genomes used in this study. The correlation results were further validated by another statistical tool called hierarchical clustering, where mutations were grouped in clusters on the basis of their similarity. We identified several positive and negative correlations among comutations in SARS-CoV-2 isolates from around the world which might contribute to viral pathogenesis. The negative correlations among some of the mutations in SARS-CoV-2 identified in this study warrant further investigations. Further analysis of mutations such as T85I in nsp2 and Q57H in ORF3a protein revealed that these mutations tend to destabilize the protein relative to the wild type, whereas P323L in RdRp is neutral and has a stabilizing effect. Thus, we have identified several comutations which can be further characterized to gain insights into SARS-CoV-2 evolution.

Cyclodextrin Derivative Enhances the Ophthalmic Delivery of Poorly Soluble Azithromycin.

Azithromycin (AZM), a macrolide antibiotic used for the treatment of chlamydial conjunctivitis, is less effective for the treatment of this disease due to its poor bioavailability (38%). Various alternatives have been developed for improving the physicochemical properties (i.e., solubility) of the AZM without much success. To overcome the problems associated with AZM, an inclusion complex employing a modified cyclodextrin, i.e., sulfobutylether-ß-cyclodextrin (SBE-ß-CD), was prepared and characterized by phase solubility studies and PXRD techniques. The results portrayed the formation of an inclusion complex of AZM with SBE-ß-CD in 1:2 molar stoichiometric ratios. This inclusion complex was later incorporated into a polymer matrix to prepare an in situ gel. Various combinations of Carbopol 934P and hydroxypropyl methylcellulose (HPMC K4M) polymers were used and evaluated by rheological and in vitro drug release studies. The optimized formulation (F4) containing Carbopol 934P (0.2% w/v) and HPMC K4M (0.2% w/v) was evaluated for clarity, pH, gelling capacity, drug content, rheological properties, in vitro drug release pattern, ocular irritation test, and antimicrobial efficacy. Finally, owing to the improved antimicrobial efficacy and increased residence time, the AZM:SBE-ß-CD in situ gel was found to be a promising formulation for the efficient treatment of bacterial ocular disease.

In silico characterization of mutations circulating in SARS-CoV-2 structural proteins.

SARS-CoV-2 has recently emerged as a pandemic that has caused more than 2.4 million deaths worldwide. Since the onset of infections, several full-length sequences of viral genome have been made available which have been used to gain insights into viral dynamics. We utilised a meta-data driven comparative analysis tool for sequences (Meta-CATS) algorithm to identify mutations in 829 SARS-CoV-2 genomes from around the world. The algorithm predicted sixty-one mutations among SARS-CoV-2 genomes. We observed that most of the mutations were concentrated around three protein coding genes viz nsp3 (non-structural protein 3), RdRp (RNA-directed RNA polymerase) and Nucleocapsid (N) proteins of SARS-CoV-2. We used various computational tools including normal mode analysis (NMA), C-α discrete molecular dynamics (DMD) and all-atom molecular dynamic simulations (MD) to study the effect of mutations on functionality, stability and flexibility of SARS-CoV-2 structural proteins including envelope (E), N and spike (S) proteins. PredictSNP predictor suggested that four mutations (L37H in E, R203K and P344S in N and D614G in S) out of seven were predicted to be neutral whilst the remaining ones (P13L, S197L and G204R in N) were predicted to be deleterious in nature thereby impacting protein functionality. NMA, C-α DMD and all-atom MD suggested some mutations to have stabilizing roles (P13L, S197L and R203K in N protein) where remaining ones were predicted to destabilize mutant protein. In summary, we identified significant mutations in SARS-CoV-2 genomes as well as used computational approaches to further characterize the possible effect of highly significant mutations on SARS-CoV-2 structural proteins.Communicated by Ramaswamy H. Sarma.

Peptide Utility (PU) search server: A new tool for peptide sequence search from multiple databases.

