Emerging therapeutic developments in neurodegenerative diseases: A clinical investigation.

Despite a century of intensive research, there is still a lack of disease-modifying treatments for neurodegenerative diseases that pose a threat to human society. A well-documented knowledge and resource gap has impeded the translation of fundamental research into promising therapies. In addition, the analysis of extensive preclinical data to allow the improved selection of therapeutic technologies and clinical candidates for further development is challenging. To address this need, we describe technologies that have emerged over the past decade that have enabled the development of novel, high-quality, cost-effective treatments for major neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Moreover, we benchmark emerging technologies that have been adopted by top pharmaceutical companies looking to bridge the gap between drug discovery and drug development in neurodegenerative disease.

Targeting cyclin-dependent kinase 6 by vanillin inhibits proliferation of breast and lung cancer cells: Combined computational and biochemical studies.

Cyclin-dependent kinase 6 (CDK6) is a member of serine/threonine kinase family, and its overexpression is associated with cancer development. Thus, it is considered as a potential drug target for anticancer therapies. This study showed the CDK6 inhibitory potential of vanillin using combined experimental and computational methods. Structure-based docking and 200 ns molecular dynamics simulation studies revealed that the binding of vanillin stabilizes the CDK6 structure and provides mechanistic insights into the binding mechanism. Enzyme inhibition and fluorescence-binding studies showed that vanillin inhibits CDK6 with an half maximal inhibitory concentration = 4.99 µM and a binding constant (K) 4.1 × 107 M-1 . Isothermal titration calorimetry measurements further complemented our observations. Studies on human cancer cell lines (MCF-7 and A549) showed that vanillin decreases cell viability and colonization properties. The protein expression studies have further revealed that vanillin reduces the CDK6 expression and induces apoptosis in the cancer cells. In conclusion, our study presents the CDK6-mediated therapeutic implications of vanillin for anticancer therapies.

Identification of natural compounds as potent inhibitors of SARS-CoV-2 main protease using combined docking and molecular dynamics simulations.

Coronavirus disease 2019 (COVID-19) has emerged from China and globally affected the entire population through the human-to-human transmission of a newly emerged virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The genome of SARS-CoV-2 encodes several proteins that are essential for multiplication and pathogenesis. The main protease (Mpro or 3CLpro) of SARS-CoV-2 plays a central role in its pathogenesis and thus is considered as an attractive drug target for the drug design and development of small-molecule inhibitors. We have employed an extensive structure-based high-throughput virtual screening to discover potential natural compounds from the ZINC database which could inhibit the Mpro of SARS-CoV-2. Initially, the hits were selected on the basis of their physicochemical and drug-like properties. Subsequently, the PAINS filter, estimation of binding affinities using molecular docking, and interaction analyses were performed to find safe and potential inhibitors of SARS-CoV-2 Mpro. We have identified ZINC02123811 (1-(3-(2,5,9-trimethyl-7-oxo-3-phenyl-7H-furo[3,2-g]chromen-6-yl)propanoyl)piperidine-4-carboxamide), a natural compound bearing appreciable affinity, efficiency, and specificity towards the binding pocket of SARS-CoV-2 Mpro. The identified compound showed a set of drug-like properties and preferentially binds to the active site of SARS-CoV-2 Mpro. All-atom molecular dynamics (MD) simulations were performed to evaluate the conformational dynamics, stability and interaction mechanism of Mpro with ZINC02123811. MD simulation results indicated that Mpro with ZINC02123811 forms a stable complex throughout the trajectory of 100 ns. These findings suggest that ZINC02123811 may be further exploited as a promising scaffold for the development of potential inhibitors of SARS-CoV-2 Mpro to address COVID-19.

Identification and structure-activity relationship (SAR) studies of carvacrol derivatives as potential anti-malarial against Plasmodium falciparum falcipain-2 protease.

