Arginine reprograms metabolism in liver cancer via RBM39.

Metabolic reprogramming is a hallmark of cancer. However, mechanisms underlying metabolic reprogramming and how altered metabolism in turn enhances tumorigenicity are poorly understood. Here, we report that arginine levels are elevated in murine and patient hepatocellular carcinoma (HCC), despite reduced expression of arginine synthesis genes. Tumor cells accumulate high levels of arginine due to increased uptake and reduced arginine-to-polyamine conversion. Importantly, the high levels of arginine promote tumor formation via further metabolic reprogramming, including changes in glucose, amino acid, nucleotide, and fatty acid metabolism. Mechanistically, arginine binds RNA-binding motif protein 39 (RBM39) to control expression of metabolic genes. RBM39-mediated upregulation of asparagine synthesis leads to enhanced arginine uptake, creating a positive feedback loop to sustain high arginine levels and oncogenic metabolism. Thus, arginine is a second messenger-like molecule that reprograms metabolism to promote tumor growth.

Down the membrane hole: Ion channels in protozoan parasites.

Parasitic diseases caused by protozoans are highly prevalent around the world, disproportionally affecting developing countries, where coinfection with other microorganisms is common. Control and treatment of parasitic infections are constrained by the lack of specific and effective drugs, plus the rapid emergence of resistance. Ion channels are main drug targets for numerous diseases, but their potential against protozoan parasites is still untapped. Ion channels are membrane proteins expressed in all types of cells, allowing for the flow of ions between compartments, and regulating cellular functions such as membrane potential, excitability, volume, signaling, and death. Channels and transporters reside at the interface between parasites and their hosts, controlling nutrient uptake, viability, replication, and infectivity. To understand how ion channels control protozoan parasites fate and to evaluate their suitability for therapeutics, we must deepen our knowledge of their structure, function, and modulation. However, methodological approaches commonly used in mammalian cells have proven difficult to apply in protozoans. This review focuses on ion channels described in protozoan parasites of clinical relevance, mainly apicomplexans and trypanosomatids, highlighting proteins for which molecular and functional evidence has been correlated with their physiological functions.

Metabolomics approach for phenolic compounds profiling of soursop (Annona muricata L.) fruit during postharvest storage.

INTRODUCTION: Soursop (Annona muricata L.) is a crop with medicinal properties and numerous bioactive compounds. Ripening is a complex process that regulates fruit quality and changes in metabolite content, such as flavonoids, polyphenols, and organic acids. OBJECTIVES: This study aimed to analyze the phenolic profiling of soursop fruit ripening. METHODS: The metabolic changes in different days of storage of soursop fruits were investigated using a semi-metabolomic approach based on ultra-performance liquid chromatography coupled to electrospray ionization quadrupole-time of flight mass spectrometry (UPLC-ESI-QTOF-MS). Further, multivariate analysis such as supervised partial least squares discriminant analysis (PLS-DA) was conducted to identify differential metabolites. RESULTS: A total of 68 metabolites were identified in soursop fruit during postharvest storage. A higher number of metabolites were identified in the Day zero (D0) compared to the Day one (D1), Day three (D3), and Day five (D5), belonging to flavonoids, other polyphenols, phenolic acids, and organic acids. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the pathways of flavone and flavonol biosynthesis, flavonoid biosynthesis, and biosynthesis of secondary metabolites were mostly enriched. Additionally, we included all the compounds and their postharvest storage in the public Phenolics profile database. CONCLUSIONS: Here, we show that the stage of ripening has a significant effect on the phenolic content, highlighting the point of cut (D0) and the onset of senescence (D5). The findings of this study provide new insights into the soursop fruit quality and may contribute to the identification of metabolic markers for its storage.

Molecularly imprinted nanoparticle-based assay (MINA) for microcystin-LR detection in water.

