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Our studies show Coomassie Brilliant Blue G-250 as a promising chemical chaperone that stabilises the α-helical native human insulin conformers, disrupting its aggregation. Furthermore, it also increases the insulin secretion....

Dengue virus (DENV) encodes a unique protease (NS3/NS2B) essential for its maturation and infectivity and, it has become a key target for anti-viral drug design to treat dengue and other flavivirus related infections. Present investigation established that some of the drug molecules currently used mainly in cancer treatment are susceptible to bind non-active site (allosteric site/ cavity) of the NS3 protease enzyme of dengue virus. Computational screening and molecular docking analysis found that dabrafenib, idelalisib and nintedanib can bind at the allosteric site of the enzyme. The binding of the molecules to the allosteric site found to be stabilized via pi-cation and hydrophobic interactions, hydrogen-bond formation and π-stacking interaction with the molecules. Several interacting residues of the enzyme were common in all the five serotypes. However, the interaction/stabilizing forces were not uniformly distributed; the π-stacking was dominated with DENV3 proteases, whereas, a charged/ionic interaction was the major force behind interaction with DENV2 type proteases. In the allosteric cavity of protease from DENV1, the residues Lys73, Lys74, Thr118, Glu120, Val123, Asn152 and Ala164 were involved in active interaction with the three molecules (dabrafenib, idelalisib and nintedanib). Molecular dynamics (MD) analysis further revealed that the molecules on binding to NS3 protease caused significant changes in structural fluctuation and gained enhanced stability. Most importantly, the binding of the molecules effectively perturbed the protein conformation. These changes in the protein conformation and dynamics could generate allosteric modulation and thus may attenuate/alter the NS3 protease functionality and mobility at the active site. Experimental studies may strengthen the notion whether the binding reduce/enhance the catalytic activity of the enzyme, however, it is beyond the scope of this study.

Aggregation of intrinsically disordered as well as the ordered proteins under certain premises or physiological conditions leads to pathological disorder. Here we have presented a detailed investigation on the effect of a porous metallic (Au) and a non-metallic (Si) nanomaterial on the formation of ordered (fiber-like/amyloid) and disordered (amorphous) aggregates of proteins. Porous nanogold (PNG) was found to reduce the amyloid aggregation of insulin but does not have much impact on the lag phase in the aggregation kinetics, whereas porous nano-silica (PNS) was found both to decrease the amount of aggregation as well as prolong the lag phase of amyloid fiber formation from insulin. On the other hand, both the porous nanoparticles are found to decrease the extent of amorphous aggregation (with slight improvement for PNS) of pathogenic huntingtin (Htt) protein in Huntington's disease cell model. This is a noted direct observation in controlling and understanding protein aggregation diseases which may help us to formulate nanotherapeutic drugs for future clinical applications.

Parkinson’s disease (PD) is linked to α-synuclein (aS) aggregation and deposition of amyloid in the substantia nigra region of the brain tissues. Recent reports suggested that oligomeric assembly structure could be neurotoxic to neuronal cells. In the current investigation we produced two distinct classes of aS oligomers and link the protein conformation state and stability to neuronal cell toxicity. Natural oligomers (NO) enriched with alpha-helical folds are produced in storage of aS at below −20°C for 7 days. Induced oligomer (IO), often observed in the aggregation pathway of aS were made incubating the protein solution at 37°C. Natural oligomers remained stable and did not transform into β-sheet rich amyloid fiber and exhibited higher toxicity (80% cell death) compared to induced oligomers. Natural oligomers were ovular shape and the size ranged between 4-5.5 nm. It maintained significant number (∼ 60%) of residues in α-helical conformational space. However, initiation of hydrophobic zipping with beta sheet conformation was evidenced in induced oligomer (IO) and a lesser number residues (45%) remained with preference to α-helical secondary structure. Hydrophobic collapse leads the transformation of IO into thermodynamically most stable β-sheet rich amyloid fibril. Molten globule like secondary structure stabilized by H-bonding in natural oligomers caused enhanced stability and cellular toxicity compared to induced oligomer. Thus off-pathway/natural oligomers could be plausible reason of neuronal cell death and possible cause of Parkinson’s disease.

