Salt Bridge Research Articles

Identification and pharmacological characterization of pH-sensitive chloride channels in the fall armyworm, Spodoptera frugiperda.

The pH-sensitive chloride channels (pHCls) are unique to invertebrates and play crucial roles in fluid regulation, food selection, and intake. In this study, we identified and isolated two cDNAs encoding the SfpHCl1 and SfpHCl2 subunits from the fall armyworm, Spodoptera frugiperda. Both subunits exhibited similar expression patterns. When expressed in Xenopus laevis oocytes, SfpHCl1 and SfpHCl2 formed functional chloride channels with reversal potentials indicative of chloride selectivity. Shifts in extracellular pH from acidic to alkaline conditions induced inward currents in both SfpHCl1 and SfpHCl2, with EC50 values of pH 8.24 and 8.49, respectively. Zinc ions (Zn2⁺) and the insecticide emamectin benzoate (EB) also activated concentration-dependent inward currents in these channels, whether expressed individually or co-expressed. Notably, SfpHCl1 and SfpHCl2 channels exhibited significant differences in their activation and deactivation time constants. Molecular docking simulations indicated that Zn2⁺ binds to both SfpHCl1 and SfpHCl2 through three hydrogen bonds, while EB interacts with these channels via hydrogen bonds and a salt bridge. These findings elucidate the biophysical and pharmacological characteristics of pH-sensitive, zinc-gated chloride channels, which, being exclusive to invertebrates, present a promising target for the development of highly specific insecticides.

Gum arabic-stabilized emulsion systems: Underlying mechanisms for enhancing storage and digestion stability of curcumin.

Although the benefits of gum arabic (GA) in developing emulsion systems are well known, the mechanism underlying its stability-enhancing effect of GA in emulsion systems stabilized with curcumin remains unclear. This study used GA-stabilized emulsion system, containing 10wt% medium-chain triglycerides and 0.1% curcumin. Results demonstrated that when the GA concentration exceeded 0.5wt%, the interface layer reached saturation, maintaining a consistent thickness of approximately 34.8nm. The curcumin-containing emulsion demonstrated good stability during storage at room temperature and heating treatment, effectively protecting the curcumin. However, during in vitro intestinal digestion, the formation of salt and protein bridges caused the aggregation of oil droplets, resulting in an increase in the droplet size to 975.2nm, while the curcumin content remained stable. Additionally, the in vitro release rate of encapsulated curcumin (44.0%) was significantly higher than that of free curcumin (19.5%). In conclusion, this study elucidated the stability-enhancing mechanism of GA-stabilized emulsions for curcumin and highlighted the potential of GA in developing emulsion systems for the encapsulation of these compounds.

Menaquinone-specific turnover by Mycobacterium tuberculosis cytochrome bd is redox regulated by the Q-loop disulfide bond.

Cytochrome bd from Mycobacterium tuberculosis (Mtbd) is a menaquinol oxidase that has gained interest as an antibiotic target because of its importance in survival under infectious conditions. Mtbd contains a characteristic disulfide bond that has been hypothesized to allow for Mtbd activity regulation at the enzymatic level, possibly helping M.tuberculosis to rapidly adapt to the hostile environment of the phagosome. Here, the role of the disulfide bond and quinone specificity have been determined by reconstitution of a minimal respiratory chain and the single-particle cryo-EM structure in the disulfide-reduced form. Mtbd was shown to be specific for menaquinone, while regulation by reduction of the Q-loop disulfide bond decreased oxidase activity up to 90%. Structural analysis shows that a salt bridge unique to Mtbd keeps the Q-loop partially structured in its disulfide-reduced form, which could facilitate the rapid activation of Mtbd upon exposure to reactive oxygen species. We signify Mtbd as the first redox sensory terminal oxidase and propose that this helps M.tuberculosis in the defense against reactive oxygen species encountered during infection.

