Cristalización de proteínas en el diseño de fármacos en los últimos 50 años

We live in an era where we expect to be able to visit our doctor and obtain a pill to cure any ailment from which we suffer. Yet, this is still not the case. Many of the current cures are still derived from natural sources although new drugs are increasingly the result of intelligent design. In this process, X-ray protein crystallography now plays a major and effective role in the discovery of new treatments. The developments that have made this possible have evolved during the past fifty years. The methods for crystallizing macromolecules and determining their structures by X-ray crystallography have been automated and the speed for X-ray data acquisition is several orders of magnitude faster. Fifty years ago it took several years to solve a single structure. Now, several protein–ligand complexes can be determined in single day. High-throughput crystallography is considered to be a great asset to the drug discovery process, providing a fast way to tailor drug candidates to their targets by analysing their binding mode in detail. Crystallization remains the main challenge.Vivimos en una época en la que esperamos ir al médico y obtener una pastilla para curar cualquier dolencia que padezcamos; por desgracia, esta expectativa no es real. Aunque muchos de los remedios en uso provienen de fuentes naturales, la mayoría de los nuevos medicamentos son el resultado de la investigación científica. En el proceso de diseño y descubrimiento de fármacos, la cristalografía de proteínas juega un papel central. Los conocimientos que han hecho esto posible han venido evolucionando desde hace cincuenta años aproximadamente. Los métodos de cristalización de macromoléculas y la determinación de sus estructuras a través de la cristalografía de rayos X han sido automatizados y miniaturizados y la velocidad de la adquisición de datos de difracción ha aumentado en varios órdenes de magnitud. Si hace cincuenta años la resolución de una sola estructura podría llevar varios años, actualmente se pueden determinar las estructuras de varios complejos proteína-ligando en un solo día. La cristalografía de alto rendimiento hoy día es un gran recurso en el proceso del descubrimiento de fármacos pues proporciona una manera rápida y precisa de adaptar los fármacos candidatos a las dianas mediante el análisis de su modo de unión. La cristalización sigue siendo el principal desafío

Transthyretin complexes with curcumin and bromo-estradiol: Evaluation of solubilizing multicomponent mixtures

Crystallographic structure determination of protein–ligand complexes of transthyretin (TTR) has been hindered by the low affinity of many compounds that bind to the central cavity of the tetramer. Because crystallization trials are carried out at protein and ligand concentration that approach the millimolar range, low affinity is less of a problem than the poor solubility of many compounds that have been shown to inhibit amyloid fibril formation. To achieve complete occupancy in co-crystallization experiments, the minimal requirement is one ligand for each of the two sites within the TTR tetramer. Here we present a new strategy for the co-crystallization of TTR using high molecular weight polyethylene glycol instead of high ionic strength precipitants, with ligands solubilized in multicomponent mixtures of compounds. This strategy is applied to the crystallization of TTR complexes with curcumin and 16a-bromo-estradiol. Here we report the crystal structures with these compounds and with the ferulic acid that results from curcumin degradation

Halogen Bonding Controls Selectivity of FRET Substrate Probes for MMP-9

SummaryMatrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases that catalyze cleavage of extracellular matrix and nonmatrix proteins. MMPs play a role in tissue remodeling, and their uncontrolled activity is associated with number of diseases, including tumor metastasis. Thus, there is a need to develop methods to monitor MMP activity, and number of probes has been previously described. The key problem many probes encounter is the issue of selectivity, since 23 human MMPs, despite playing different physiological roles, have structurally similar active sites. Here, we introduce the halogen bonding concept into the probe design and show that the probe containing iodine exhibits an unprecedented selectivity for MMP-9. We provide structure-based explanation for the selectivity, confirming that it is due to formation of the halogen bond that supports catalysis, and we highlight the value of exploring halogen bonding in the context of selective probe design

Discovery of a new selective inhibitor of A Disintegrin And Metalloprotease 10 (ADAM-10) able to reduce the shedding of NKG2D ligands in Hodgkin's lymphoma cell models

Hodgkin's lymphoma (HL) is the most common malignant lymphoma in young adults in the western world. This disease is characterized by an overexpression of ADAM-10 with increased release of NKG2D ligands, involved in an impaired immune response against tumor cells. We designed and synthesized two new ADAM-10 selective inhibitors, 2 and 3 based on previously published ADAM-17 selective inhibitor 1. The most promising compound was the thiazolidine derivative 3, with nanomolar activity for ADAM-10, high selectivity over ADAM-17 and MMPs and good efficacy in reducing the shedding of NKG2D ligands (MIC-B and ULBP3) in three different HL cell lines at non-toxic doses. Molecular modeling studies were used to drive the design and X-ray crystallography studies were carried out to explain the selectivity of 3 for ADAM-10 over MMPs

