A non-clinical and clinical IUCLID database for 530 pharmaceuticals (part I): Methodological aspects of its development.

A new IUCLID database is provided containing results from non-clinical animal studies and human information for 530 approved drugs. The database was developed by extracting data from pharmacological reviews of repeat-dose, carcinogenicity, developmental, and reproductive toxicity studies. In the database, observed and no-observed effects are linked to the respective effect levels, including information on severity/incidence and transiency/reversibility. It also includes some information on effects in humans, that were extracted from relevant sections of standard product labels of the approved drugs. The database is complemented with a specific ontology for reporting effects that was developed as an improved version of the Ontology Lookup Service's mammalian and human phenotype ontologies and includes different hierarchical levels. The developed ontology contains novel and unique standardized terms, including ontological terms for reproductive and endocrine effects. The database aims to facilitate correlation and concordance analyses based on the link between observed and no-observed effects and their respective effect levels. In addition, it offers a robust dataset on drug information for the pharmaceutical industry and research. The reported ontology supports the analyses of toxicological information, especially for reproductive and endocrine endpoints and can be used to encode legacy data or develop additional ontologies. The new database and ontology can be used to support the development of alternative non-animal approaches, to elucidate mechanisms of toxicity, and to analyse human relevance. The new IUCLID database is provided free of charge at https://iuclid6.echa.europa.eu/us-fda-toxicity-data.

Discovery of the 4-aminopiperidine-based compound EM127 for the site-specific covalent inhibition of SMYD3.

Recent findings support the hypothesis that inhibition of SMYD3 methyltransferase may be a therapeutic avenue for some of the deadliest cancer types. Herein, active site-selective covalent SMYD3 inhibitors were designed by introducing an appropriate reactive cysteine trap into reversible first-generation SMYD3 inhibitors. The 4-aminopiperidine derivative EM127 (11C) bearing a 2-chloroethanoyl group as reactive warhead showed selectivity for Cys186, located in the substrate/histone binding pocket. Selectivity towards Cys186 was retained even at high inhibitor/enzyme ratio, as shown by mass spectrometry. The mode of interaction with the SMYD3 substrate/histone binding pocket was revealed by crystallographic studies. In enzymatic assays, 11C showed a stronger SMYD3 inhibitory effect compared to the reference inhibitor EPZ031686. Remarkably, 11C attenuated the proliferation of MDA-MB-231 breast cancer cell line at the same low micromolar range of concentrations that reduced SMYD3 mediated ERK signaling in HCT116 colorectal cancer and MDA-MB-231 breast cancer cells. Furthermore, 11C (5 µM) strongly decreased the steady-state mRNA levels of genes important for tumor biology such as cyclin dependent kinase 2, c-MET, N-cadherin and fibronectin 1, all known to be regulated, at least in part, by SMYD3. Thus, 11C is as a first example of second generation SMYD3 inhibitors; this agent represents a covalent and a site specific SMYD3 binder capable of potent and prolonged attenuation of methyltransferase activity.

Identification of NAPRT Inhibitors with Anti-Cancer Properties by In Silico Drug Discovery.

Depriving cancer cells of sufficient NAD levels, mainly through interfering with their NAD-producing capacity, has been conceived as a promising anti-cancer strategy. Numerous inhibitors of the NAD-producing enzyme, nicotinamide phosphoribosyltransferase (NAMPT), have been developed over the past two decades. However, their limited anti-cancer activity in clinical trials raised the possibility that cancer cells may also exploit alternative NAD-producing enzymes. Recent studies show the relevance of nicotinic acid phosphoribosyltransferase (NAPRT), the rate-limiting enzyme of the Preiss-Handler NAD-production pathway for a large group of human cancers. We demonstrated that the NAPRT inhibitor 2-hydroxynicotinic acid (2-HNA) cooperates with the NAMPT inhibitor FK866 in killing NAPRT-proficient cancer cells that were otherwise insensitive to FK866 alone. Despite this emerging relevance of NAPRT as a potential target in cancer therapy, very few NAPRT inhibitors exist. Starting from a high-throughput virtual screening approach, we were able to identify and annotate two additional chemical scaffolds that function as NAPRT inhibitors. These compounds show comparable anti-cancer activity to 2-HNA and improved predicted aqueous solubility, in addition to demonstrating favorable drug-like profiles.

Differential modulation of SIRT6 deacetylase and deacylase activities by lysine-based small molecules.

Sirtuin 6 (SIRT6) is an NAD+-dependent deacetylase regulating important functions: modulators of its enzymatic activity have been considered as possible therapeutic agents. Besides the deacetylase activity, SIRT6 also has NAD+-dependent deacylase activity, whereby it regulates the secretion of cytokines and proteins. We identified novel SIRT6 modulators with a lysine-based structure: compound 1 enhances SIRT6 deacylase while inhibiting the deacetylase activity. As expected based on the biological effects of SIRT6 deacetylase activity, compound 1 increased histone 3 lysine 9 acetylation and the activity of glycolytic enzymes. Moreover, the fact that compound 1 enhanced SIRT6 deacylase activity was accompanied by an increased TNF-α release. In conclusion, new SIRT6 modulators with a lysine-like structure were identified, with differential effects on specific SIRT6 activities. The novel SIRT6 modulator concomitantly inhibits deacetylase and enhances deacylase activity.