Proteins are essential building blocks in humans that have garnered huge attention from researchers worldwide due to their numerous therapeutic applications. To date, different computational tools have been developed to extract pre-existing information on these biological molecules, but most of these tools suffer from limitations such as non-user friendly interface, redundancy of data, etc. To overcome these limitations, a user-friendly interface, the Peptide Utility (PU) webserver (https://chain-searching.herokuapp.com/) has been developed for searching and analyzing homologous and identical protein/peptide sequences that can be searched from approximately 0.4 million sequences (structural and sequence information) in both online and offline modes. The PU web server can also be used to study different types of interactions in PDBSum, identifying the most dominating interface residues, the most prevalent interactions, and the interaction preferences of different residues. The webserver would also pave way for the design of novel therapeutic peptides and folds by identifying conserved residues in the three-dimensional structure space of proteins.

Architectural and functional details of CF IA proteins involved in yeast 3'-end pre-mRNA processing and its significance for eukaryotes: A concise review.

In eukaryotes, maturation of pre-mRNA relies on its precise 3'-end processing. This processing involves co-transcriptional steps regulated by sequence elements and other proteins. Although, it holds tremendous importance, defect in the processing machinery will result in erroneous pre-mRNA maturation leading to defective translation. Remarkably, more than 20 proteins in humans and yeast share homology and execute this processing. The defects in this processing are associated with various diseases in humans. We shed light on the CF IA subunit of yeast Saccharomyces cerevisiae that contains four proteins (Pcf11, Clp1, Rna14 and Rna15) involved in this processing. Structural details of various domains of CF IA and their roles during 3'-end processing, like cleavage and polyadenylation at 3'-UTR of pre-mRNA and other cellular events are explained. Further, the chronological development and important discoveries associated with 3'-end processing are summarized. Moreover, the mammalian homologues of yeast CF IA proteins, along with their key roles are described. This knowledge would be helpful for better comprehension of the mechanism associated with this marvel; thus opening up vast avenues in this area.

Tuberculosis: An Overview of the Immunogenic Response, Disease Progression, and Medicinal Chemistry Efforts in the Last Decade toward the Development of Potential Drugs for Extensively Drug-Resistant Tuberculosis Strains.

Tuberculosis (TB) is a slow growing, potentially debilitating disease that has plagued humanity for centuries and has claimed numerous lives across the globe. Concerted efforts by researchers have culminated in the development of various strategies to combat this malady. This review aims to raise awareness of the rapidly increasing incidences of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, highlighting the significant modifications that were introduced in the TB treatment regimen over the past decade. A description of the role of pathogen-host immune mechanisms together with strategies for prevention of the disease is discussed. The struggle to develop novel drug therapies has continued in an effort to reduce the treatment duration, improve patient compliance and outcomes, and circumvent TB resistance mechanisms. Herein, we give an overview of the extensive medicinal chemistry efforts made during the past decade toward the discovery of new chemotypes, which are potentially active against TB-resistant strains.

Fluorescent quantum dots: An insight on synthesis and potential biological application as drug carrier in cancer.

Quantum dots (QDs) are nanocrystals of semiconducting material possessing quantum mechanical characteristics with capability to get conjugated with drug moieties. The particle size of QDs varies from 2 to 10 nm and can radiate a wide range of colours depending upon their size. Their wide and diverse usage of QDs across the world is due to their adaptable properties like large quantum yield, photostability, and adjustable emission spectrum. QDs are nanomaterials with inherent electrical characteristics that can be used as drug carrier vehicle and as a diagnostic in the field of nanomedicine. Scientists from various fields are aggressively working for the development of single platform that can sense, can produce a microscopic image and even be used to deliver a therapeutic agent. QDs are the fluorescent nano dots with which the possibilities of the drug delivery to a targeted site and its biomedical imaging can be explored. This review is mainly focused on the different process of synthesis of QDs, their application especially in the areas of malignancies and as a theranostic tool. The attempt is to consolidate the data available for the use of QDs in the biomedical applications.

SARS-CoV-2: Insights into its structural intricacies and functional aspects for drug and vaccine development.

Globally, SARS-CoV-2 has emerged as threat to life and economy. Researchers are trying to find a cure against this pathogen but without much success. Several attempts have been made to understand the atomic level details of SARS-CoV-2 in the past few months. However, one review with all structural details for drug and vaccine development has been missing. Hence, this review aims to summarize key functional roles played by various domains of SARS-CoV-2 genome during its entry into the host, replication, repression of host immune response and overall viral life cycle. Additionally, various proteins of SARS-CoV-2 for finding a potent inhibitor have also been highlighted. To mitigate this deadly virus, an understanding of atomic level information, pathogenicity mechanisms and functions of different proteins in causing the infection is imperative. Thus, these structural details would finally pave the way for development of a potential drug/vaccine against the disease caused by SARS-CoV-2.