Thermal properties and non-bonded interactions in human PMP2 variants: A molecular dynamics study

Charcot-Marie-Tooth disease has been known for a long time, affecting population worldwide. Many studies on this disease have been conducted, including clinical as well as computational, but no effective medical cure has yet been found. This disease is caused due to mutation in the human peripheral myelin protein 2 (PMP2) responsible for myelin formation and maintenance. In this research work, we have done a comparative molecular dynamics study on the PMP2 protein and its M114T mutant variant, focusing on non-bonded interactions and thermal properties in a biologically relevant environment. The primary objective was to explore the structural differences between the two proteins at temperatures of 305 K, 310 K, and 315 K. Using the NAMD software for molecular dynamics simulations, various analyses were performed, including RMSD estimation, counting number of hydrogen bond formation, salt bridge occupancy assessment, estimation of vdW and electrostatic interaction energies in the system, and estimation of thermal diffusivity of the proteins as well as specific heat capacity (Cv) of the system. The results suggested slight difference between the structure of the two proteins. In the mutant, a slight increase in RMSD values was observed, suggesting a minor reduction in structural stability compared to the wild-type protein. The number of hydrogen bonds formed was generally higher in the wild-type protein, with slightly more differences observed at 305 K and 315 K than at 310 K. Analysis of salt bridge occupancy revealed notable differences in the formation patterns between the two proteins. The mutation was found to have a greater impact on salt bridge formation. In addition to this, the vdW and electrostatic interaction energies were found to be lower in the system with mutant variant, with both energies decreasing gradually as temperature increased, indicating a more robust interaction profile in the system with wild-type protein. Also, the estimated value of thermal diffusivity indicated that the mutant was slightly more efficient at conducting heat than the wild-type protein; the thermal diffusivity of both proteins was much lower than that of water. Finally, a non-linear (concave nature) change in Cv with temperature was observed in both protein systems. The work was further extended to verify the Maxwell-Boltzmann distribution law which confirmed the proper distribution of kinetic energy among particles, and the temperature fluctuation was found to follow a Gaussian distribution during the NVE simulation, which was as expected.

Cryo-EM SPRstructures of Salmonella typhimurium ArnC; the key enzyme in lipid-A modification conferring polymyxin resistance.

Polymyxins are last-resort antimicrobial peptides administered clinically against multi-drug resistant bacteria, specifically in the case of Gram-negative species. However, an increasing number of these pathogens employ a defense strategy that involves a relay of enzymes encoded by the pmrE (ugd) loci and the arnBCDTEF operon. The pathway modifies the lipid-A component of the outer membrane (OM) lipopolysaccharide (LPS) by adding a 4-amino-4-deoxy-l-arabinose (L-Ara4N) headgroup, which renders polymyxins ineffective. Here, we report the cryo-EM SPR structures of glycosyltransferase ArnC from Salmonella typhimurium determined in apo and UDP-bound forms at resolutions 2.75 Å and 3.8 Å, respectively. The structure of the ArnC protomer comprises three distinct regions: an N-terminal glycosyltransferase domain, transmembrane region, and the interface helices (IHs). ArnC forms a tetramer with C2 symmetry, where the C-terminal strand inserts into the adjacent protomer. This tetrameric state is further stabilized by two distinct interfaces formed by ArnC that form a network of hydrogen bonds and salt bridges. The binding of UDP induces conformational changes that stabilize the loop between residues H201 to S213, and part of the putative catalytic pocket formed by IH1 and IH2. The surface property analysis revealed a hydrophobic cavity formed by TM1 and TM2 in the apo state, which is disrupted upon UDP binding. The comparison of ArnC structures to their homologs GtrB and DPMS suggests the key residues involved in ArnC catalytic activity.