In an effort to develop a potent anti-malarial agent against Plasmodium falciparum, a structure-guided virtual screening using an in-house library comprising 652 compounds was performed. By docking studies, we identified two compounds (JMI-105 and JMI-346) which formed significant non-covalent interactions and fit well in the binding pocket of PfFP-2. We affirmed this observation by MD simulation studies. As evident by the biochemical analysis, such as enzyme inhibition assay, Surface Plasmon Resonance (SPR), live-cell imaging and hemozoin inhibition, JMI-105 and JMI-346 at 25 µM concentration showed an inhibitory effect on purified PfFP-2. JMI-105 and JMI-346 inhibited the growth of CQS (3D7; IC50 = 8.8 and 13 µM) and CQR (RKL-9; IC50 = 14.3 and 33 µM) strains of P. falciparum. Treatment with compounds resulted in defect in parasite growth and development. No significant hemolysis or cytotoxicity towards human cells was observed suggesting that these molecules are non-toxic. We pursued, structural optimization on JMI-105 and in the process, SAR oriented derivatives (5a-5l) were synthesized and evaluated for growth inhibition potential. JMI-105 significantly decreased parasitemia and prolonged host survival in a murine model with P. berghei ANKA infection. The compounds (JMI-105 and JMI-346) against PfFP-2 have the potential to be used as an anti-malarial agent.

Structure, function and therapeutic implications of OB-fold proteins: A lesson from past to present.

Oligonucleotide/oligosaccharide-binding (OB)-fold proteins play essential roles in the regulation of genome and its correct transformation to the subsequent generation. To maintain the genomic stability, OB-fold proteins are implicated in various cellular processes including DNA replication, DNA repair, cell cycle regulation and maintenance of telomere. The diverse functional spectrums of OB-fold proteins are mainly due to their involvement in protein-DNA and protein-protein complexes. Mutations and consequential structural alteration in the OB-fold proteins often lead to severe diseases. Here, we have investigated the structure, function and mode of action of OB-fold proteins (RPA, BRCA2, DNA ligases and SSBs1/2) in cellular pathways and their relationship with diseases and their possible use in therapeutic intervention. Due to the crucial role of OB-fold proteins in regulating the key physiological process, a detailed structural understanding in the context of underlying mechanism of action and cellular complexity offers a new avenue to target OB-proteins for therapeutic intervention.

Synthesis and SAR studies of novel 1,2,4-oxadiazole-sulfonamide based compounds as potential anticancer agents for colorectal cancer therapy.

A diverse series of 1,2,4-oxadiazoles based substituted compounds were designed, synthesized and evaluated as anticancer agents targeting carbonic anhydrase IX (CAIX). Initial structure-activity analysis suggested that the thiazole/thiophene-sulfonamide conjugates of 1,2,4-oxadiazoles exhibited potent anticancer activities with low µM potencies. Compound OX12 exhibited antiproliferative activity (IC50 = 11.1 µM) along with appreciable inhibition potential for tumor-associated CAIX (IC50 = 4.23 µM) isoform. Therefore, OX12 was structurally optimized and its SAR oriented derivatives (OX17-27) were synthesized and evaluated. This iteration resulted in compound OX27 with an almost two-fold increase in antiproliferative effect (IC50 = 6.0 µM) comparable to the clinical drug doxorubicin and significantly higher potency against CAIX (IC50 = 0.74 µM). Additionally, OX27 treatment decreases the expression of CAIX, induces apoptosis and ROS production, inhibited colony formation and migration of colon cancer cells. Our studies provide preclinical rational for the further optimization of identified OX27 as a suitable lead for the possible treatment of CRC.

Virtual high-throughput screening of natural compounds in-search of potential inhibitors for protection of telomeres 1 (POT1).

Communicated by Ramaswamy H. Sarma.

Exploring the aggregation-prone regions from structural domains of human TDP-43.

