Matías Zúñiga-Bustos

Gold nanostructures have emerged as promising platforms for the development of targeted therapies against Alzheimer's disease (AD) owing to their biocompatibility, chemical stability, and versatile surface functionalization. Among them, anisotropic gold nanorods (GNR) provide a unique nano–bio interface that can actively regulate the organization and biological performance of surface-bound ligands. Here, we designed PEGylated GNR functionalized with the D-enantiomeric peptide D3 (rprtrlhthrnr), a β-sheet breaker that selectively recognizes toxic amyloid-β (Aβ) oligomers. The aspect ratio of the GNR was engineered to extend their localized surface plasmon resonance into the second near-infrared (NIR-II) biological window, providing a platform with future theranostic potential. Physicochemical characterization confirmed successful peptide conjugation and high colloidal stability, while computational and spectroscopic analyses demonstrated that the GNR-PEG-D3 interface is stabilized by the synergistic contribution of covalent Au–S anchoring and distributed non-covalent interactions, preserving peptide accessibility for Aβ recognition. As a result, GNR-PEG-D3 exhibited significantly greater inhibition of Aβ fibrillization than the free peptide, redirected the aggregation pathway toward less ordered and less toxic species, and reduced β-sheet formation. Furthermore, the nanosystem exhibited biocompatibility and significantly ameliorated Aβ-induced paralysis in a Caenorhabditis elegans in vivo model of Alzheimer's disease. Overall, this study demonstrates that the anisotropic architecture of GNR acts as an active design element rather than a passive carrier, optimizing the spatial presentation of peptide D3 and the modulation of the Aβ aggregation process. These findings establish nanoparticle geometry as a key parameter for the rational design of next-generation peptide-based nanotherapeutics for AD.

Gingipain B is a protease released by Porphyromonas gingivalis , which contributes to the development of diseases such as periodontitis, Alzheimer's disease, rheumatoid arthritis and cancer. Therefore, it could be a key target for the design of inhibitors that could be used in new drug therapies. In this search for inhibitors, it is interesting to study their potentiation by Zn ²⁺ , mainly because this divalent cation can be a common additive in toothpastes or mouthwashes. To understand this potentiation process, the structural aspects that determine the effect of Zn ²⁺ on the different potentiation of gingipain B inhibition by three inhibitors with benzamidine group were studied. For this purpose, molecular dynamics simulations of the enzyme/inhibitor complex, in the presence or absence of Zn ²⁺ , were performed to identify the residues involved in the interaction with the inhibitor or with the cation. From the simulations it is observed that the compounds that are potentiated by Zn interact with the ion via water molecules, which are oriented towards the carbonyl functional group. In addition, a higher solvent exposure of the catalytic site and a lower flexibility of a region adjacent to cysteine 244, which participates in catalysis, are observed in the simulations. The structural and dynamic results obtained through molecular dynamics simulations support the hypothesis that the potentiation of gingipain B inhibition by Zn ²⁺ is associated with ion‐mediated ligand stabilization, leading to a decrease in the number of water molecules in the second hydration shell, as well as polarization of the molecules in the vicinity of Zn ²⁺ . Taken together, these findings open new avenues for the design of inhibitors whose activity can be modulated or enhanced by the presence of Zn ²⁺ , with potential applications in the development of innovative therapeutic strategies.

DDX3X is a cellular DEAD-box ATP-dependent RNA helicase known to play pivotal roles during the replication cycle of different viruses including some flaviviruses. Whether DDX3X plays a role during replication of Zika virus (ZIKV), a mosquito-borne flavivirus with a broad tissue tropism, has not been explored in detail. Here, we show that DDX3X is required for efficient ZIKV replication in a human microglia cell line but no other cell lines. Mechanistically, we provide evidence showing that DDX3X is recruited to the viral replication compartments where it binds to the 5’UTR of the ZIKV RNA and promotes viral protein synthesis in an ATP-dependent manner. We also show that DDX3X binds to the viral RNA-dependent RNA polymerase NS5 and interferes with viral RNA synthesis in an ATP-independent manner. Such an effect was not observed during replication of Dengue 2 virus in human microglia, revealing specific roles for DDX3X in the regulation of the translation-replication cycle of ZIKV in human microglia. IMPORTANCE: Zika virus emerged as a major threat to humans due to its pandemic potential and its association with birth defects and neurological complications. The lack of available vaccines or specific treatments makes the understanding of Zika virus-host interactions a priority. Here, we demonstrate a mechanism by which DDX3X, a cellular ATP-dependent RNA helicase, promotes Zika virus replication by regulating the translation-replication cycle in a human microglia cell line. Considering the threat of this virus to the human population and the wide range of RNA viruses that usurp this host protein to accomplish the replication cycle, DDX3X continuously rises as a potential and valuable target for pharmacological intervention.