Scaling analysis of the swirling wake of a porous disc: application to wind turbines

We report a comprehensive study of the wake of a porous disc, the design of which has been modified to incorporate a swirling motion at an inexpensive cost. The swirl intensity is passively controlled by varying the internal disc geometry, i.e. the pitch angle of the blades. A swirl number is introduced to characterise the competition between the linear (drag) and the azimuthal (swirl) momenta on the wake recovery. Assuming that swirl dominates the near wake and non-equilibrium turbulence theory applies, new scaling laws of the mean wake properties are derived. To assess these theoretical predictions, an in-depth analysis of the aerodynamics of these original porous discs has been conducted experimentally. It is found that, at the early stage of wake recovery, the swirling motion induces a low-pressure core, which controls the mean velocity deficit properties and the onset of self-similarity. The measurements collected in the swirling wake of the porous discs support the new scaling laws proposed in this work. Finally, it is shown that, as far as swirl is injected in the wake, the characteristics of the mean velocity deficit profiles match very well those of both laboratory-scale and real-scale wind-turbine data extracted from the literature. Overall, our results emphasise that, by setting the initial conditions of the wake recovery, swirl is a key ingredient to be taken into account in order to faithfully replicate the mean wake of wind turbines.

Molecular Docking Studies and In Vitro Activity of Pancreatic Lipase Inhibitors from Yak Milk Cheese.

Pancreatic lipase serves as a primary trigger for hyperlipidemia and is also a crucial target in the inhibition of hypercholesterolemia. By synthesizing anti-hypercholesterolemic drugs such as atorvastatin, which are used to treat hypercholesterolemia, there were some side effects associated with the long-term use of statins. Based on this idea, in the present study, we identified peptides that inhibited PL by virtual screening and in vitro activity assays. In addition, to delve into the underlying mechanisms, we undertook a dual investigative approach involving both molecular docking analyses and molecular dynamics simulations. The results showed that peptides RK7, KQ7, and TL9, all with molecular weights of <1000 Da and a high proportion of hydrophobic amino acids, inhibited PL well. Molecular docking and molecular dynamics showed that peptides RK7, KQ7, and TL9 bound to important amino acid residues of PL, such as Pro and Leu, through hydrogen bonding, hydrophobic interactions, salt bridges, and π-π stacking to occupy the substrate-binding site, which inhibited PL and identified them as potential PL inhibitors. In vitro tests showed that the IC50 of RK7 and KQ7 on PL were 0.690 mg/mL and 0.593 mg/mL, respectively, and the inhibitory effects of RK7 and KQ7 on PL were significantly enhanced after simulated gastrointestinal digestion. Our results suggested that peptides RK7 and KQ7 from yak milk cheese can be identified as a novel class of potential PL inhibitors.

Charge Reversal of the Uppermost Arginine in Sliding Helix S4-I Affects Gating of Cardiac Sodium Channel.

Several mutations of the uppermost arginine, R219, in the voltage-sensing sliding helix S4I of cardiac sodium channel Nav1.5 are reported in the ClinVar databases, but the clinical significance of the respective variants is unknown (VUSs). AlphaFold 3 models predicted a significant downshift of S4I in the R219C VUS. Analogous downshift S4I, upon its in silico deactivation, resulted in a salt bridge between R219 and the uppermost glutamate, E161, in helix S2I. To understand how salt bridge elimination affects biophysical characteristics, we generated mutant channel R219E, expressed it in the HEK293-T cells, and employed the patch-clamp method in a whole-cell configuration. Mutation R219E did not change the peak current density but shortened time to the peak current at several potentials, significantly enhanced activation, enhanced steady-state inactivation and steady-state fast inactivation, and slowed recovery from inactivation. Taken together, these data suggest that mutation R219E destabilized the resting state of Nav1.5. Cardiac syndromes associated with mutations R219P/H/C/P or E161Q/K are consistent with the observed changes of biophysical characteristics of mutant channel R219E suggesting pathogenicity of the respective VUSs, as well as ClinVar-reported VUSs involving arginine or glutamate in homologous positions of several Nav1.5 paralogs.

