Biology subjects

Pacheco-Garcia, J. L.

Publications and source records attributed to Pacheco-Garcia, J. L..

3 recordsLinked to original sources

bioRxiv2026

Albumin-coated VS1 nanocrystals enable STARD3 inhibition and potentiate fluoropyrimidine therapy in colorectal cancer

Poor aqueous solubility remains a major obstacle to the translational development of targeted anticancer compounds. VS1, a first-in-class inhibitor of the cholesterol-transfer protein STARD3, has emerged as a promising chemosensitizing agent in colorectal cancer (CRC), but its clinical applicability is limited by its poor water solubility. Here, we combine structural biology, nanotechnology, and functional pharmacology to establish STARD3 inhibition as a delivery-enabled strategy to potentiate fluoropyrimidine therapy. To define the molecular basis of STARD3 inhibition, we solved the crystal structure of VS1 bound to the STARD3 ligand-binding domain at 2.1 [A] resolution, revealing direct occupation of the sterol-binding cavity. Molecular dynamics simulations confirmed a stable binding mode and identified the {Omega}1 loop as a dynamic gate regulating ligand binding and dissociation. To overcome the formulation barrier of VS1, we engineered carrier-free, albumin-coated nanocrystals through sonication-assisted nanocrystallization followed by surfactant exchange with human serum albumin. The resulting rod-shaped nanocrystals displayed nanometric size, narrow size distribution, sustained release, and improved aqueous dispersibility, increasing the apparent solubility of VS1 by more than 14-fold while preserving its molecular integrity and crystallinity. Biologically, VS1 selectively potentiated 5-fluorouracil (5-FU) in CRC cells, with synergistic effects restricted to 5-FU-sensitive models and associated with enhanced reactive oxygen species accumulation. Albumin-coated formulation retained the chemosensitizing activity of the free compound. In HCT-116 xenografts, combined treatment with albumin-coated VS1 nanocrystals and 5-FU significantly reduced tumor growth, prolonged tumor doubling time, and increased intratumoral necrosis without exacerbating systemic toxicity. Together, these findings establish that albumin-coated nanocrystals can overcome the delivery limitations of an insoluble STARD3 inhibitor and provide a formulation-enabled strategy to enhance fluoropyrimidine therapy in colorectal cancer. HIGHLIGHTSO_LIA 2.1 [A] crystal structure shows VS1 in the STARD3 sterol-binding cavity C_LIO_LIAlbumin-coated nanocrystals make the insoluble STARD3 inhibitor VS1 deliverable C_LIO_LINanocrystallization raises VS1 apparent solubility >14-fold with sustained release C_LIO_LIVS1 synergizes with 5-FU selectively in 5-FU-sensitive colorectal cancer lines C_LIO_LIAlbumin-nanocrystal VS1 plus 5-FU curbs xenograft growth without added toxicity C_LI

cancer biology↗

bioRxiv2025

Active site tyrosine residues in human NQO1 homodimer are critical for non-synchronous enzyme catalysis at the two active sites.

AbstractHuman NQO1 is a flavoenzyme essential for the redox metabolization of many substances and associated with wide-impacting diseases such as cancer and Alzheime[r]s. Recent X-ray crystallographic studies have proposed that a few residues at the active site of NQO1 (including Tyr126 and Tyr128) may control enzyme catalysis and functional negative cooperativity. In this work, we use rapid mixing pre-steady state kinetics and hydrogen-deuterium exchange followed by mass spectrometry (HDX-MS) to evaluate experimentally the role of Tyr126 and Tyr128 in NQO1 functionality by generating mutants to Phe, Ala and Glu. Mutations to Phe caused mild effects, whereas those to Ala significantly decreased hydride transfer efficiency and those to Glu virtually abolished NQO1 activity. Interestingly, structural stability studies by HDX-MS showed significant perturbations particularly affecting the binding site of NADH/NAD+ in the less conservative mutations (particularly to Glu). Mutations of Tyr126 and Tyr128 seem to also modulate the non-synchronous catalysis in the two active sites (negative cooperativity) as well as the selectivity for NADH/NADPH as coenzymes. Our work experimentally demonstrates the critical role of Tyr126 and Tyr128 in the flavin reductive half-reaction of the catalytic cycle of NQO1 in the negative cooperativity, and also suggests that phosphorylation of these two Tyr residues might shut down NQO1 activity reversibly.

biophysics↗

bioRxiv2022

Effect of naturally-occurring mutations on the stability and function of cancer-associated NQO1: comparison of experiments and computation.

Recent advances in DNA sequencing technologies are revealing a large variability in the human genome. Our capacity to establish genotype-phenotype correlations in such large-scale is, however, limited. This task is particularly challenging due to the multifunctional nature of proteins. Here we describe an extensive analysis of the stability and function of naturally-occurring variants (found in the COSMIC and gnomAD databases) of the cancer-associated human NAD(P)H: quinone oxidoreductase 1 (NQO1). First, we performed in silico saturation mutagenesis studies (>5000 substitutions) aimed to identify regions in NQO1 important for stability and function. We then experimentally characterized twenty-two naturally-occurring variants in terms of protein levels during bacterial expression, solubility, thermal stability and coenzyme binding. These studies showed a good overall correlation between experimental analysis and computational predictions; also the magnitude of the effects of the substitutions are similarly distributed in variants from the COSMIC and gnomAD databases. Outliers in these experimental-computational genotype-phenotype correlations remain, and we discuss these on the grounds and limitations of our approaches. Our work represents a further step to characterize the mutational landscape of NQO1 in the human genome and may help to improve high-throughput in silico tools for genotype-phenotype correlations in multifunctional proteins associated with disease.

biophysics↗