TDP-43 (transactive- response DNA binding protein) amazes structural biologist as its aberrant ubiquitinated cytosolic inclusions is largely involved in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). An important question in TDP-43 research is to identify the structural region mediating the formation of cytoplasmic pathological aggregates. In this study, we attempted to delineate the aggregation-prone sequences of the structural domain of TDP-43. Here, we investigated the self-assembly of peptides of TDP-43 using aggregation prediction algorithms, Zipper DB and AMYLPRED2. The three aggregation-prone peptides identified were from N-terminal domain (24GTVLLSTV31), and RNA recognition motifs, RRM1 (128GEVLMVQV135) and RRM2 (247DLIIKGIS254). Furthermore, the amyloid fibril forming propensities of these peptides were analyzed through different biophysical techniques and molecular dynamics simulation. Our study shows the different aggregation ability of conserved stretches in structural domain of TDP-43 that will possibly induce full-length aggregation of TDP-43 in vivo. The peptide form RRM2 demonstrates the higher intrinsic amyloid forming propensity and suggests that RRM2 might form the structural core of TDP-43 aggregation seen in vivo. The results of this study would help in designing peptide based inhibitors of TDP-43 aggregation.

Implication of sulfonylurea derivatives as prospective inhibitors of human carbonic anhydrase II.

Selective carbonic anhydrase (CA) inhibitors have gained a lot of importance owing to the implication of specific isoforms of CA in certain diseases like glaucoma, leukemia, cystic fibrosis, and epilepsy. A novel class of sulfonylurea derivatives was synthesized from corresponding sulfonyl chlorides and amines. Compounds with different pendant moieties in the sulfonylurea derivatives show significant interactions with human carbonic anhydrase II (CAII). In vitro evaluation of the sulfonylurea derivatives revealed that three compounds possess admirable inhibitory activity against CAII. Compounds containing methyl (G2), isopropyl (G4) and o-tosyl (G5) groups displayed IC50 (109-137 µm) for CAII. Fluorescence binding and cytotoxicity studies revealed that these compounds are showing good binding affinity (18-34 µM) to CAII and non- toxic to human cells. Further, molecular docking studies of G2, G4 and G5 with CAII showed that these compounds fit nicely in the active site of CAII. Molecular dynamics simulation studies of these compounds complexed with CAII showed that essential interactions were maintained up to 50 ns of simulation. These results indicate the promising nature of the sulfonylurea scaffold towards CAII inhibition and opens scope of hit to-lead optimization for discovery of effective drugs against CAII-associated disorders.

Exploring molecular insights into the interaction mechanism of cholesterol derivatives with the Mce4A: A combined spectroscopic and molecular dynamic simulation studies.

Mammalian cell entry protein (Mce4A) is a member of MCE-family, and is being considered as a potential drug target of Mycobacterium tuberculosis infection because it is required for invasion and latent survival of pathogen by utilizing host's cholesterol. In the present study, we performed molecular docking followed by 100 ns MD simulation studies to understand the mechanism of interaction of Mce4A to the cholesterol derivatives and probucol. The selected ligands, cholesterol, 25-hydroxycholesterol, 5-cholesten-3ß-ol-7-one and probucol bind to the predicted active site cavity of Mce4A, and complexes remain stable during entire simulation of 100 ns. In silico studies were further validated by fluorescence-binding studies to calculate actual binding affinity and number of binding site(s). The non-toxicity of all ligands was confirmed on human monocytic cell (THP1) by MTT assay. This work provides a deeper insight into the mechanism of interaction of Mce4A to cholesterol derivatives, which may be further exploited to design potential and specific inhibitors to ameliorate the Mycobacterium pathogenesis.

QSAR based therapeutic management of M. tuberculosis.

Mycobacterium tuberculosis is responsible for severe mortality and morbidity worldwide but, under-developed and developing countries are more prone to infection. In search of effective and wide-spectrum anti-tubercular agents, interdisciplinary approaches are being explored. Of the several approaches used, computer based quantitative structure activity relationship (QSAR) have gained momentum. Structure-based drug design and discovery implies a combined knowledge of accurate prediction of ligand poses with the good prediction and interpretation of statistically validated models derived from the 3D-QSAR approach. The validated models are generally used to screen a small combinatorial library of potential synthetic candidates to identify hits which further subjected to docking to filter out compounds as novel potential emerging drug molecules to address multidrug-resistant tuberculosis. Several newer models are integrated to QSAR methods which include different types of chemical and biological data, and simultaneous prediction of pharmacological activities including toxicities and/or other safety profiles to get new compounds with desired activity. In the process, several newer molecules have been identified which are now being assessed for their clinical efficacy. Present review deals with the advances made in the field highlighting overall future prospects of the development of anti-tuberculosis drugs.