Human insulin (HI) is an essential protein hormone and its biological activity mostly depends on folded and active conformation in the monomeric state. The present investigation established that Coomassie Brilliant Blue G-250 (CBBG), a small multicyclic hydroxyl compound can reversibly bind to the hormonal protein dimer and maintained most of α-helical folds crucial for biological function of the enzyme. The solution-state 1D NMR and isothermal calorimetric analysis showed a sub-micromolar binding affinity of the molecule to HI. 2D NOESY NMR established that the HI dimer undergoes residue level local conformational change upon binding to CBBG. The chemical shift perturbation and the NOE parameters of active protons of amino acid residues throughout the polypeptides further suggested that CBBG upon binding the protein stabilize α-helixes of both the A and B subunits of the hormonal protein. The changes in Gibb’s free energy (∆G) of the binding was of ~−11.1 kcal/mol and suggested a thermodynamically favourable process. The changes in enthalpy (∆H) and entropy term (T∆S) were −57.2 kcal/mol and −46.1 kcal/mol, respectively. The negative changes in entropy and the NOE transfer effectiveness of several residues in the presence of CBBG molecules indicated that the binding was an enthalpy driven favourable equilibrium process. The NMR-based atomic resolution data and molecular docking studies confirmed that the CBBG binds to HI at the dimeric stage and prevents the availability of the crucial residue segments that partake directly in further oligomerization and subsequent fibrillation. Extended computational analysis based on chemical shift perturbation of protons of active residues further established receptor-ligand based pharmacophore model comprised of 5 hydrophobic and a hydrogen bond acceptor features that can anchor the residues at the A and B chains of HI and inhibit the partial unfolding and hydrophobic collapse to nucleate the fibrillation. Taken together, the results demonstrated that CBBG and their close analogues might be useful to develop a formulation that will maintain the active and functional form of the hormonal protein for a significantly longer time.TOC

In Alzheimer’s disease (AD), amyloid β (Aβ) protein plays a detrimental role in neuronal injury and death. Recent in vitro and in vivo studies suggest that soluble oligomers of the Aβ peptide are neurotoxic. Structural properties of the oligomeric assembly, however, are largely unknown. Our present investigation established that the 40-residue-long Aβ peptide (Aβ40) became more helical, ordered, and compact in the oligomeric state, and both the helical and β-sheet components were found to increase significantly in the early event of oligomerization. The band-selective two-dimensional NMR analysis suggested that majority of the residues from sequence 12 to 22 gained a higher-ordered secondary structure in the oligomeric condition. The presence of a significant amount of helical conformation was confirmed by Raman bands at 1650 and 1336 cm–1. Other residues remained mostly in the extended polyproline II (PPII) and less compact β-conformation space. In the event of maturation of the oligomers into an amyloid fiber, both the helical content and the PPII-like structural components declined and ∼72% residues attained a compact β-sheet structure. Interestingly, however, some residues remained in the collagen triple helix/extended 2.51-helix conformation as evidenced by the amide III Raman signature band at 1272 cm–1. Molecular dynamics analysis using an optimized potential for liquid simulation force field with the peptide monomer indicated that some of the residues may have preferences for helical conformation and this possibly contributed in the event of oligomer formation, which eventually became a β-sheet-rich amyloid fiber.

Lysozyme, like many other well-folded globular proteins, under stressful conditions produces nanoscale oligomer assembly and amyloid-like fibrillar aggregates. With engaging Raman microscopy, we made a critical structural analysis of oligomer and other assembly structures of lysozyme obtained from hen egg white and provided a quantitative estimation of a protein secondary structure in different states of its fibrillation. A strong amide I Raman band at 1660 cm-1 and a N-Cα-C stretching band at ∼930 cm-1 clearly indicated the presence of a substantial amount of α-helical folds of the protein in its oligomeric assembly state. In addition, analysis of the amide III region and Raman difference spectra suggested an ample presence of a PPII-like secondary structure in these oligomers without causing major loss of α-helical folds, which is found in the case of monomeric samples. Circular dichroism study also revealed the presence of typical α-helical folds in the oligomeric state. Nonetheless, most of the Raman bands associated with aromatic residues and disulfide (-S-S-) linkages broadened in the oligomeric state and indicated a collapse in the tertiary structure. In the fibrillar state of assembly, the amide I band became much sharper and enriched with the β-sheet secondary structure. Also, the disulfide bond vibration in matured fibrils became much weaker compared to monomer and oligomers and thus confirmed certain loss/cleavage of this bond during fibrillation. The Raman band of tryptophan and tyrosine residues indicated that some of these residues experienced a greater hydrophobic microenvironment in the fibrillar state than the protein in the oligomeric state of the assembly structure.

Triple negative breast cancer (TNBC) is one of the most common invasive malignancies among women, associated with poor prognosis. Standard chemotherapy targets all dividing cells, resulting in dose-limiting toxicities. In this study, we demonstrated a strategy of encapsulating a hydrophobic synthetic compound, nifetepimine, having anticancer properties, in poly (lactic-co-glycolic acid) nanoparticles to increase selectivity of drug to cancerous cells with minimum toxicity towards normal cells. Nanoencapsulated nifetepimine (30-100nm) having loading and encapsulation efficiency of 7.45% and 75% respectively, was successfully internalized inside TNBC cells upon sustained release resulting in apoptosis. An in vivo bio-distribution study indicated that nanonifetepimine selectively accumulated into breast tumor sites of mice, primarily due to prolonged blood circulation time and binding of nifetepimine to epidermal growth factor receptor that remains overexpressed in most of the TNBC tumors. Moreover, we observed significant reduction in breast tumor volume with improved survival implying high tumor targetability of nanonifetepimine.

