Biophysical insight into anti-amyloidogenic nature of novel ionic Co(II)(phen)(H2O)4]+[glycinate]- chemotherapeutic drug candidate against human lysozyme aggregation.

In the recent past, there has been an ever-increasing interest in the search for metal-based therapeutic drug candidates for protein misfolding disorders (PMDs) particularly neurodegenerative disorders such as Alzheimer's, Parkinson's, Prion's diseases, and amyotrophic lateral sclerosis. Also, different amyloidogenic variants of human lysozyme (HL) are involved in hereditary systemic amyloidosis. Metallo-therapeutic agents are extensively studied as antitumor agents, however, they are relatively unexplored for the treatment of non-neuropathic amyloidoses. In this work, inhibition potential of a novel ionic cobalt(II) therapeutic agent (CoTA) of the formulation [Co(phen)(H2O)4]+[glycinate]- is evaluated against HL fibrillation. Various biophysical techniques viz., dye-binding assays, dynamic light scattering (DLS), differential scanning calorimetry (DSC), electron microscopy, and molecular docking experiments validate the proposed mechanism of inhibition of HL fibrillation by CoTA. The experimental corroborative results of these studies reveal that CoTA can suppress and slow down HL fibrillation at physiological temperature and pH. DLS and 1-anilino-8-naphthalenesulfonate (ANS) assay show that reduced fibrillation in the presence of CoTA is marked by a significant decrease in the size and hydrophobicity of the aggregates. Fluorescence quenching and molecular docking results demonstrate that CoTA binds moderately to the aggregation-prone region of HL (Kb = 6.6 × 104 M-1), thereby, inhibiting HL fibrillation. In addition, far-UV CD and DSC show that binding of CoTA to HL does not cause any change in the stability of HL. More importantly, CoTA attenuates membrane damaging effects of HL aggregates against RBCs. This study identifies inorganic metal complexes as a therapeutic intervention for systemic amyloidosis.

Biophysical insight into the anti-fibrillation potential of Glyburide for its possible implication in therapeutic intervention of amyloid associated diseases.

Protein aggregation is an underlying cause of many neurodegenerative diseases. Also, the overlapping pathological disturbances between neurodegenerative diseases and type-2 diabetes mellitus have urged the scientific community to explore potential of already available anti-diabetic medications in impeding amyloid formation too. Recent study brief out promising potential of an anti-diabetic drug Glyburide(GLY) as an inhibitor of amyloid fibrillation utilizing several biophysical techniques, computational methods and imaging tools. The mechanism of interaction was elucidated and the structural alterations in human serum albumin(HSA) as well as the microenvironment changes of its fluorophores(tryptophan, tyrosine) upon interacting with GLY were studied by spectroscopic techniques like Circular dichroism and synchronous fluorescence. Binding studies detailing about the GLY-HSA complex distance and the energy transfer efficiency was obtained by Fluorescence resonance energy transfer. For aggregation inhibition studies, the existence and size of aggregates formed in HSA and their inhibition by GLY was determined by Turbidity assay, Dynamic light scattering and Rayleigh light scattering along with dye binding assays. The ThT kinetics measurements analysis suggested that GLY deaccelerates fibrillation by decrement of apparent rate(Kapp) constant. The inhibitory effect of GLY might be attributed to native structure stabilization of HSA by obstruction into ß-sheet conversion as confirmed by CD spectroscopy results. Amyloid inhibition and suppression of amyloid-induced hemolysis by GLY was further delineated by TEM and SEM analysis respectively. All these findings for the first time report the new facet of the anti-amyloidogenic potential of GLY, making it a promising candidate to treat neurodegenerative diseases too in the near future.

Mechanistic insight into inhibition of amyloid fibrillation of human serum albumin by Vildagliptin.

