Challenges in distinguishing functional proteins from polyproteins in databases: implications for drug discovery.

This opinion article addresses a major issue in molecular biology and drug discovery by highlighting the complications that arise from combining polyproteins and their functional products within the same database entry. This problem, exemplified by the discovery of novel inhibitors for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease, has an influence on our ability to retrieve precise data and hinders the development of targeted therapies. It also emphasizes the need for improved database practices and underscores their significance in advancing scientific research. Furthermore, it emphasizes the need of learning from the SARS-CoV-2 pandemic in order to improve global preparedness for future health crises.

Mining large databases to find new leads with low similarity to known actives: application to find new DPP-IV inhibitors.

Aim: Fragment-based drug design or bioisosteric replacement is used to find new actives with low (or no) similarity to existing ones but requires the synthesis of nonexisting compounds to prove their predicted bioactivity. Protein-ligand docking or pharmacophore screening are alternatives but they can become computationally expensive when applied to very large databases such as ZINC. Therefore, fast strategies are necessary to find new leads in such databases. Materials & methods: We designed a computational strategy to find lead molecules with very low (or no) similarity to existing actives and applied it to DPP-IV. Results: The bioactivity assays confirm that this strategy finds new leads for DPP-IV inhibitors. Conclusion: This computational strategy reduces the time of finding new lead molecules.

Activity and selectivity cliffs for DPP-IV inhibitors: Lessons we can learn from SAR studies and their application to virtual screening.

The inhibition of dipeptidyl peptidase-IV (DPP-IV) has emerged over the last decade as one of the most effective treatments for type 2 diabetes mellitus, and consequently (a) 11 DPP-IV inhibitors have been on the market since 2006 (three in 2015), and (b) 74 noncovalent complexes involving human DPP-IV and drug-like inhibitors are available at the Protein Data Bank (PDB). The present review aims to (a) explain the most important activity cliffs for DPP-IV noncovalent inhibition according to the binding site structure of DPP-IV, (b) explain the most important selectivity cliffs for DPP-IV noncovalent inhibition in comparison with other related enzymes (i.e., DPP8 and DPP9), and (c) use the information deriving from this activity/selectivity cliff analysis to suggest how virtual screening protocols might be improved to favor the early identification of potent and selective DPP-IV inhibitors in molecular databases (because they have not succeeded in identifying selective DPP-IV inhibitors with IC50 ≤ 100 nM). All these goals are achieved with the help of available homology models for DPP8 and DPP9 and an analysis of the structure-activity studies used to develop the noncovalent inhibitors that form part of some of the complexes with human DPP-IV available at the PDB.

Resveratrol Potently Counteracts Quercetin Starvation-Induced Autophagy and Sensitizes HepG2 Cancer Cells to Apoptosis.

SCOPE: Resveratrol (RSV) has been described as a potent antioxidant, antisteatotic, and antitumor compound, and it has also been identified as a potent autophagy inducer. On the other hand, quercetin (QCT) is a dietary flavonoid with known antitumor, anti-inflammatory, and antidiabetic effects. Additionally, QCT increases autophagy. To study the hypothetical synergistic effect of both compounds, we test the combined effect of QCT and RSV on the autophagy process in HepG2 cells. METHODS AND RESULTS: Autophagy is studied by western blotting, real-time RT-PCR, and cellular staining. Our results clearly indicate a bifunctional molecular effect of RSV. Both polyphenols are individually able to promote autophagy. Strikingly, when RSV is combined with QCT, it promotes a potent reduction of QCT-induced autophagy and influences proapoptotic signaling. CONCLUSION: RSV acts differentially on the autophagic process depending on the cellular energetic state. We further characterize the molecular mechanisms related to this effect, and we observe that AMP-activated protein kinase (AMPK) phosphorylation, heme oxygenase 1 (HO-1) downregulation, lysosomal membrane permeabilization (LMP), and Zinc (Zn2+ ) dynamics could be important modulators of such RSV-related effects and could globally represent a promising strategy to sensitize cancer cells to QCT treatment.

Ephedrine as a lead compound for the development of new DPP-IV inhibitors.