Microcystins (MCs) are highly toxic peptides produced by cyanobacteria during algal blooms. Microcystin-leucine-arginine (MC-LR) is the most toxic and common MC variant with major effects on human and animal health upon exposure. MC-LR detection has become critical to ensure water safety, therefore robust and reliable analytical methods are needed. This work reports the development of a simple and optimized Molecularly Imprinted Nanoparticle-Based Assay (MINA) for MC-LR detection in water. Molecularly Imprinted Nanoparticles (MINs) were prepared by solid-phase polymerization on glass beads conjugated to MC-LR through (3-aminopropyl) triethoxysilane (APTES) via amide bonding. APTES-modified glass beads were obtained under optimized conditions to maximize the density of surface amino groups available for MC-LR conjugation. Two quinary mixtures of acrylic monomers differing in charge, polarity, and functionality were selected from molecular docking calculations and used to obtain MINs for MC-LR recognition using N,N'-methylene-bis-acrylamide (BIS) as the crosslinking agent. MINs were immobilized by physical adsorption onto 96-well polystyrene microplate and evaluated as per their rebinding capacity toward the analyte by using a covalent conjugate between MC-LR and the enzyme horseradish peroxidase (HRP). Experimental conditions for the MINs immobilization protocol, HRP-MC-LR concentration, and composition of the blocking solution were set to maximize the colorimetric response of the MINs compared to non-treated wells. Optimized conditions were then applied to conduct competitive MINAs with the HRP-MC-LR conjugate and the free analyte, which confirmed the preferential binding of MC-LR to the immobilized MINs for analyte concentrations ranging from 1 × 10-5 nmol L-1 to 100 nmol L-1. The best competitive MINA showed a limit of detection of 2.49 × 10-4 nmol L-1 and coefficients of variation less than 10% (n = 6), which are auspicious for the use of MINs as analytical tools for MC-LR detection below the permissible limits issued by WHO for safe water consumption (1.00 nmol L-1). This assay also proved to be selective to the analyte in cross-reactivity studies with two analogous microcystins (MC-RR and MC-YR). Analyses of lagoon and drinking water samples enriched with MC-LR revealed strong matrix effects that reduce the MINA response to the analyte, thus suggesting the need for sample pretreatment methods in future development in this subject.

Cell internalization kinetics and surface charge accessibility of surface-modified PAMAM dendrimers.

Surface-modified PAMAM dendrimers have important applications in drug delivery, yet a gap remains about the role that surface functionalization plays on their cell internalization capacity. We examined the cell internalization kinetics of PAMAM dendrimers that were surface-modified with acetyl, folate and poly(ethylene glycol), as model functional groups differing in size, charge, and chemical functionality. Dendrimers with 25% functionalization were internalized by HEK cells, but with slower rates and lower maximum uptakes than the native dendrimer between 1-6 h of incubation. Dendrimers with 50% functionalization exhibited negligible internalization capacities at all incubation times. Molecular dynamics simulations revealed that the solvent accessibility of the cationic surface charges is a key factor affecting cell internalization, unlike the total charge, functionality or size of surface-modified PAMAM dendrimers. These findings provide valuable insights to assist the design of PAMAM-based systems for drug delivery applications.

Insight into the Role of Active Site Protonation States and Water Molecules in the Catalytic Inhibition of DPP4 by Vildagliptin.

Vildagliptin (VIL) is an antidiabetic drug that inhibits dipeptidyl peptidase-4 (DPP4) through a covalent mechanism. The molecular bases for this inhibitory process have been addressed experimentally and computationally. Nevertheless, relevant issues remain unknown such as the roles of active site protonation states and conserved water molecules nearby the catalytic center. In this work, molecular dynamics simulations were applied to examine the structures of 12 noncovalent VIL-DPP4 complexes encompassing all possible protonation states of three noncatalytic residues (His126, Asp663, Asp709) that were inconclusively predicted by different computational tools. A catalytically competent complex structure was only achieved in the system with His126 in its ε-form and nonconventional neutral states for Asp663/Asp709. This complex suggested the involvement of one water molecule in the catalytic process of His740/Ser630 activation, which was confirmed by QM/MM simulations. Our findings support the suitability of a novel water-mediated mechanism in which His740/Ser630 activation occurs concertedly with the nucleophilic attack on VIL and the imidate protonation by Tyr547. Then, the restoration of His740/ Tyr547 protonation states occurs via a two-water hydrogen bonding network in a low-barrier process, thus describing the final step of the catalytic cycle for the first time. Additionally, two hydrolytic mechanisms were proposed based on the hydrogen bonding networks formed by water molecules and the catalytic residues along the inhibitory mechanism. These findings are valuable to unveil the molecular features of the covalent inhibition of DPP4 by VIL and support the future development of novel derivatives with improved structural or mechanistic profiles.