Mucormycosis Amid COVID-19 Crisis: Pathogenesis, Diagnosis, and Novel Treatment Strategies to Combat the Spread.

The havoc unleashed by COVID-19 pandemic has paved way for secondary ominous fungal infections like Mucormycosis. It is caused by a class of opportunistic pathogens from the order Mucorales. Fatality rates due to this contagious infection are extremely high. Numerous clinical manifestations result in damage to multiple organs subject to the patient's underlying condition. Lack of a proper detection method and reliable treatment has made the management of this infection troublesome. Several reports studying the behavior pattern of Mucorales inside the host by modulation of its defense mechanisms have helped in understanding the pathogenesis of this angio-invasive infection. Many recent advances in diagnosis and treatment of this fungal infection have not been much beneficial. Therefore, there is a need to foster more viable strategies. This article summarizes current and imminent approaches that could aid effective management of these secondary infections in these times of global pandemic. It is foreseen that the development of newer antifungal drugs, antimicrobial peptides, and nanotechnology-based approaches for drug delivery would help combat this infection and curb its spread.

Comparative structure, dynamics and evolution of acyl-carrier proteins from Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii.

Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria.

NMR structure and dynamics of inhibitory repeat domain variant 12, a plant protease inhibitor from Capsicum annuum, and its structural relationship to other plant protease inhibitors.

Although several plant protease inhibitors have been structurally characterized using X-ray crystallography, very few have been studied using NMR techniques. Here, we report an NMR study of the solution structure and dynamics of an inhibitory repeat domain (IRD) variant 12 from the wound-inducible Pin-II type proteinase inhibitor from Capsicum annuum. IRD variant 12 (IRD12) showed strong anti-metabolic activity against the Lepidopteran insect pest, Helicoverpa armigera. The NMR-derived three-dimensional structure of IRD12 reveals a three-stranded anti-parallel ß-sheet rigidly held together by four disulfide bridges and shows structural homology with known IRDs. It is interesting to note that the IRD12 structure containing ∼75% unstructured part still shows substantial amount of rigidity of N-H bond vectors with respect to its molecular motion.Communicated by Ramaswamy H. Sarma.

Applications of NMR to structure determination of RNAs large and small.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to investigate the structure and dynamics of RNA, because many biologically important RNAs have conformationally flexible structures, which makes them difficult to crystallize. Functional, independently folded RNA domains, range in size between simple stem-loops of as few as 10-20 nucleotides, to 50-70 nucleotides, the size of tRNA and many small ribozymes, to a few hundred nucleotides, the size of more complex RNA enzymes and of the functional domains of non-coding transcripts. In this review, we discuss new methods for sample preparation, assignment strategies and structure determination for independently folded RNA domains of up to 100 kDa in molecular weight.

NMR structure of an acyl-carrier protein from Borrelia burgdorferi.

Nearly complete resonance assignment and the high-resolution NMR structure of the acyl-carrier protein from Borrelia burgdorferi, a target of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure-determination pipeline, are reported. This protein was chosen as a potential target for drug-discovery efforts because of its involvement in fatty-acid biosynthesis, an essential metabolic pathway, in bacteria. It was possible to assign >98% of backbone resonances and >92% of side-chain resonances using multidimensional NMR spectroscopy. The NMR structure was determined to a backbone r.m.s.d. of 0.4 Å and contained four α-helices and two 3(10)-helices. A structure-homology search revealed that this protein is highly similar to the acyl-carrier protein from Aquifex aeolicus.

Temperature-dependent oligomerization in M-crystallin: lead or lag toward cataract, an NMR perspective.