Protein aggregation leads to several human pathologies such as Alzheimer's disease (AD), type 2 diabetes (T2D), Parkinson's disease (PD), etc. Due to the overlap in the mechanisms of type 2 diabetes and brain disorders, common effective pharmacological interventions to treat both T2D and AD is under extensive research. Therefore, major aim of research is to repurpose already established treatment of diabetes to cure AD as well. This study evaluates mechanistic insight into anti-amyloidogenic potential of anti-diabetic drug Vildagliptin (VLD) on human serum albumin fibrillation (HSA) by using biophysical, calorimetric, imaging techniques along with hemolytic assay. Dynamic light scattering (DLS) and Rayleigh light scattering (RLS) results showed presence of few small-sized aggregates in the presence of VLD which are formed by deaccelerating the amyloidogenesis as shown by thioflavin T (ThT) fluorescence and Congo red (CR) binding assay. Further, Isothermal titration calorimetry (ITC), steady state fluorescence quenching, molecular docking results revealed that VLD form complex with amyloid facilitating state of HSA and consequently mask the hydrophobic residues involved in amyloidogenesis as evident from decrease in ANS fluorescence. Differential scanning calorimetry (DSC) results confirm that VLD stabilizes the amyloid facilitating state of HSA. In addition, SEM images demonstrated that VLD alleviates the hemolytic effect induced by fibrils of HSA. This study reports VLD as a potential inhibitor of amyloid fibrillation and provides promising results to repurpose VLD as a drug candidate for the cure of Alzheimer's diseases along with diabetes.

Computational studies on phylogeny and drug designing using molecular simulations for COVID-19.

Since the first appearance of a novel coronavirus pneumonia (NCP) caused by a novel human coronavirus, and especially after the infection started its rapid spread over the world causing the COVID-19 (coronavirus disease 2019) pandemics, a very substantial part of the scientific community is engaged in the intensive research dedicated to finding of the potential therapeutics to cure this disease. As repurposing of existing drugs represents the only instant solution for those infected with the virus, we have been working on utilization of the structure-based virtual screening method to find some potential medications. In this study, we screened a library of 646 FDA approved drugs against the receptor-binding domain of the SARS-CoV-2 spike (S) protein and the main protease of this virus. Scoring functions revealed that some of the anticancer drugs (such as Pazopanib, Irinotecan, and Imatinib), antipsychotic drug (Risperidone), and antiviral drug (Raltegravir) have a potential to interact with both targets with high efficiency. Further we performed molecular dynamics simulations to understand the evolution in protein upon interaction with drug. Also, we have performed a phylogenetic analysis of 43 different coronavirus strains infecting 12 different mammalian species.Communicated by Ramaswamy H. Sarma.

Evaluation of ThT augmentation and RLS inner filter effect caused by highly fluorescent coumarin derivative and establishing it as true inhibitor of amyloid fibrillation.

Screening of inhibitors that slow down or suppress amyloid fibrils formation relies on some simple but sensitive spectroscopy techniques. Thioflavin T (ThT) fluorescence assay is one of the most common, amyloid specific and sensitive method. However, if an inhibitor is itself fluorescent in the ThT fluorescence range, its screening becomes complicated and require complementary assays. One of such molecules, 6, 7-dihydroxycoumarin (6, 7-DHC, also known as aesculetin, esculetin, and cichorigenin) is fluorescent in the ThT emission range and absorbs in the ThT excitation range. Therefore, it can produce a subtractive effect attributed to primary inner filter effect and/or additive effect due to its self-fluorescence in ThT assay. Our study shows that 6, 7-DHC produces an additive effect in ThT fluorescence, which is minimized at high concentration of ThT and decrease in ThT fluorescence is solely due to its inhibitory effect against HSA fibrillation. These ThT fluorescence-based results are verified through other complementary assays, such as Rayleigh and dynamic light scattering and amyloid-specific Congo red binding assay. Furthermore, hydrophobicity reduction is studied through Nile red (NR) and kinetics through far-UV circular dichroism (far-UV CD) in place of the most commonly employed ThT assay owing to extremely high fluorescence of 6, 7-DHC during initial incubation period.