Bacillus mojavensis enhances the antioxidant defense mechanism of soursop (Annona muricata L.) fruits during postharvest storage.

Rapid softening of soursop (Annona muricata L.) fruit results in postharvest losses. Bacillus genus is one of the most studied antagonistic biological control agents against postharvest diseases. Nevertheless, information about how this bacterium acts on the fruits is still not understood. The objective of this study aims to gain an insight into the effect of Bacillus mojavensis on the activity and gene expression of antioxidant defense enzymes in soursop fruits during postharvest storage. Our findings indicate different responses in the fruits inoculated with B. mojavensis at biochemical and molecular levels. On day one, fruits inoculated with B. mojavensis presented a mean value of 79.09 GAE/100 gFW in total phenols, and higher superoxide dismutase (SOD) and catalase (CAT) activities (1.35 and 1.78-fold higher, respectively). On the other hand, on the third day of storage, the ferric reducing/antioxidant power (FRAP) reached its highest level, including an increase in the expression of SOD, and PPO genes by 18.7-fold and 4.5-fold in fruits inoculated with B. mojavensis. Finally, on the fifth day of storage, soursop fruits inoculated with B. mojavensis had the highest mean values for 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH·), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS· +), with values of 194.68 EAA/100 gFW, and 172.33 EAA/100 gFW, respectively. Indeed, higher polyphenol oxidase (PPO), and peroxidase (POD) activities (2.17-fold and 1.27-fold higher, respectively) were recorded compared to the control fruits. We show that depending on the stage of ripening, the antagonist bacteria B. mojavensis enhanced the antioxidant capacity, enzymatic activity, and gene expression of soursop fruits.

The old and the new about the contractile vacuole of Trypanosoma cruzi.

Osmoregulation is a conserved cellular process required for the survival of all organisms. In protists, the need for robust compensatory mechanisms that can maintain cell volume and tonicity within physiological range is even more relevant, as their life cycles are often completed in different environments. Trypanosoma cruzi, the protozoan pathogen responsible for Chagas disease, is transmitted by an insect vector to multiple types of mammalian hosts. The contractile vacuole complex (CVC) is an organelle that senses and compensates osmotic changes in the parasites, ensuring their survival upon ionic and osmotic challenges. Recent work shows that the contractile vacuole is also a key component of the secretory and endocytic pathways, regulating the selective targeting of surface proteins during differentiation. Here we summarize our current knowledge of the mechanisms involved in the osmoregulatory processes that take place in the vacuole, and we explore the new and exciting functions of this organelle in cell trafficking and signaling.

Rational Discovery of Microtubule-Stabilizing Peptides.

Microtubule (MT) stabilization is an attractive pharmacological strategy to hamper the progress of neurodegenerative diseases. In this regard, seeking peptides with MT-stabilizing properties has awoken great interest. This work reports the rational discovery of two structurally related MT-stabilizing octapeptides using a combination of protein-peptide docking, conventional molecular dynamics, Gaussian accelerated molecular dynamics (GaMD), and tubulin polymerization assays. FASTA sequences for ∼1000 peptides were crafted from single and double mutants of davunetide (NAP) and docked against the Taxol (TX) site on an octameric MT model representing a portion of the MT wall. Docked peptides were rescored after MM minimization and binding free energy refinement through single-point MM/GBSA calculations. The 60 best-ranked peptides were subjected to 50 ns MD simulations on peptide-MT complexes at the terminal TX site in the octameric Tau-MT model resulting in 11 complexes with occupancies greater than 99% and peptide-protein binding free energies less than -40 kcal/mol. Selected peptides were then examined through 300 ns GaMD simulations in complexes containing two identical ligands at the terminal and intermediate TX sites in the Tau-MT model to account for the differential association of MT-binding peptides to different regions of the MT structure. Six candidates showed a favorable MT-binding potential based on the analysis of interaction frequencies and relative mobilities of the complex components, suggesting a pivotal role of Arg278, Gln281, and Arg369 residues for peptides recognition. Four candidates were predicted to preserve an adequate balance of longitudinal and lateral interactions between tubulin dimers in peptide-MT complexes such that MT-stabilizing effects could be expected. MT polymerization experiments confirmed that four peptides (HAPVSIHQ, NYPVSIHQ, NWPVSIWQ, HAPVSIIQ) exhibit MT-stabilizing activity in vitro with NWPVSIWQ (P43) and HAPVSIIQ (P52) being the most active. Tryptophan quenching assays verified that P43 and P52 bind to nonpolymeric tubulin, whereas viability experiments on HEK cells confirmed their safety to pursue future pharmacological studies. The results herein presented are valuable to making progress in the rational design of MT-stabilizing peptides.