Unbinding of Alpha Chain of Hemoglobin in Sickle and Normal Structures

Abstract Sickle cell disease, a genetic disorder, is caused by a mutation of glutamic acid into valine in the β chain of hemoglobin at the sixth residue, resulting in structural change of the entire hemoglobin molecule into a sickle shape. We investigated the atomic level interaction between the α chain (chain A) and the remaining three chains to identify the structural modification in sickle hemoglobin using the molecular dynamics simulations. H-bond, solvent accessible surface area (SASA), hydrophobic interactions, salt bridges of sickle and normal hemoglobin have been estimated. The estimated parameters from sickle hemoglobin are compared to normal hemoglobin structure. Steered molecular dynamics (SMD) is utilized to estimate the force required in breaking hydrogen bonds in given chains. The SMD simulations at different pulling velocities show that the decoupling forces depend on values of pulling force. This relation is linear, 6780 pN to 12345 pN with pulling velocities of 0.00020 nm/ps to 0.00040 nm/ps in sickle hemoglobin. Much higher force of 8738 pN to 16557 pN in normal is required in normal hemoglobin with same spring constants values from k = 500 to 1100 kcal mol-1 nm-2 and same pulling velocities.

Dissecting the Binding Affinity of Anti-SARS-CoV-2 Compounds to Human Transmembrane Protease, Serine 2: A Computational Study.

The human transmembrane protease, serine 2 (TMPRSS2), essential for SARS-CoV-2 entry, is a key antiviral target. Here, we computationally profiled the TMPRSS2-binding affinities of 15 antiviral compounds. Molecular dynamics (MD) simulations for the docked complexes revealed that three compounds exited the substrate-binding cavity (SBC), suggesting noncompetitive inhibition. Of the remaining compounds, five charged ones exhibited reduced binding stability due to competing electrostatic interactions and increased solvent exposure, while seven neutral compounds showed stronger binding affinity driven by van der Waals (vdW) interactions compensating for unfavorable electrostatic effects (including electrostatic interactions and desolvation penalties). Positive and negative hotspot residues were identified as uncharged and charged, respectively, both lining the SBC. Despite forming diverse interactions with compounds, the burial of positive hotspots led to strong vdW interactions that overcompensated for unfavorable electrostatic effects, whereas negative hotspots incurred high desolvation penalties, negating any favorable contributions. Charged residues at the SBC's outer rim can reduce binding affinity significantly when forming hydrogen bonds or salt bridges. These findings underscore the importance of enhancing vdW interactions with uncharged residues and minimizing the unfavorable electrostatic effects of charged residues, providing valuable insights for designing effective TMPRSS2 inhibitors.

Design, synthesis and cytotoxic activity of novel lipophilic cationic derivatives of diosgenin and sarsasapogenin.

Novel lipophilic cationic derivatives including quaternary ammonium salt and triphenylphosphine series were designed and synthesized using diosgenin (1) and sarsasapogenin (2) as substrates to improve the cytotoxicity and selectivity. Most of the derivatives showed higher cytotoxicity against all cancer cell lines tested, compound 13 exhibited the most superior activity against A549 cells with an IC50 value of 0.95μM, which was 34-fold of diosgenin. Preliminary cellular mechanism studies elucidated that compound 13 might arrest cell cycle at G0/G1 phase, trigger apoptosis via up-regulating the expression of Bax, down-regulating the expression of Bcl-2 and caspase-3, and induce an increase in the generation of intracellular reactive oxygen species (ROS) in A549 cells. In addition, molecular docking analysis revealed that compound 13 could occupy the active site of p38α-MAPK well and interact to the surrounding amino acids by salt bridge and conjugation. These results suggested that compound 13 had the potential to serve as an antitumor lead agent, probably exert antitumor effect through mitochondrial pathway and p38α MAPK pathway.

Studies on inhibition of α-glucosidase using debittered formulation of Bacopa monnieri juice: Enzyme inhibition kinetics, interaction strategy, and molecular docking approach