Probing the Nongeneralized Amyloid Inhibitory Mechanism of Hydrophobic Chaperone.

Increasing prevalence of protein misfolding disorders urges the search for effective therapies. Although several antiaggregation molecules have been identified, their molecular process of aggregation and clinical trials are underway. The present study is focused on the mechanism through which phenyl butyrate (PB), a chemical chaperone, triggers inhibition of human serum albumin (HSA) fibrillation. Turbidity and Rayleigh light scattering (RLS) measurements reveal the marked presence of aggregates in HSA that were confirmed as amyloid fibrils by thioflavin T (ThT) and Congo red (CR) and were subsequently inhibited by PB in a dose dependent manner. ThT fluorescence kinetics reveals a decrease in the apparent rate constant, Kapp, in the presence of PB without triggering a lag phase in HSA suggesting PB's interference with the elongation phase. Dynamic light scattering (DLS) results display a reduction in the aggregate size in the presence of PB. Isothermal titration calorimetry (ITC) data reveals strong binding of PB at site II both at 25 °C (Kb ≈ 1.94 × 105 M-1) and 65 °C (Kb ≈ 2.90 × 104 M-1), mediated by hydrogen bonding. Overall, our finding establishes that PB stabilizes partially unfolded HSA molecules through hydrogen bonding, thereby preventing establishment of hydrogen bonds between them and hindering their progression into amyloid fibrils. This is in contrast to its chaperone effect manifested with other proteins.

Unravelling the inhibitory and cytoprotective potential of diuretics towards amyloid fibrillation.

Protein misfolding and deposition of aggregated proteins inside as well as outside of the cells have been associated with several neurotoxic and neurodegenerative disorders like Alzheimer's, Parkinson's and familial amyloid polyneuropathy etc. that could be controlled by anti-aggregation methodologies employing either inhibition or disaggregation of toxic aggregates. Also, the Alzheimer's disease develops in later life is somehow related to the high mid-life blood pressure. Therefore the present work targets the amyloid inhibiting potential of diuretics (a class of antihypertensive drugs) - Indapamide (INDP) and Hydrochlorothiazide (HCTZ) against human serum albumin (HSA) and human lysozyme (HL) fibrillogenesis. The effect of both INDP and HCTZ on the kinetics of amyloid formation of HSA and HL was illustrated and various biophysical techniques like Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, Congo red measurements and circular dichroism (CD) measurements depicted the inhibitory action of both INDP and HCTZ in a dose dependent manner. Transmission Electronic Microscopy (TEM) confirmed the absence of fibrillar structures when HSA and HL were co-incubated with INDP and HCTZ. In addition, molecular docking results revealed that both the drugs interacts with HSA and HL through hydrophobic interactions as well as hydrogen bonding, and also showed non-hemolytic activity on human RBCs demonstrated by the Hemolytic assay. Thus, both INDP and HCTZ could be propitious as a therapeutic agent and aid in the cure of amyloid related diseases.

An insight into the inhibition of fibrillation process verses disaggregation of preformed fibrils of bovine serum albumin by isoprenaline hydrochloride.

This study is based on the analysis of the recent trend of medication in neurodegenerative diseases. Due to the asymptomatic nature of the diseases, medication delays. Therefore, mechanism of medication assists in removal of the symptoms. Therefore, in order to find out remedy for complete prevention of the disease we have considered "inhibition verses disaggregation" study. Various biophysical techniques such as turbidity measurement (TM), Thioflavin T (ThT) binding assays, circular dichroism (CD), transmission electron microscopy (TEM) etc. has been performed. Isoprenaline hydrochloride (ISO) was a good candidate for inhibition and disaggregation of preformed fibrils of BSA. Therefore, it is concluded that inhibition of fibrillation process was more momentous, effective procedure in restricting the aggregation by stabilizing the native conformation of BSA than the removal of preformed amyloid fibrils under in vitro condition. Forwarding ahead, to understand the efficiency of the two processes under in vivo condition, this study can be applied on animal models so that we can look forward on human beings as well for the development of vaccines. This study is concerned about the applied aspect of research in future so that we can hope for prevention of the disease instead of only removal of the symptoms.