AIM: Extracts from Ephedra species have been reported to be effective as antidiabetics. A previous in silico study predicted that ephedrine and five ephedrine derivatives could contribute to the described antidiabetic effect of Ephedra extracts by inhibiting dipeptidyl peptidase IV (DPP-IV). Finding selective DPP-IV inhibitors is a current therapeutic strategy for Type 2 diabetes mellitus management. Therefore, the main aim of this work is to experimentally determine whether these alkaloids are DPP-IV inhibitors. Materials & methods: The DPP-IV inhibition of Ephedra's alkaloids was determined via a competitive-binding assay. Then, computational analyses were used in order to find out the protein-ligand interactions and to perform a lead optimization. RESULTS: Our results show that all six molecules are DPP-IV inhibitors, with IC50 ranging from 124 µM for ephedrine to 28 mM for N-methylpseudoephedrine. CONCLUSION: Further computational analysis shows how Ephedra's alkaloids could be used as promising lead molecules for designing more potent and selective DPP-IV inhibitors.

Tools for in silico target fishing.

Computational target fishing methods are designed to identify the most probable target of a query molecule. This process may allow the prediction of the bioactivity of a compound, the identification of the mode of action of known drugs, the detection of drug polypharmacology, drug repositioning or the prediction of the adverse effects of a compound. The large amount of information regarding the bioactivity of thousands of small molecules now allows the development of these types of methods. In recent years, we have witnessed the emergence of many methods for in silico target fishing. Most of these methods are based on the similarity principle, i.e., that similar molecules might bind to the same targets and have similar bioactivities. However, the difficult validation of target fishing methods hinders comparisons of the performance of each method. In this review, we describe the different methods developed for target prediction, the bioactivity databases most frequently used by these methods, and the publicly available programs and servers that enable non-specialist users to obtain these types of predictions. It is expected that target prediction will have a large impact on drug development and on the functional food industry.

Grape seed procyanidin extract reduces the endotoxic effects induced by lipopolysaccharide in rats.

Acute inflammation is a response to injury, infection, tissue damage, or shock. Bacterial lipopolysaccharide (LPS) is an endotoxin implicated in triggering sepsis and septic shock, and LPS promotes the inflammatory response, resulting in the secretion of proinflammatory and anti-inflammatory cytokines such as the interleukins (IL-6, IL-1ß, and IL-10) and tumor necrosis factor-α by the immune cells. Furthermore, nitric oxide and reactive oxygen species levels increase rapidly, which is partially due to the activation of inducible nitric oxide synthase in several tissues in response to inflammatory stimuli. Previous studies have shown that procyanidins, polyphenols present in foods such as apples, grapes, cocoa, and berries, have several beneficial properties against inflammation and oxidative stress using several in vitro and in vivo models. In this study, the anti-inflammatory and antioxidant effects of two physiological doses and two pharmaceutical doses of grape seed procyanidin extract (GSPE) were analyzed using a rat model of septic shock by the intraperitoneal injection of LPS derived from Escherichia coli. The high nutritional (75mg/kg/day) and the high pharmacological doses (200mg/kg/day) of GSPE showed anti-inflammatory effects by decreasing the proinflammatory marker NOx in the plasma, red blood cells, spleen, and liver. Moreover, the high pharmacological dose also downregulated the genes Il-6 and iNos; and the high nutritional dose decreased the glutathione ratio (GSSG/total glutathione), further illustrating the antioxidant capability of GSPE. In conclusion, several doses of GSPE can alleviate acute inflammation triggered by LPS in rats at the systemic and local levels when administered for as few as 15 days before the injection of endotoxin.

Identification of novel human dipeptidyl peptidase-IV inhibitors of natural origin (Part II): in silico prediction in antidiabetic extracts.