Evaluation of human microtubule affinity-regulating kinase 4 inhibitors: fluorescence binding studies, enzyme, and cell assays.

Human microtubule affinity-regulating kinase 4 (MARK4) is considered as an encouraging drug target for the design and development of inhibitors to cure several life-threatening diseases such as Alzheimer disease, cancer, obesity, and type-II diabetes. Recently, we have reported four ligands namely, BX-912, BX-795, PKR-inhibitor, and OTSSP167 (hydrochloride) which bind preferentially to the two different constructs of human MARK4 containing kinase domain. To ensure the role of ubiquitin-associated (UBA) domain in the ligand binding, we made a newer construct of MARK4 which contains both kinase and UBA domains, named as MARK4-F3. We observed that OTSSP167 (hydrochloride) binds to the MARK4-F3 with a binding constant (K) of 3.16 × 106, M-1 (±.21). However, UBA-domain of MARK4-F3 doesn't show any interaction with ligands directly as predicted by the molecular docking. To validate further, ATPase inhibition assays of all three constructs of MARK4 in the presence of mentioned ligands were carried out. An appreciable correlation between the binding experiments and ATPase inhibition assays of MARK4 was observed. In addition, cell-proliferation inhibition activity for all four ligands on the Human embryonic kidney (HEK-293) and breast cancer cell lines (MCF-7) was performed using MTT assay. IC50 values of OTSSP167 for HEK-293 and MCF-7 were found to be 58.88 (±1.5), and 48.2 (±1.6), respectively. OTSSP167 among all four inhibitors, showed very good enzyme inhibition activity against three constructs of MARK4. Moreover, all four inhibitors showed anti-neuroblastoma activity and anticancer properties. In conclusion, OTSSP167 may be considered as a promising scaffold to discover novel inhibitors of MARK4.

Human microtubule affinity-regulating kinase 4 is stable at extremes of pH.

MAP/microtubule affinity-regulating kinase 4 (MARK4) is a member of adenosine monophosphate-activated protein kinases, directly associated with cancer and neurodegenerative diseases. Here, we have cloned, expressed, and purified two variants of MARK4 [the kinase domain (MARK4-F2), and kinase domain along with 59 N-terminal residues (MARK4-F1)] and compared their stability at varying pH range. Structural and functional changes were observed by incubating both forms of MARK4 in buffers of different pH. We measured the secondary structure of MARK4 using circular dichroism and tertiary structure by measuring intrinsic fluorescence and absorbance properties along with the size of proteins by dynamic light scattering. We observed that at extremes of pH (below pH 3.5 and above pH 9.0), MARK4 is quite stable. However, a remarkable aggregate formation was observed at intermediate pH (between pH 3.5 and 9.0). To further validate this result, we have modeled both forms of MARK4 and performed molecular dynamics simulation for 15 ns. The spectroscopic observations are in excellent agreement with the findings of molecular dynamics simulation. We also performed ATPase activity at varying pH and found a significant correlation of structure of MARK4 with its enzyme activity. It is interesting to note that both forms of MARK4 are showing a similar pattern of structure changes with reference to pH.

Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA.

Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme that catalyzes the reversible hydration of CO2 to produce HCO3- and proton. CAV is primarily involved in several biosynthetic processes such as ureagenesis, gluconeogenesis and lipogenesis by providing bicarbonate ion. Here, we report a new strategy for cloning, expression and purification for CAVA in the bacterial system followed by its biophysical characterization. The cDNA of CAVA, a 801 nucleotide long that encodes a 267-amino acid polypeptide of molecular mass of 30-kDa (excluding signal peptide), was sub-cloned in the expression vector pET21c and transformed into Escherichia coli strain BL21 (DE3) for expression. The recombinant protein was purified in two steps by Ni-NTA and DEAE weak anion-exchange chromatography under native condition from the supernatant, while inclusion bodies (IBs) were used to get protein under the denatured condition with a relatively high yield. CAVA was purified under denatured conditions in a single step using Ni-NTA chromatography. SDS-PAGE showed a band of 30-kDa, which was further confirmed as CAVA by Western blot and MALDI-TOF/MS. We further performed enzyme activity to ensure that both forms of purified proteins are enzymatically active. Measurements of secondary structure of the native, denatured and renatured proteins were carried out using circular dichroism. The purified protein can be further used for structural and biochemical studies.