Background: Tubulin plays a pivotal role in cell division and other essential cellular processes, making it a key pharmacological target for cancer therapy, antiparasitic treatments, and neurodegenerative diseases. Numerous compounds have been developed to regulate microtubule polymerization through tubulin binding; however, most have shown significant limitations, including adverse side effects, poor bioavailability and limited specificity. In recent years, peptide-based therapies have gained considerable attention, particularly for their ability to modulate protein–protein interaction while offering improved selectivity and safety profiles. Methods: In this study, we employed an integrated computational–experimental approach combining molecular docking, molecular dynamics simulations, and MM-GBSA free energy calculations to design and evaluate 14 peptides derived from the αβ-tubulin dimer interface. Results: The peptide NH₂-P14-COOH emerged as the most promising candidate, displaying the stronger inhibition of tubulin polymerization activity (IC₅₀ = 11.24 ± 3.82 μM), selective cytotoxicity against NCI-H1299 lung carcinoma cells (IC₅₀ = 45.64 ± 3.20 μM), and no significant toxicity toward non-cancerous EA.hy926 endothelial cells (IC₅₀ > 100 μM). Flow cytometry analysis confirmed that NH₂-P14-COOH induces apoptosis, supporting a mechanism of action based on microtubule disruption. Conclusions: These findings highlight NH₂-P14-COOH as a selective antimitotic peptide with a favorable therapeutic index and demonstrate the potential of structure-guided peptide design for the development of novel microtubule-targeting agents with reduced off-target toxicity.

The dual inhibition of microtubule dynamics and immune checkpoint signaling represents a promising strategy in cancer therapy by simultaneously disrupting cell proliferation and immune evasion.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is recognized as an essential modulator of transient receptor potential (TRP) channels. Specifically, it influences the vanilloid receptor I (TRPV1), a pain receptor activated by a wide range of stimuli, including the binding of phospholipids, such as PIP2. The primary PIP2-binding site in TRPV1 has been identified through advanced techniques, revealing that the PIP2 binds to a specific pocket composed of positively charged residues located predominantly within the proximal C-terminus region. Additionally, a conserved segment with positively charged amino acids, K431 and R432, situated at the beginning of TRPV1’s S1 transmembrane domain, has attracted considerable attention from the TRP research community. To date, our knowledge of this site’s function and the subsequent effects following PIP2 binding is still emerging. In this work, MD simulations were conducted using coarse-grained models to investigate the binding dynamics of PIP2 on both WT and various mutated forms of TRPV1 channels. Our findings indicate that the K431A and R432A mutations significantly reduce the frequency of PIP2 contacts, suggesting that these mutated residues are part of a “peripheral binding pocket.” This pocket seems to play a crucial role in facilitating the entry of PIP2 to the TRPV1 channel’s primary binding site. Furthermore, our research has shown that these highly conserved residues within the TRPV subfamily are also structurally conserved across other TRP subfamilies, such as TRPM and TRPC, a detail not evident from sequence alignment alone. Consequently, we propose the existence of a structurally conserved peripheral PIP2-binding site shared among the diverse members of the TRP family, which can be categorized into distinct subfamilies.

This study evaluated the antioxidant capacity of the oxidation products of three flavonols using oxygen radical absorbance capacity—fluorescein assay (ORAC-FL), oxygen radical absorbance capacity—pyrogallol red assay (ORAC-PGR), and the cellular antioxidant activity (CAA) assay in human dermal fibroblast (HFF) cells, with 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) as a free radical generator under controlled pH and solvent conditions. At pH 2 in a polar aprotic solvent, BZF-OH (benzofuranone-OH) compounds were formed, while methoxylated analogs were obtained at pH 7 in a polar protic solvent. The products generated at pH 2 exhibited significantly higher antioxidant capacities, demonstrating the influence of the reaction environment on modulating antioxidant properties. The antioxidant activity was observed to reflect the combined action of the flavonol precursor and its oxidation products. This led to the proposal of the Gross Antioxidant Capacity (GAC) concept to integrate the contribution of all generated species. Since chemical assays such as ORAC do not fully capture the complexity of biological systems, they should be complemented with cellular approaches for a more accurate evaluation. Additionally, BZF-OH compounds were analyzed as potential cyclooxygenase-2 (COX-2) inhibitors through docking and molecular dynamics simulations, where BZF-Quer-OH showed binding affinities comparable to celecoxib, a selective COX-2 inhibitor. These findings were complemented by an analysis of COX-2 expression in RAW 264.7 cells treated with lipopolysaccharide (LPS), where treatment with the antioxidants significantly inhibited COX-2 expression. In the case of the oxidation products, only the oxidation product of rhamnetin showed a reduction in COX-2 expression compared to the LPS-treated control. Together, these results highlight that flavonol-derived oxidation products not only retain significant antioxidant capacity but may also possess anti-inflammatory properties, opening new perspectives for the development of innovative therapies targeting oxidative stress and chronic inflammation.