Bacopa monnieri juice (BMJ) is traditionally used, reported, and scientifically validated for memory enhancement. However, its efficacy against diabetes is less explored. The extreme bitterness of BMJ restricts its commercial applications. This study investigates the reduction of bitterness of BMJ followed by evaluation for its α-glucosidase inhibitory activity. Initially, debittering of 30 % (v/v) BMJ using ZnSO4 (15 mM) was optimized by time-intensity analysis and molecular docking of ZnSO4 as well as bacoside A3, the main active compound in BMJ, with TAS2R14 taste receptor. The study indicated 5 hydrogen bonds to be involved in binding with bacoside A3 with binding energy of −11.82 Kcal/mol, while hydrogen bond, salt bridges and metal complexes were involved in binding of ZnSO4 with binding energy of −6.65 Kcal/mol. Subsequently, BMJ, ZnSO4 and BMJ + ZnSO4 (debittered juice) were also found to be potent inhibitors of α-glucosidase in dose-dependent manner. These inhibitors showed parabolic mixed inhibition of α-glucosidase, altered the secondary structure, and quenching of fluorescence. In silico studies revealed hydrogen bonding and hydrophobic interactions between inhibitors and α-glucosidase with lowest binding energy of −15.53 and −7.54 Kcal/mol being recorded for bacoside A3 and ZnSO4, respectively. Molecular docking of other bioactive compounds in BMJ such as apigenin, luteolin, quercetin and bacopasaponin C also showed lower binding energy than the standard drug, acarbose (−5.84). This study inferred the binding of bacoside A3 at the active site of α-glucosidase and of ZnSO4 with other sites on the protein. The study proposes a debittered BMJ formulation to control hyperglycemia.

Thermal performance of DispersedInorganic magnetic hybrid nanomaterials into mixed convective flow through flexible porous disks

AbstractThis study applies advanced AI techniques, including machine learning algorithms, to explore the numerically unsteady laminar flow of viscous and incompressible fluids in coaxially swirled porous disks, with applications in engineering sciences. Our focus encompasses the effects of magnetic hybrid nanomaterials and the dynamic behaviors associated with expanding/contracting and injection/suction. Utilizing single‐phase simulations, we address nonlinear coupled ordinary differential equations set against appropriate boundary conditions. Key parameters of our study include permeability and relaxation, as well as the influence of chemical reactions and mixed convection on fluid behaviors. The thermophysical properties of Al2O3/Cu nanoparticles have been by varying their morphological aspects. For the hybrid nanofluids flow, the aggregation of nanoparticle volume fraction has been designed critically in conjunction with an energy and mass transfer equation. Because dimensionless ordinary differential equations are employed, the obtained expression is transmuted using the obliging transformation technique. The desired nonlinear system of ODEs is implemented using an accurate numerical method. Our findings reveal significant impacts of chemical reaction parameters on the Sherwood number and a marked increase in the skin friction coefficient, Nusselt number, and Sherwood number as nanoparticle volume fraction rises from 2% to 7%.

The structural organisation of pentraxin-3 and its interactions with heavy chains of inter-α-inhibitor regulate crosslinking of the hyaluronan matrix.

Pentraxin-3 (PTX3) is an octameric protein, comprised of eight identical protomers, that has diverse functions in reproductive biology, innate immunity and cancer. PTX3 interacts with the large polysaccharide hyaluronan (HA) to which heavy chains (HCs) of the inter-α-inhibitor (IαI) family of proteoglycans are covalently attached, playing a key role in the (non-covalent) crosslinking of HC•HA complexes. These interactions stabilise the cumulus matrix, essential for ovulation and fertilisation in mammals, and are also implicated in the formation of pathogenic matrices in the context of viral lung infections. To better understand the physiological and pathological roles of PTX3 we have analysed how its quaternary structure underpins HA crosslinking via its interactions with HCs. A combination of X-ray crystallography, cryo-electron microscopy (cryo-EM) and AlphaFold predictive modelling revealed that the C-terminal pentraxin domains of the PTX3 octamer are arranged in a central cube, with two long extensions on either side, each formed from four protomers assembled into tetrameric coiled-coil regions, essentially as described by (Noone et al., 2022; doi:10.1073/pnas.2208144119). From crystallography and cryo-EM data, we identified a network of inter-protomer salt bridges that facilitate the assembly of the octamer. Small angle X-ray scattering (SAXS) validated our model for the octameric protein, including the analysis of two PTX3 constructs: a tetrameric 'Half-PTX3' and a construct missing the 24 N-terminal residues (Δ1-24-PTX3). SAXS determined a length of ∼520 Å for PTX3 and, combined with 3D variability analysis of cryo-EM data, defined the flexibility of the N-terminal extensions. Biophysical analyses revealed that the prototypical heavy chain HC1 does not interact with PTX3 at pH 7.4, consistent with our previous studies showing that, at this pH, PTX3 only associates with HC•HA complexes if they are formed in its presence. However, PTX3 binds to HC1 at acidic pH, and can also be incorporated into pre-formed HC•HA complexes under these conditions. This provides a novel mechanism for the regulation of PTX3-mediated HA crosslinking (e.g., during inflammation), likely mediated by a pH-dependent conformational change in HC1. The PTX3 octamer was found to associate simultaneously with up to eight HC1 molecules and, thus, has the potential to form a major crosslinking node within HC•HA matrices, i.e., where the physical and biochemical properties of resulting matrices could be tuned by the HC/PTX3 composition.