Purification and characterization of RGA2, a Rho2 GTPase-activating protein from Tinospora cordifolia.

Rho GTPases activating protein 2 (RGA2) is primarily involved in the modulation of numerous morphological events in eukaryotes. It protects plants by triggering the defense system which restricts the pathogen growth. This is the first report on the isolation, purification and characterization of RGA2 from the stems of Tinospora cordifolia, a medicinal plant. The RGA2 was purified using simple two-step process using DEAE-Hi-Trap FF and Superdex 200 chromatography columns, with a high yield. The purity of RGA2 was confirmed by SDS-PAGE and identified by MALDI-TOF/MS. The purified protein was further characterized for its secondary structural elements using the far-UV circular dichroism measurements. Our purification procedure is simple two-step process with high yield which can be further used to produce RGA2 for structural and functional studies.

Structure-based functional annotation of putative conserved proteins having lyase activity from Haemophilus influenzae.

Haemophilus influenzae is a small pleomorphic Gram-negative bacteria which causes several chronic diseases, including bacteremia, meningitis, cellulitis, epiglottitis, septic arthritis, pneumonia, and empyema. Here we extensively analyzed the sequenced genome of H. influenzae strain Rd KW20 using protein family databases, protein structure prediction, pathways and genome context methods to assign a precise function to proteins whose functions are unknown. These proteins are termed as hypothetical proteins (HPs), for which no experimental information is available. Function prediction of these proteins would surely be supportive to precisely understand the biochemical pathways and mechanism of pathogenesis of Haemophilus influenzae. During the extensive analysis of H. influenzae genome, we found the presence of eight HPs showing lyase activity. Subsequently, we modeled and analyzed three-dimensional structure of all these HPs to determine their functions more precisely. We found these HPs possess cystathionine-ß-synthase, cyclase, carboxymuconolactone decarboxylase, pseudouridine synthase A and C, D-tagatose-1,6-bisphosphate aldolase and aminodeoxychorismate lyase-like features, indicating their corresponding functions in the H. influenzae. Lyases are actively involved in the regulation of biosynthesis of various hormones, metabolic pathways, signal transduction, and DNA repair. Lyases are also considered as a key player for various biological processes. These enzymes are critically essential for the survival and pathogenesis of H. influenzae and, therefore, these enzymes may be considered as a potential target for structure-based rational drug design. Our structure-function relationship analysis will be useful to search and design potential lead molecules based on the structure of these lyases, for drug design and discovery.

Structure, function and applications of carbonic anhydrase isozymes.

The carbonic anhydrases enzymes (CAs, EC 4.2.1.1) are zinc containing metalloproteins, which efficiently catalyse the reversible conversion of carbon dioxide to bicarbonate and release proton. These enzymes are essentially important for biological system and play several important physiological and patho-physiological functions. There are 16 different alpha-carbonic anhydrase isoforms studied, differing widely in their cellular localization and biophysical properties. The catalytic domains of all CAs possess a conserved tertiary structure fold, with predominately ß-strands. We performed an extensive analysis of all 16 mammalian CAs for its structure and function in order to establish a structure-function relationship. CAs have been a potential therapeutic target for many diseases. Sulfonamides are considered as a strong and specific inhibitor of CA, and are being used as diuretics, anti-glaucoma, anti-epileptic, anti-ulcer agents. Currently CA inhibitors are widely used as a drug for the treatment of neurological disorders, anti-glaucoma drugs, anti-cancer, or anti-obesity agents. Here we tried to emphasize how CAs can be used for drug discovery, design and screening. Furthermore, we discussed the role of CA in carbon capture, carbon sensor and metabolon. We hope this review provide many useful information on structure, function, mechanism, and applications of CAs in various discipline.