Nanomaterials, specifically silver nanoparticles (AgNPs), have demonstrated great potential in biomedical applications due to their unique properties, such as antimicrobial activity and conductivity. One promising strategy to improve their biocompatibility and functional specificity is through the functionalization of AgNPs with peptides. By attaching peptides to the surface of AgNPs, their interaction with biological systems can be enhanced and tailored for specific applications. This computational study uses classical molecular dynamics and enhancement sampling techniques to investigate the interaction between AgNPs and RADA16-I peptides, as well as their derivative CLKRADA16-I. It utilizes classical molecular dynamics and enhanced sampling methods to gain insights into the structural information and details of their interaction. Furthermore, this study addresses the need for a better understanding of the interaction between composite materials made of nanoparticles and peptides. Our results demonstrate that the incorporation of the CLK motif significantly augments both structural stability and the binding affinity of peptides to silver nanoparticles. Through computational simulations, we observed that peptides modified with the CLK motif (CLKRADA16-I) exhibit a higher binding affinity toward a silver surface model, with the adsorption energy increasing by up to 4.2 kcal mol⁻¹ relative to unmodified peptides. This calculated interaction energy boosts adsorption and surface coverage, facilitating a packed and more effective peptide coating on the silver nanoparticles. These findings pave the way for the advancement of AgNPs as versatile agents in nanomedicine, particularly necessitating precise molecular recognition and robust bioactive scaffolding. Our study enhances the understanding of nanoparticle–peptide conjugates and their implications for designing next-generation nanomaterials.

Chagas disease is a prevalent health problem in Latin America which has received insufficient attention worldwide. Current treatments for this disease, benznidazole and nifurtimox, have limited efficacy and may cause side effects. A recent study proposed investigating a wide range of nitroindazole and indazolone derivatives as feasible treatments. Therefore, it is proposed that adding a nitro group at the 5-position of the indazole and indazolone structure could enhance trypanocidal activity by inducing oxidative stress through activation of the nitro group by NTRs (nitroreductases). The study results indicate that the nitro group advances free radical production, as confirmed by several analyses. Compound 5a (5-nitro-2-picolyl-indazolin-3-one) shows the most favorable trypanocidal activity (1.1 ± 0.3 µM in epimastigotes and 5.4 ± 1.0 µM in trypomastigotes), with a selectivity index superior to nifurtimox. Analysis of the mechanism of action indicated that the nitro group at the 5-position of the indazole ring induces the generation of reactive oxygen species (ROS), which causes apoptosis in the parasites. Computational docking studies reveal how the compounds interact with critical residues of the NTR and FMNH₂ (flavin mononucleotide reduced) in the binding site, which is also present in active ligands. The lipophilicity of the studied series was shown to influence their activity, and the nitro group was found to play a crucial role in generating free radicals. Further investigations are needed of derivatives with comparable lipophilic characteristics and the location of the nitro group in different positions of the base structure.

The development of new compounds to treat Chagas disease is imperative due to the adverse effects of current drugs and their low efficacy in the chronic phase. This study aims to investigate nitroisoxazole derivatives that produce oxidative stress while modifying the compounds’ lipophilicity, affecting their ability to fight trypanosomes. The results indicate that these compounds are more effective against the epimastigote form of T. cruzi, with a 52 ± 4% trypanocidal effect for compound 9. However, they are less effective against the trypomastigote form, with a 15 ± 3% trypanocidal effect. Additionally, compound 11 interacts with a higher number of amino acid residues within the active site of the enzyme cruzipain. Furthermore, it was also found that the presence of a nitro group allows for the generation of free radicals; likewise, the large size of the compound enables increased interaction with aminoacidic residues in the active site of cruzipain, contributing to trypanocidal activity. This activity depends on the size and lipophilicity of the compounds. The study recommends exploring new compounds based on the nitroisoxazole skeleton, with larger substituents and lipophilicity to enhance their trypanocidal activity.