Influence of Viscosity and Thermal Conductivity in Boger Nanofluid Flow through Porous Disk: Finite Difference Analysis

Influence of Viscosity and Thermal Conductivity in Boger Nanofluid Flow through Porous Disk: Finite Difference Analysis

Effect of a Steaming Treatment on the Alpha-Glucosidase Inhibitory Components in the Brown Alga Sargassum fusiforme.

The brown alga Sargassum fusiforme (SF) is historically consumed as a food material in Japan. A steaming process is often required for SF products on the market due to their moderate hardness and astringent taste. This investigation aimed to elucidate the effect of steaming on the anti-diabetic activity of SF and its related chemical components. Acetone extracts of SF were prepared after it were steamed for 0, 1, 2, or 4 h (SF-0h, SF-1h, SF-3h, and SF-4h, respectively). Alpha-glucosidase inhibitory profiles of each SF extract were made based on activity-guided separation. The active fractions were collected and NMR was applied for a further chemical composition analysis. Our results suggested that total polyphenol levels decreased drastically after steaming, which resulted in a drop in α-glucosidase inhibitory activity. The fatty acid, pheophytin a, and pyropheophytin a contents were elevated significantly after steaming, which contributed to the majority of the activity of steamed SF (SF-1h). However, prolonging the steaming time did not significantly affect the activity of SF further since the content of free fatty acids in steamed SF (SF-2h and SF-4h) almost did not change with a longer time of steaming. Moreover, palmitic acid, 8-octadecenoic acid, and tetradecanoic acid were identified as the top three important fatty acids for the inhibition of α-glucosidase by steamed SF. Further molecular docking results revealed that these fatty acids could interact with residues of α-glucosidase via hydrogen bonds, salt bridges, and hydrophobic interactions. In conclusion, steaming altered the α-glucosidase inhibitory properties of SF by changing the contents of polyphenols, fatty acids, and chlorophyll derivatives.

Functional Roles of the Charged Residues of the C- and M-Gates in the Yeast Mitochondrial NAD+ Transporter Ndt1p.

Mitochondrial carriers transport organic acids, amino acids, nucleotides and cofactors across the mitochondrial inner membrane. These transporters consist of a three-fold symmetric bundle of six transmembrane α-helices that encircle a pore with a central substrate binding site, whose alternating access is controlled by a cytoplasmic and a matrix gate (C- and M-gates). The C- and M-gates close by forming two different salt-bridge networks involving the conserved motifs [YF][DE]XX[KR] on the even-numbered and PX[DE]XX[KR] on the odd-numbered transmembrane α-helices, respectively. We have investigated the effects on transport of mutating the C-gate charged residues of the yeast NAD+ transporter Ndt1p and performed molecular docking with NAD+ and other substrates into structural models of Ndt1p. Double-cysteine substitutions and swapping the positions of the C-gate charged-pair residues showed that all of them contribute to the high transport rate of wild-type Ndt1p, although no single salt bridge is essential for activity. The in silico docking results strongly suggest that both the C-gate motif mutations and our previously reported M-gate mutations affect gate closing, whereas those of the M-gate also affect substrate binding, which is further supported by molecular dynamics. In particular, NAD+ most likely interferes with the cation-π interaction between R303-W198, which has been proposed to exist in the Ndt1p M-gate in the place of one of the salt bridges. These findings contribute to understanding the roles of the charged C- and M-gate residues in the transport mechanism of Ndt1p.