The oligomerization and/or aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from abnormal disease states like Alzheimer's and Parkinson's disease to the production of inclusion bodies, stability, and delivery of protein drugs. In the case of eye-lens proteins, oligomerization is implicated in cataract formation. In the present study, we have investigated the temperature driven oligomerization of M-crystallin, a close homologue of eye-lens proteins, using NMR spectroscopy and dynamic-light scattering (DLS). The NMR data primarily included R(1), R(2) relaxation rates and nOes of the backbone amide groups recorded at three different temperatures, 25, 20, and 15° C. The major outcome of the study is the two fold increase in the overall tumbling time (τ(c)) of M-crystallin on lowering the temperature from 25 to 15° C. An extrapolation of τ(c) to a further lower temperature (5° C) may lead to a τ(c) of ∼19 ns that would correspond to a τ(c) value of a tetrameric M-crystallin. These results also validate the observed changes in the hydrodynamic radius of M-crystallin, determined using DLS data. Further, the temperature-dependent protein dynamics of M-crystallin reveal considerable variation at/near the Ca(2+)-binding sites. A concerted analysis of the temperature dependent relaxation parameters and DLS data reveals that the self-association of the protein is not only a monomer-dimer equilibrium, but also goes to tetramers or other multimeric states. These higher states may co-exist in fast exchange with the monomeric and dimeric M-crystallin at milli-molar to sub-millimolar concentrations and at lower temperature.

Root-mean-square-deviation-based rapid backbone resonance assignments in proteins.

We have shown that the methodology based on the estimation of root-mean-square deviation (RMSD) between two sets of chemical shifts is very useful to rapidly assign the spectral signatures of (1)H(N), (13)C(α), (13)C(ß), (13)C', (1)H(α) and (15)N spins of a given protein in one state from the knowledge of its resonance assignments in a different state, without resorting to routine established procedures (manual and automated). We demonstrate the utility of this methodology to rapidly assign the 3D spectra of a metal-binding protein in its holo-state from the knowledge of its assignments in apo-state, the spectra of a protein in its paramagnetic state from the knowledge of its assignments in diamagnetic state and, finally, the spectra of a mutant protein from the knowledge of the chemical shifts of the corresponding wild-type protein. The underlying assumption of this methodology is that, it is impossible for any two amino acid residues in a given protein to have all the six chemical shifts degenerate and that the protein under consideration does not undergo large conformational changes in going from one conformational state to another. The methodology has been tested using experimental data on three proteins, M-crystallin (8.5 kDa, predominantly ß-sheet, for apo- to holo-state), Calbindin (7.5 kDa, predominantly α-helical, for diamagnetic to paramagnetic state and apo to holo) and EhCaBP1 (14.3 kDa, α-helical, the wild-type protein with one of its mutant). In all the cases, the extent of assignment is found to be greater than 85%.

Complete backbone assignment of a Ca2+-binding protein of the betagamma-crystallin superfamily from Methanosarcina acetivorans, at two denaturant concentrations.

We report here almost complete backbone assignment of a Ca(2+)-binding protein of the betagamma-crystallin superfamily from Methanosarcina acetivorans, at two denaturant (GdmCl) concentrations, using double and triple resonance experiments. These NMR assignments will be useful to understand the unfolding path of this protein.

Interaction of follicle-stimulating hormone (FSH) receptor binding inhibitor-8: a novel FSH-binding inhibitor, with FSH and its receptor.

Follicle-stimulating hormone (FSH) receptor binding inhibitor (FRBI-8) is a novel octapeptide purified from human ovarian follicular fluid. In vitro, it inhibits the binding of FSH to granulosa cells and in vivo, it induces atresia in developing follicles in rodents. This peptide, when administered to marmosets and bonnet monkeys, altered the circulating progesterone levels. This study was carried out to elucidate structure of the FRBI-8 and understand its mechanism of inhibiting interaction of FSH to its receptors. Homology modeling predicted that the FRBI-8 adopts a turn and random coil. This is further confirmed by circular dichroism and NMR. Docking studies of the FRBI-8 with reported FSH-FSHR hormone binding (FSHR(HB)) domain complex using ZDOCK algorithm revealed that the FRBI-8 binds to FSHbetaL2-FSHR(HB) binding interface which is otherwise known to be crucial for activation of signal transduction cascade. FRBI-8 analogs were designed by replacing the acidic amino acid residues at positions 2, 5 and 6 with Ala, individually. Docking studies revealed that D6A mutant (FRBI-8(D6A)) had a higher binding affinity than the native FRBI-8. In vitro radioreceptor assay with FRBI-8(D6A) showed 50% lower IC(50) compared with the FRBI-8, confirming the in silico observations. Thus, the study reveals that both FRBI-8 and FRBI-8(D6A) interfered with the binding of FSH to its receptor.