The human proteome contains a significant number of intrinsically disordered proteins (IDPs). They show unusual structural features that enable them to participate in diverse cellular functions and play significant roles in cell signaling and reorganization processes. In addition, the actions of IDPs, their functional cooperativity, conformational alterations and folding often accompany binding to a target macromolecule. Applying bioinformatics approaches and with the aid of statistical methodologies, we investigated the statistical parameters of binding regions (BRs) found in disordered human proteome. In this report, we detailed the bioinformatics analysis of binding regions found in the IDPs. Statistical models for the occurrence of BRs, their length distribution and percent occupancy in the parent proteins are shown. The frequency of BRs followed a Poisson distribution pattern with increasing expectancy with the degree of disorderedness. The length of the individual BRs also followed Poisson distribution with a mean of 6 residues, whereas, percentage of residues in BR showed a normal distribution pattern. We also explored the physicochemical properties such as the grand average of hydropathy (GRAVY) and the theoretical isoelectric points (pIs). The theoretical pIs of the BRs followed a bimodal distribution as in the parent proteins. However, the mean acidic/basic pIs were significantly lower/higher than that of the proteins, respectively. We further showed that the amino acid composition of BRs was enriched in hydrophobic residues such as Ala, Val, Ile, Leu and Phe compared to the average sequence content of the proteins. Sequences in a BR showed conformational adaptability mostly towards flexible coil structure and followed by helix, however, the ordered secondary structural conformation was significantly lower in BRs than the proteins. Combining and comparing these statistical information of BRs with other methods may be useful for high-throughput functional annotation of proteins, drug target identification and drug discovery linking protein disorder.

Induction of apoptosis is the target of choice for modern chemotherapeutic treatment of cancer, where lack of potent "target-specific" drugs has led to extensive research on anticancer compounds from natural sources. In our study, we have used astrakurkurone, a triterpene isolated from wild edible mushroom, Astraeus hygrometricus. We have discussed the structure and stability of astrakurkurone employing single-crystal X-ray crystallography and studied its potential apoptogenicity in hepatocellular carcinoma (HCC) cells. Our experiments reveal that it is cytotoxic against the HCC cell lines (Hep 3B and Hep G2) at significantly low doses. Further investigations indicated that astrakurkurone acts by inducing apoptosis in the cells, disrupting mitochondrial membrane potential and inducing the expression of Bcl-2 family proteins, for example, Bax, and the downstream effector caspases 3 and 9. A molecular docking study also predicted direct interactions of the drug with antiapoptotic proteins Bcl-2 and Bcl-xL. Thus, astrakurkurone could become a valuable addition to the conventional repertoire of future anticancer drugs. © 2019 IUBMB Life, 1-11, 2019.

An analog of coomassie brilliant blue-R (CBB-R) was recently found to act as an antagonist to ATP-sensitive purinergic receptors (P2X7R) and has potential to be used in medicine. With the aim of understanding its transportation and distribution through blood, in this investigation, we measured the binding parameters of CBB-R with bovine hemoglobin (BHG). The molecule specifically bound to a single binding site of the protein with a stoichiometric ratio of 1 : 1 and the observed binding constant Ka was 3.5, 2.5, 2.0 and 1.5 × 10(5) M(-1) at 20 °C, 27 °C, 37 °C and 45 °C, respectively. The measured respective ΔG(0) values of the binding at four temperatures were -30.45, -22.44, -18.04 and -11.95 kJ mol(-1). The ΔH(0) (change in enthalpy) and ΔS(0) (change in entropy) values were -23.6 kJ mol(-1) and -70.66 J mol(-1) respectively in the binding process. The negative value of ΔH(0) and ΔS(0) indicated that the binding of the molecule was thermodynamically favorable. The best energy structure in the molecular docking analysis revealed that CBB-R preferred to be intercalated in the cavity among the α2, β1 and β2 subunits and the binding location was 7.4 Å away from Trp37 in the β2 subunit. The binding of the molecule with the protein was stabilized by hydrogen bonds involving the side chain of two amino acid residues. The residues were Lys104 and Glu101 in the β2 subunit. The binding was further stabilized via hydrogen bond formation between the amide group of the peptide backbone (residue Tyr145 of the β1 subunit) and CBB-R. A shift of the amide I (-C=O stretching) band frequency of ∼8 cm(-1) to low energy was ascribed to the hydrogen bond interaction involving the polypeptide carbonyl of the protein and the CBB-R molecule. In addition, two π-cation interactions between Lys99 of the α2 subunit and Lys104 of the β2 subunit and CBB-R contributed favorably in the binding processes. No substantial change in the soret and Q absorption bands of BHG could be observed in the presence of CBB-R. It indicated that the oxygen binding domain or the heme proximity was not blocked or substantially perturbed due to the binding of CBB-R. The circular dichroism and the molecular dynamics analysis further established that the binding interaction caused no significant alteration in the protein long range secondary structure.