Drimane Sesquiterpene Alcohols with Activity against Candida Yeast Obtained by Biotransformation with Cladosporium antarcticum.

Fungal biotransformation is an attractive synthetic strategy to produce highly specific compounds with chemical functionality in regions of the carbon skeleton that are not easily activated by conventional organic chemistry methods. In this work, Cladosporium antarcticum isolated from sediments of Glacier Collins in Antarctica was used to obtain novel drimane sesquiterpenoids alcohols with activity against Candida yeast from drimendiol and epidrimendiol. These compounds were produced by the high-yield reduction of polygodial and isotadeonal with NaBH4 in methanol. Cladosporium antarcticum produced two major products from drimendiol, identified as 9α-hydroxydrimendiol (1, 41.4 mg, 19.4% yield) and 3ß-hydroxydrimendiol (2, 74.8 mg, 35% yield), whereas the biotransformation of epidrimendiol yielded only one product, 9ß-hydroxyepidrimendiol (3, 86.6 mg, 41.6% yield). The products were purified by column chromatography and their structure elucidated by NMR and MS. The antifungal activity of compounds 1-3 was analyzed against Candida albicans, C. krusei and C. parapsilosis, showing that compound 2 has a MIC lower than 15 µg/mL against the three-pathogenic yeast. In silico studies suggest that a possible mechanism of action for the novel compounds is the inhibition of the enzyme lanosterol 14α-demethylase, affecting the ergosterol synthesis.

Drimane Sesquiterpene Aldehydes Control Candida Yeast Isolated from Candidemia in Chilean Patients.

Drimys winteri J.R. (Winteraceae) produce drimane sesquiterpenoids with activity against Candida yeast. In this work, drimenol, polygodial (1), isotadeonal (2), and a new drimane α,ß-unsaturated 1,4-dialdehyde, named winterdial (4), were purified from barks of D. winteri. The oxidation of drimenol produced the monoaldehyde drimenal (3). These four aldehyde sesquiterpenoids were evaluated against six Candida species isolated from candidemia patients in Chilean hospitals. Results showed that 1 displays fungistatic activity against all yeasts (3.75 to 15.0 µg/mL), but irritant effects on eyes and skin, whereas its non-pungent epimer 2 has fungistatic and fungicide activities at 1.9 and 15.0 µg/mL, respectively. On the other hand, compounds 3 and 4 were less active. Molecular dynamics simulations suggested that compounds 1-4 are capable of binding to the catalytic pocket of lanosterol 14-alpha demethylase with similar binding free energies, thus suggesting a potential mechanism of action through the inhibition of ergosterol synthesis. According to our findings, compound 2 appears as a valuable molecular scaffold to pursue the future development of more potent drugs against candidiasis with fewer side effects than polygodial. These outcomes are significant to broaden the alternatives to treat fungal infections with increasing prevalence worldwide using natural compounds as a primary source for active compounds.

Salpianthus macrodontus Extracts, a Novel Source of Phenolic Compounds with Antibacterial Activity against Potentially Pathogenic Bacteria Isolated from White Shrimp.