Copper mediated amyloid-β binding to Transthyretin

Transthyretin (TTR), a homotetrameric protein that transports thyroxine and retinol both in plasma and in cerebrospinal (CSF) fluid provides a natural protective response against Alzheimer’s disease (AD), modulates amyloid-β (Aβ) deposition by direct interaction and co-localizes with Aβ in plaques. TTR levels are lower in the CSF of AD patients. Zn2+, Mn2+and Fe2+transform TTR into a protease able to cleave Aβ. To explain these activities, monomer dissociation or conformational changes have been suggested. Here, we report that when TTR crystals are exposed to copper or iron salts, the tetramer undergoes a significant conformational change that alters the dimer-dimer interface and rearranges residues implicated in TTR’s ability to neutralize Aβ. We also describe the conformational changes in TTR upon the binding of the various metal ions. Furthermore, using bio-layer interferometry (BLI) with immobilized Aβ(1–28), we observe the binding of TTR only in the presence of copper. Such Cu2+-dependent binding suggests a recognition mechanism whereby Cu2+modulates both the TTR conformation, induces a complementary Aβ structure and may participate in the interaction. Cu2+-soaked TTR crystals show a conformation different from that induced by Fe2+, and intriguingly, TTR crystals grown in presence of Aβ(1–28) show different positions for the copper sites from those grown its absence

Distribution of \u3ci\u3eBaylisascaris procyonis\u3c/i\u3e in Raccoons (\u3ci\u3eProcyon lotor\u3c/i\u3e) in Florida, USA

Baylisascaris procyonis, or raccoon roundworm, is an intestinal nematode parasite of raccoons (Procyon lotor) that is important to public and wildlife health. Historically, the parasite was uncommon in the southeastern US; however, the range of B. procyonis has expanded to include Florida, US. From 2010 to 2016, we opportunistically sampled 1,030 raccoons statewide. The overall prevalence was 3.7% (95% confidence interval=2.5–4.8%) of sampled individuals, and infection intensity ranged from 1 to 48 (mean±standard deviation 9.9±4.0). We found raccoon roundworm in 9/56 (16%) counties sampled, and the percent positive ranged from 1.1% to 13.3% of specimens collected per county. Including previously published data, B. procyonis was detected in 11 Florida counties. We used logistic regression to estimate the contribution of raccoon demographic variables and the presence of the endoparasite Macracanthorhynchus ingens to B. procyonis detection in Florida. Following the model selection process we found housing density, M. ingens presence, and urbanicity to be predictive of raccoon roundworm presence. We also found substantial among-county variation. Raccoon sex and age were not useful predictors. Public health officials, wildlife rehabilitators, wildlife managers, and others should consider any Florida raccoon to be potentially infected with B. procyonis, particularly in areas where housing density is high

Preterm birth is not associated with asymptomatic/mild SARS-CoV-2 infection per se: Pre-pregnancy state is what matters

Evidence for the real impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on preterm birth is unclear, as available series report composite pregnancy outcomes and/or do not stratify patients according to disease severity. The purpose of the research was to determine the real impact of asymptomatic/mild SARS-CoV-2 infection on preterm birth not due to maternal respiratory failure. This case-control study involved women admitted to Sant Anna Hospital, Turin, for delivery between 20 September 2020 and 9 January 2021. The cumulative incidence of Coronavirus disease-19 was compared between preterm birth (case group, n = 102) and full-term delivery (control group, n = 127). Only women with spontaneous or medically-indicated preterm birth because of placental vascular malperfusion (pregnancy-related hypertension and its complications) were included. Current or past SARS-CoV-2 infection was determined by nasopharyngeal swab testing and detection of IgM/IgG antibodies in blood samples. A significant difference in the cumulative incidence of Coronavirus disease-19 between the case (21/102, 20.5%) and the control group (32/127, 25.1%) (P= 0.50) was not observed, although the case group was burdened by a higher prevalence of three known risk factors (body mass index > 24.9, asthma, chronic hypertension) for severe Coronavirus disease-19. Logistic regression analysis showed that asymptomatic/mild SARS-CoV-2 infection was not an independent predictor of spontaneous and medically-indicated preterm birth due to pregnancy-related hypertension and its complications (0.77; 95% confidence interval, 0.41-1.43). Pregnant patients without comorbidities need to be reassured that asymptomatic/mild SARS-CoV-2 infection does not increase the risk of preterm delivery. Preterm birth and severe Coronavirus disease-19 share common risk factors (i.e., body mass index > 24.9, asthma, chronic hypertension), which may explain the high rate of indicated preterm birth due to maternal conditions reported in the literature