A multiparametric analysis of the synergistic impact of anti-Parkinson's drugs on the fibrillation of human serum albumin.

Protein aggregation have been associated with several human neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases. There are several small molecules that can reduce aggregation of proteins. The present study aimed to test the hypothesis that the application of more than one inhibitor either simultaneously or consecutively may result in more efficient inhibition of protein aggregation. To this end, the anti-amyloidogenic behaviour of benserazide hydrochloride (BH) and levodopa (LD) individually and in combination (BH + LD) was investigated using various biophysical, microscopic, and computational techniques. BH, LD, and BH + LD treatments showed inhibitory effects on protein aggregation and had the ability to minimise the amyloid-induced cytotoxicity in human neuroblastoma cell line (SH-SY5Y). The two drugs in combination showed synergism (combination index, CI < 1) between them. These drugs also destabilised the preformed fibrils of human serum albumin (HSA). Our studies consistently showed that the BH + LD treatment showed highest efficacy towards inhibition and disaggregation of amyloid fibrils in comparison to treatment with BH and LD individually. Therefore, application of drugs in combination against fibrillogenesis may represent a new route for development of means for prevention or delaying of the aggregation-related diseases.

Deciphering the enhanced inhibitory, disaggregating and cytoprotective potential of promethazine towards amyloid fibrillation.

Increasing evidence proposed that amyloid deposition by proteins play a crucial role in an array of neurotoxic and degenerative disorders like Parkinson's disease, systemic amyloidosis etc, that could be controlled by anti-aggregation methodologies which either inhibit or disaggregate such toxic aggregates. The present work targets the amyloid inhibiting and disaggregating potential of promethazine (PRM) against human insulin (HI) and human lysozyme (HL) fibrillogenesis. Biophysical techniques like Rayleigh scattering measurements (RLS), Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, circular dichroism (CD) and dynamic light scattering (DLS) measurements illustrated the inhibitory action of PRM. The half maximal inhibitory concentration (IC50) of PRM for HI and HL was estimated to be 114.81±1.21µM and 186.20±1.03µM, respectively. Microscopic techniques revealed the absence of fibrillar structures when HI and HL was co-incubated with PRM. Cytoprotective behavior of PRM was investigated by cell based cytotoxicity assay performed on SH-SY5Y neuronal cell lines. The half maximal disaggregation concentration (DC50) was calculated as 21.37±0.89µM and 45.70±0.76µM, signifying that PRM is much potent to disaggregate pre formed fibrils rather than to inhibit fibrillation. Thus, PRM could be beneficial as therapeutic agent that can aid in the cure of amyloid related diseases.

Interaction of catecholamine precursor l-Dopa with lysozyme: A biophysical insight.

The current study comprises of an inclusive biophysical study, enlightening the binding of L-3, 4-dihydroxyphenylalanine (l-Dopa) with human lysozyme (HL) and hen egg white lysozyme (HEWL). Spectroscopic and molecular docking tools have been utilized to study the interaction of l-Dopa with both HL and HEWL. Spectrofluorimetric measurements exhibited that l-Dopa quenched the HL and HEWL intrinsic fluorescence. A binding constant (Kb) of ∼104M-1 for both HL and HEWL was obtained, asserting a significant binding. Negative value of ΔG affirmed that the reaction between proteins and l-Dopa was spontaneous. Far-UV CD spectra revealed a boost to the proteins helical content in the presence of l-Dopa. Furthermore, DLS measurements displayed the decrease in hydrodynamic radii (Rh) of HL and HEWL in the presence of l-Dopa. Molecular docking studies established that l-Dopa formed complexes with both the proteins through hydrogen bonding and hydrophobic interaction. The present study characterizing the l-Dopa interaction with lysozyme could be noteworthy in realizing both pharmaco-dynamics and/or -kinetics of drugs used in various diseases.