This study aimed to evaluate the antibacterial activity in vitro of Salpianthus macrodontus and Azadirachta indica extracts against potentially pathogenic bacteria for Pacific white shrimp. Furthermore, the extracts with higher inhibitory activity were analyzed to identify compounds responsible for bacterial inhibition and evaluate their effect on motility and biofilm formation. S. macrodontus and A. indica extracts were prepared using methanol, acetone, and hexane by ultrasound. The minimum inhibitory concentration (MIC) of the extracts was determined against Vibrio parahaemolyticus, V. harveyi, Photobacterium damselae and P. leiognathi. The polyphenol profile of those extracts showing the highest bacterial inhibition were determined. Besides, the bacterial swimming and swarming motility and biofilm formation were determined. The highest inhibitory activity against the four pathogens was found with the acetonic extract of S. macrodontus leaf (MIC of 50 mg/mL for Vibrio spp. and 25 mg/mL for Photobacterium spp.) and the methanol extract of S. macrodontus flower (MIC of 50 mg/mL for all pathogens tested). Both extracts affected the swarming and swimming motility and the biofilm formation of the tested bacteria. The main phenolic compounds related to Vibrio bacteria inhibition were naringin, vanillic acid, and rosmarinic acid, whilst hesperidin, kaempferol pentosyl-rutinoside, and rhamnetin were related to Photobacterium bacteria inhibition.

BMP7 overexpression in adipose tissue induces white adipogenesis and improves insulin sensitivity in ob/ob mice.

BACKGROUND/OBJECTIVES: During obesity, hypertrophic enlargement of white adipose tissue (WAT) promotes ectopic lipid deposition and development of insulin resistance. In contrast, WAT hyperplasia is associated with preservation of insulin sensitivity. The complex network of factors that regulates white adipogenesis is not fully understood. Bone morphogenic protein 7 (BMP7) can induce brown adipogenesis, but its role on white adipogenesis remains to be elucidated. Here, we assessed BMP7-mediated effects on white adipogenesis in ob/ob mice. METHODS: BMP7 was overexpressed in either WAT or liver of ob/ob mice using adeno-associated viral (AAV) vectors. Analysis of gene expression, histological and morphometric alterations, and metabolites and hormones concentrations were carried out. RESULTS: Overexpression of BMP7 in adipocytes of subcutaneous and visceral WAT increased fat mass, the proportion of small-size adipocytes and the expression of adipogenic and mature adipocyte genes, suggesting induction of adipogenesis irrespective of fat depot. These changes were associated with reduced hepatic steatosis and improved insulin sensitivity. In contrast, liver-specific overproduction of BMP7 did not promote WAT hyperplasia despite BMP7 circulating levels were similar to those achieved after genetic engineering of WAT. CONCLUSIONS: This study unravels a new autocrine/paracrine role of BMP7 on white adipogenesis and highlights that BMP7 may modulate WAT plasticity and increase insulin sensitivity.

On the Microtubule-Stabilizing Properties of a Tau Oligopeptide.

Preserving the integrity of neuronal microtubules (MTs) has emerged as a promising strategy to inhibit the progression of neurodegenerative disorders such as Alzheimer's disease. Such a goal could be achieved by peptides that mimic the functional role of Tau, an MT-associated protein that stabilizes MTs by dynamically binding to their outer surface. This work examines the binding properties and MT-stabilizing potential of a 27-amino acid Tau oligopeptide from 300 ns Gaussian-accelerated molecular dynamics simulations and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculations on octameric MT models bound to two equivalent and independent Tau peptides. Bound peptides adopted extended conformations that are highly consistent with cryo-electron microscopy reports for full-length Tau bound to MTs. Anchoring points in three consecutive tubulin subunits were identified, with a relevant contribution of the Ser419-Val435 region to α-tubulin. Tau peptides strengthen the longitudinal protein-protein contacts within the MT lattice and exert a cooperative MT-stabilizing effect in MT complexes simultaneously bonded to taxol or peloruside A. Ser phosphorylation results in a larger peptide mobility, altered interaction profiles, and MT destabilization, which are in line with the loss of MT integrity resulting from the post-translational hyperphosphorylation of Tau. Our results shed light on the MT-stabilizing potential of Tau-mimetic peptides to act as novel neuroprotective agents targeting MTs.

NanoMIPs Design for Fucose and Mannose Recognition: A Molecular Dynamics Approach.