BACKGROUND: Natural extracts play an important role in traditional medicines for the treatment of diabetes mellitus and are also an essential resource for new drug discovery. Dipeptidyl peptidase IV (DPP-IV) inhibitors are potential candidates for the treatment of type 2 diabetes mellitus, and the effectiveness of certain antidiabetic extracts of natural origin could be, at least partially, explained by the inhibition of DPP-IV. METHODOLOGY/PRINCIPAL FINDINGS: Using an initial set of 29,779 natural products that are annotated with their natural source and an experimentally validated virtual screening procedure previously developed in our lab (Guasch et al.; 2012) [1], we have predicted 12 potential DPP-IV inhibitors from 12 different plant extracts that are known to have antidiabetic activity. Seven of these molecules are identical or similar to molecules with described antidiabetic activity (although their role as DPP-IV inhibitors has not been suggested as an explanation for their bioactivity). Therefore, it is plausible that these 12 molecules could be responsible, at least in part, for the antidiabetic activity of these extracts through their inhibitory effect on DPP-IV. In addition, we also identified as potential DPP-IV inhibitors 6 molecules from 6 different plants with no described antidiabetic activity but that share the same genus as plants with known antidiabetic properties. Moreover, none of the 18 molecules that we predicted as DPP-IV inhibitors exhibits chemical similarity with a group of 2,342 known DPP-IV inhibitors. CONCLUSIONS/SIGNIFICANCE: Our study identified 18 potential DPP-IV inhibitors in 18 different plant extracts (12 of these plants have known antidiabetic properties, whereas, for the remaining 6, antidiabetic activity has been reported for other plant species from the same genus). Moreover, none of the 18 molecules exhibits chemical similarity with a large group of known DPP-IV inhibitors.

Identification of novel human dipeptidyl peptidase-IV inhibitors of natural origin (part I): virtual screening and activity assays.

BACKGROUND: There has been great interest in determining whether natural products show biological activity toward protein targets of pharmacological relevance. One target of particular interest is DPP-IV whose most important substrates are incretins that, among other beneficial effects, stimulates insulin biosynthesis and secretion. Incretins have very short half-lives because of their rapid degradation by DPP-IV and, therefore, inhibiting this enzyme improves glucose homeostasis. As a result, DPP-IV inhibitors are of considerable interest to the pharmaceutical industry. The main goals of this study were (a) to develop a virtual screening process to identify potential DPP-IV inhibitors of natural origin; (b) to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits; and (c) to use the most active hit for predicting derivatives with higher binding affinities for the DPP-IV binding site. METHODOLOGY/PRINCIPAL FINDINGS: We predicted that 446 out of the 89,165 molecules present in the natural products subset of the ZINC database would inhibit DPP-IV with good ADMET properties. Notably, when these 446 molecules were merged with 2,342 known DPP-IV inhibitors and the resulting set was classified into 50 clusters according to chemical similarity, there were 12 clusters that contained only natural products for which no DPP-IV inhibitory activity has been previously reported. Nine molecules from 7 of these 12 clusters were then selected for in vitro activity testing and 7 out of the 9 molecules were shown to inhibit DPP-IV (where the remaining two molecules could not be solubilized, preventing the evaluation of their DPP-IV inhibitory activity). Then, the hit with the highest activity was used as a lead compound in the prediction of more potent derivatives. CONCLUSIONS/SIGNIFICANCE: We have demonstrated that our virtual-screening protocol was successful in identifying novel lead compounds for developing more potent DPP-IV inhibitors.

Grape seed-derived procyanidins decrease dipeptidyl-peptidase 4 activity and expression.

Dipeptidyl-peptidase 4 (DPP4) inhibitors are among the newest treatments against type 2 diabetes. Since some flavonoids modulate DPP4 activity, we evaluated whether grape seed-derived procyanidins (GSPEs), which are antihyperglycemic, modulate DPP4 activity and/or expression. In vitro inhibition assays showed that GSPEs inhibit pure DPP4. Chronic GSPE treatments in intestinal human cells (Caco-2) showed a decrease of DPP4 activity and gene expression. GSPE was also assayed in vivo. Intestinal but not plasmatic DPP4 activity and gene expression were decreased by GSPE in healthy and diet-induced obese animals. Healthy rats also showed glycemia improvement after oral glucose consumption but not after an intraperitoneal glucose challenge. In genetically obese rats, only DPP4 gene expression was down-regulated. Thus, procyanidin inhibition of intestinal DPP4 activity, either directly and/or via gene expression down-regulation, could be responsible for some of their effects in glucose homeostasis.

Development of docking-based 3D-QSAR models for PPARgamma full agonists.