New Results of Differential Subordination for a Specific Subclass of p-Valent Meromorphic Functions Involving a New Operator

The present article aims to significantly improve geometric function theory by making an important contribution to p-valent meromorphic and analytic functions. It focuses on subordination, which describes the relationships of analytic functions. In order to achieve this, we utilize a technique that is based on the properties of differential subordination. This approach, which is one of the most recent developments in this field, may obtain a number of conclusions about differential subordination for p-valent meromorphic functions described by the new operator IHp,q,s j,pν1,n,α,lJ(ζ) within the porous unit disk Δ. Numerous mathematical and practical issues involving orthogonal polynomials, such as system identification, signal processing, fluid dynamics, antenna technology, and approximation theory, can benefit from the results presented in this article. The knowledge and comprehension of the unit’s analytical functions and its interacting higher relations are also greatly expanded by this text.

Novel Tripeptides as Tyrosinase Inhibitors: In Silico and In Vitro Approaches.

Tyrosinase is a key enzyme responsible for the formation of melanin (a natural skin pigment with ultraviolet-protection properties). However, some people experience melanin overproduction, so new, safe, and biocompatible enzyme inhibitors are sought. New tripeptide tyrosinase inhibitors were developed using molecular modeling. A combinatorial library of tripeptides was prepared and docked to the mushroom tyrosinase crystal structure and investigated with molecular dynamics. Based on the results of calculations and expert knowledge, the three potentially most active peptides (CSF, CSN, CVL) were selected. Their in vitro properties were examined, and they achieved half-maximal inhibitory concentration (IC50) values of 136.04, 177.74, and 261.79 µM, respectively. These compounds attach to the binding pocket of tyrosinase mainly through hydrogen bonds and salt bridges. Molecular dynamics simulations demonstrated the stability of the peptid-tyrosinase complexes and highlighted the persistence of key interactions throughout the simulation period. The ability of these peptides to complex copper ions was also confirmed. The CSF peptide showed the highest chelating activity with copper. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay confirmed that none of the test tripeptides showed cytotoxicity toward the reconstructed human epidermis. Our results indicated that the developed tripeptides were non-toxic and effective tyrosinase inhibitors. They could be applied as raw materials in skin-brightening or anti-aging cosmetic products.

Exploring Binding Sites in Chagas Disease Protein TcP21 Using Integrated Mixed Solvent Molecular Dynamics Approaches.

Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a significant global health burden, particularly in Latin America, where millions are at risk. This disease predominantly affects socioeconomically vulnerable populations, aggravating economic inequality, marginalization, and low political visibility. Despite extensive research, effective treatments are still lacking, partly due to the complex biology of the parasite and its infection mechanisms. This study focuses on TcP21, a novel 21 kDa protein secreted by extracellular amastigotes, which has been implicated in T. cruzi infection via an alternative infective pathway. Although the potential of TcP21 for understanding Chagas disease is promising, further exploration is necessary, particularly in identifying potential binding sites on its surface. Computational tools offer a versatile and effective strategy for preliminary binding site assessment, facilitating a more cost-efficient allocation of experimental resources. In this study, we employed three independent computational approaches─mixed solvent molecular dynamics simulations (MSMD), fragment-based molecular docking, and pharmacophore model docking coupled with molecular dynamics simulations─to identify potential binding sites and provide comprehensive insights into TcP21. The three methodologies converged on a common site located on the external surface of the protein, characterized by key residues such as GLU55, ASP52, VAL70, ILE62, and TRP77. The protonated amino, acetamido, and phenyl groups of the pharmacophore probe were consistently observed to interact with the site via a network of salt bridges, hydrogen bonds, charge-charge interactions, and alkyl-π interactions, suggesting these groups play a significant role in ligand binding. This study does not aim to propose specific therapeutic hits but to highlight a still unknown and unexplored protein involved in T. cruzi cell invasion. In this regard, given the strong correlation between the three distinct approaches used for mapping, we consider this study offers valuable insights for further research into P21 and its role in Chagas disease.