Nanoscale molecularly imprinted polymers (nanoMIPs) are powerful molecular recognition tools with broad applications in the diagnosis, prognosis, and treatment of complex diseases. In this work, fully atomistic molecular dynamics (MD) simulations are used to assist the design of nanoMIPs with recognition capacity toward l-fucose and d-mannose as prototype disease biomarkers. MD simulations were conducted on prepolymerization mixtures containing different molar ratios of the monomers N-isopropylacrylamide (NIPAM), methacrylamide (MAM), and (4-acrylamidophenyl)(amino)methaniminium acetate (AB) and fixed molar ratios of the cross-linker ethylene glycol dimethacrylate (EGDMA) in explicit acetonitrile as the porogenic solvent. Prepolymerization mixtures containing ternary mixtures of NIPAM (50%), MAM (25%), and AB (25%) exhibit the best imprinting potential for both l-fucose and d-mannose, as they maximize (i) the stability of template-monomer plus template-cross-linker interactions, (ii) the number of functional monomers plus cross-linkers organized around the template, and (iii) the number of hydrogen bonds participating in template recognition. The studied prepolymerization mixtures exhibit an overall increased recognition capacity toward d-mannose over l-fucose, which is attributed to the higher hydrogen-bonding capacity of the former template. Our results are valuable to guide the synthesis of efficient nanoMIPs for sugar recognition and provide a computational framework extensible to any other template, monomer, or cross-linker combination, thus constituting a promising strategy for the rational design of molecularly imprinted materials.

Rational Design of Novel Glycomimetic Peptides for E-Selectin Targeting.

E-selectin is a cell-adhesion receptor with specific recognition capacity toward sialo-fucosylated Lewis carbohydrates present in leukocytes and tumor cells. E-selectin interactions mediate the progress of inflammatory processes and tumor metastasis, which aroused the interest in using this protein as a biomolecular target to design glycomimetic inhibitors for active targeting or therapeutic purposes. In this work, we report the rational discovery of two novel glycomimetic peptides targeting E-selectin based on mutations of the reference selectin-binding peptide IELLQAR. Sixteen single or double mutants at Ile1, Leu3, Leu4, and Arg7 residues were evaluated as potential candidates for E-selectin targeting using 50 ns molecular dynamics (MD) simulations. Nine peptides showing a stable association with the functional pocket were modified by adding a cysteine residue to the N-terminus to confer versatility for further chemical conjugation. Subsequent 50 ns MD simulations resulted in five cysteine-modified peptides with retained or improved E-selectin binding potential. Then, 300 ns accelerated MD (aMD) simulations were used to examine the binding properties of the best five cysteine-modified peptides. CIEELQAR and CIELFQAR exhibit the most selective association with the functional pocket of E-selectin, as revealed by potential of mean force profiles. Microscale thermophoresis experiments confirmed the E-selectin binding capacity of the selected peptides with KD values in the low micromolar range (CIEELQAR KD = 35.0 ± 1.4 µM; CIELFQAR KD = 16.4 ± 0.7 µM), which are 25-fold lower than the reported value for the native ligand sLex (KD = 878 µM). Our findings support the potential of CIEELQAR and CIELFQAR as novel E-selectin-targeting peptides with high recognition capacity and versatility for chemical conjugation, which are critical for enabling future applications in active targeting.

Interdimeric Curvature in Tubulin-Tubulin Complexes Delineates the Microtubule-Destabilizing Properties of Plocabulin.

Plocabulin is a novel microtubule (MT) destabilizer agent with potent antineoplastic activity. This compound binds to the maytansine site at the longitudinal interface between tubulin dimers and exerts a hinge-like effect that disrupts normal microtubule assembly. Plocabulin has emerged as a valuable model for the rational design of novel MT destabilizers because of its unique structural and mechanistic features. To make progress on this matter, detailed molecular-level understanding of the ligand-protein interactions responsible for plocabulin association and the conformation and energetic effects arising from plocabulin binding on the longitudinal interaction between tubulin dimers must be provided. In this work, fully atomistic MD simulations and MM/GBSA binding free-energy calculations were used to examine the association of plocabulin to one or two tubulin dimers in longitudinal arrangement. Our results revealed that plocabulin binding is favored by the addition of a second tubulin dimer and that this ligand promotes the assembly of curved tetrameric arrangements with strengthened longitudinal interdimeric interactions compared to ligand-free systems. The applicability of these findings to the rational discovery of novel MT destabilizers was tested using MD and MM/GBSA calculations as filtering tools to narrow the results of virtual screening among an FDA-approved drug database. Our results confirmed that tight-binding ligands do not necessarily exert the expected conformational and energetic effects on longitudinal tubulin-tubulin interactions, which is a matter to consider in future design strategies.