Peroxisome proliferator-activated receptor gamma (PPARγ) has become an attractive molecular target for drugs that aim to treat diabetes mellitus type II, and its therapeutic potency against skin cancer and other skin diseases is also currently being explored. To study the relationship between the structure of several PPARγ full agonists and the trans-activation activity of PPARγ, we have performed a three-dimensional quantitative structure-activity relationship (3D-QSAR) study of tyrosine-based derivatives, based on the 3D alignment of conformations obtained by docking. Highly predictive 3D-QSAR models, with Pearson-R values of 0.86 and 0.90, were obtained for the transactivation activity and binding affinity of PPARγ, respectively. These models are in good agreement with the structural characteristics of the binding pocket of PPARγ and provide some structural insights for the improvement of PPARγ full agonist bioactivities.

Procyanidins modify insulinemia by affecting insulin production and degradation.

Previous studies from our research group have suggested that procyanidins modify glycemia and insulinemia. The aim of this work was to evaluate the effects of procyanidins on ß-cell functionality in a nonpathological system. Four groups of healthy rats were studied. The animals were given daily acute doses of grape seed procyanidin extract (GSPE) for different time periods and at different daily amounts. A ß-cell line (INS-1E) was treated with 25 mg GSPE/L for 24 h to identify possible mechanisms of action for the procyanidins. In vivo experiments showed that different doses of GSPE affected insulinemia in different ways by modifying ß-cell functionality and/or insulin degradation. The islets isolated from rats that were treated with 25 mg GSPE/kg of body weight for 45 days exhibited a limited response to glucose stimulation. In addition, insulin gene expression, insulin synthesis and expression of genes related to insulin secretion were all down-regulated. In vitro studies revealed that GSPE decreased the ability of ß-cells to secrete insulin in response to glucose. GSPE increased glucose uptake in ß-cells under high-glucose conditions but impaired glucose-induced mitochondrial hyperpolarization, decreased adenosine triphosphate (ATP) synthesis and altered cellular membrane potentials. GSPE also modified Glut2, glucokinase and Ucp2 gene expression as well as altered the expression of hepatic insulin-degrading enzyme (Ide), thereby altering insulin degradation. At some doses, procyanidins changed ß-cell functionality by modifying insulin synthesis, secretion and degradation under nonpathological conditions. Membrane potentials and Ide provide putative targets for procyanidins to induce these effects.

Different papillomaviruses have different repertoires of transcription factor binding sites: convergence and divergence in the upstream regulatory region.

BACKGROUND: Papillomaviruses (PVs) infect stratified squamous epithelia in warm-blooded vertebrates and have undergone a complex evolutionary process. The control of the expression of the early ORFs in PVs depends on the binding of cellular and viral transcription factors to the upstream regulatory region (URR) of the virus. It is believed that there is a core of transcription factor binding sites (TFBS) common to all PVs, with additional individual differences, although most of the available information focuses only on a handful of viruses. RESULTS: We have studied the URR of sixty-one PVs, covering twenty different hosts. We have predicted the TFBS present in the URR and analysed these results by principal component analysis and genetic algorithms. The number and nature of TFBS in the URR might be much broader than thus far described, and different PVs have different repertoires of TFBS. CONCLUSION: There are common fingerprints in the URR in PVs that infect primates, although the ancestors of these viruses diverged a long time ago. Additionally, there are obvious differences between the URR of alpha and beta PVs, despite these PVs infect similar histological cell types in the same host, i.e. human. A thorough analysis of the TFBS in the URR might provide crucial information about the differential biology of cancer-associated PVs.

Papillomaviruses: different genes have different histories.

Papillomaviruses (PVs) infect stratified squamous epithelia in vertebrates. Some PVs are associated with different types of cancer and with certain benign lesions. It has been assumed that PVs coevolved with their hosts. However, recently it has been shown that different regions of the genome have different evolutionary histories. The PV genome has a modular nature and appeared after the addition of pre-existent blocks. This order of appearance in the PV genome is evident today in the different evolutionary rates of the different genes, with new genes--E5, E6 and E7--diverging faster than old genes--E1, E2, L2 and L1. Here, we propose an evolutionary framework aiming to integrate genome evolution, PV biology and epidemiology of PV infections.