Catalytic Role of Gln202 in the Carboligation Reaction Mechanism of Yeast AHAS: A QM/MM Study.

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate-dependent enzyme involved in the biosynthesis of valine, leucine, isoleucine, and lysine. Experimental evidence has shown that mutation of the Gln202 residue results in a decrease in the enzymatic activity, thus suggesting the main role of the carboligation catalyzed by AHAS. It has been postulated that this residue acts as an acid/base group, protonating the carbonyl oxygen from the 2-ketoacid substrate, during the carboligation reaction. However, previous studies have revealed that 2-ketoacid is not engaged in catalytically relevant interactions with ionizable groups that can act as an acid/base group during the catalysis. Therefore, it has been proposed that the carboligation reaction could occur through an intramolecular proton transfer without the assistance of an amino acid residue with acid-base properties. To decipher the role of Gln202, in this work, we studied the last two catalytic steps of the AHAS through quantum mechanics/molecular mechanics calculations using a full enzyme model of the wild-type AHAS and the Gln202Ala mutant. Our results indicate that the carboligation mechanism occurs through an intramolecular proton transfer that does not require the action of an additional acid-base group. The mechanism is composed of two steps in which the last one is rate-limiting. Our findings reveal that Gln202 stabilizes a catalytic water molecule in the reactive site through electrostatic contributions that are mostly relevant during the carboligation step, in agreement with experimental evidence. The catalytic water engages in intermolecular hydrogen bonds with the reacting species and makes a strong electronic contribution to the stabilization of the reaction intermediate (AL-ThDP).

Rational Design and In Vitro Evaluation of Novel Peptides Binding to Neuroligin-1 for Synaptic Targeting.

Neuroligin-1 (NL1) is a postsynaptic cell adhesion protein that plays a crucial role in synapsis and signaling between neurons. Due to its clustered distribution in synaptic clefts, NL1 appears as a novel potential site for synaptic targeting purposes. In this work, in silico protein topography analysis was employed to identify two prospective binding sites on the NL1 dimer surface in the 2:2 synaptic adhesion complex with ß-neurexin (PDB code 3B3Q ). Receptor-based rational design, cell-penetrating capability prediction, molecular docking, molecular dynamics simulations, and binding free energy calculations were used to identify five heptapeptides candidates with favorable predicted profiles as non cell-penetrating NL1-binding agents. Preliminary in vitro colocalization assays with NL1-transfected HEK 293 cells confirmed that peptides remain in the extracellular space without inducing detectable changes in cell morphology. The highest NL1-colocatization capability was attained by the peptide ADEAIVA, which appears as a promising candidate for the future development of specific NL1-targeting systems as part of synapse-directed therapies against central nervous system diseases.

Mechanism-Based Rational Discovery and In Vitro Evaluation of Novel Microtubule Stabilizing Agents with Non-Taxol-Competitive Activity.

Microtubules (MT) are cytoskeletal polymers of αß-tubulin dimers that play a critical role in many cellular functions. Diverse antimitotic drugs bind to MT and disrupt their dynamics acting as MT stabilizing or destabilizing agents. The occurrence of undesired side effects and drug resistance encourages the search for novel MT binding agents with chemically diverse structures and different interaction profiles compared to known active compounds. This work reports the rational discovery of seven novel MT stabilizers using a combination of molecular modeling methods and in vitro experimental assays. Virtual screening, similarity filtering, and molecular mechanics generalized Born surface area (MM/GBSA) binding free energy refinement were employed to select seven potential candidates with high predicted affinity toward the non-taxoid site for MT stabilizers on ß-tubulin. MD simulations of 150 ns on reduced MT models suggest that candidate compounds strengthen the longitudinal interactions between tubulin dimers across protofilaments, which is a primary molecular mechanism of action for known MT stabilizers. In vitro MT polymerization assays confirmed that all candidates promote MT assembly at concentrations of >50 mM and exhibit noncompetitive MT polymerization profiles when cotreating with Taxol. Preliminary HeLa cell viability assays revealed a moderate cytotoxic effect for the compounds under study at 100 µM concentration. These results support the validity of our rational discovery strategy and the use of molecular modeling methods to pursue the search and optimization of new MT targeting agents.