SIRT6 inhibitors with salicylate-like structure show immunosuppressive and chemosensitizing effects.

The NAD+-dependent deacetylase SIRT6 is an emerging cancer drug target, whose inhibition sensitizes cancer cells to chemo-radiotherapy and has pro-differentiating effects. Here we report on the identification of novel SIRT6 inhibitors with a salicylate-based structure. The new SIRT6 inhibitors show improved potency and specificity compared to the hit inhibitor identified in an in silico compound screen. As predicted based on SIRT6 biological roles, the new leads increase histone 3 lysine 9 acetylation and glucose uptake in cultured cells, while blocking TNF-α production and T lymphocyte proliferation. Notably, the new SIRT6 inhibitors effectively sensitize pancreatic cancer cells to gemcitabine. Finally, studies of compound fingerprinting and pharmacokinetics defined the drug-like properties of one of the new SIRT6 inhibitors, potentially allowing for subsequent in vivo proof-of-concept studies. In conclusion, new SIRT6 inhibitors with a salicylate-like structure were identified, which are active in cells and could potentially find applications in disease conditions, including cancer and immune-mediated disorders.

Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

Sirtuin 6 (SIRT6) is a sirtuin family member involved in a wide range of physiologic and disease processes, including cancer and glucose homeostasis. Based on the roles played by SIRT6 in different organs, including its ability to repress the expression of glucose transporters and glycolytic enzymes, inhibiting SIRT6 has been proposed as an approach for treating type 2 diabetes mellitus (T2DM). However, so far, the lack of small-molecule Sirt6 inhibitors has hampered the conduct of in vivo studies to assess the viability of this strategy. We took advantage of a recently identified SIRT6 inhibitor, compound 1, to study the effect of pharmacological Sirt6 inhibition in a mouse model of T2DM (i.e., in high-fat-diet-fed animals). The administration of the Sirt6 inhibitor for 10 d was well tolerated and improved oral glucose tolerance, it increased the expression of the glucose transporters GLUT1 and -4 in the muscle and enhanced the activity of the glycolytic pathway. Sirt6 inhibition also resulted in reduced insulin, triglycerides, and cholesterol levels in plasma. This study represents the first in vivo study of a SIRT6 inhibitor and provides the proof-of-concept that targeting SIRT6 may be a viable strategy for improving glycemic control in T2DM.-Sociali, G., Magnone, M., Ravera, S., Damonte, P., Vigliarolo, T., Von Holtey, M., Vellone, V. G., Millo, E., Caffa, I., Cea, M., Parenti, M. D., Del Rio, A., Murone, M., Mostoslavsky, R., Grozio, A., Nencioni, A., Bruzzone S. Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

1,4-Substituted Triazoles as Nonsteroidal Anti-Androgens for Prostate Cancer Treatment.

Prostate cancer (PC) is the fifth leading cause of cancer death in men, and the androgen receptor (AR) represents the primary target for PC treatment, even though the disease frequently progresses toward androgen-independent forms. Most of the commercially available nonsteroidal antiandrogens show a common scaffold consisting of two aromatic rings connected by a linear or a cyclic spacer. By taking advantage of a facile, one-pot click chemistry reaction, we report herein the preparation of a small library of novel 1,4-substituted triazoles with AR antagonistic activity. Biological and theoretical evaluation demonstrated that the introduction of the triazole core in the scaffold of nonsteroidal antiandrogens allowed the development of small molecules with improved overall AR-antagonist activity. In fact, compound 14d displayed promising in vitro antitumor activity toward three different prostate cancer cell lines and was able to induce 60% tumor growth inhibition of the CW22Rv1 in vivo xenograft model. These results represent a step toward the development of novel and improved AR antagonists.

Synthesis and biological characterization of 3-(imidazol-1-ylmethyl)piperidine sulfonamides as aromatase inhibitors.

The most frequently used treatment for hormone receptor positive breast cancer in post-menopausal women are aromatase inhibitors. In order to develop new aromatase inhibitors, we designed and synthesized new imidazolylmethylpiperidine sulfonamides using the structure of the previously identified aromatase inhibitor SYN 20028567 as starting lead. By this approach, three new aromatase inhibitors with IC50 values that are similar to that of letrozole and SYN 20028567 were identified.

Quinazolinedione SIRT6 inhibitors sensitize cancer cells to chemotherapeutics.

The NAD(+)-dependent sirtuin SIRT6 is highly expressed in human breast, prostate, and skin cancer where it mediates resistance to cytotoxic agents and prevents differentiation. Thus, SIRT6 is an attractive target for the development of new anticancer agents to be used alone or in combination with chemo- or radiotherapy. Here we report on the identification of novel quinazolinedione compounds with inhibitory activity on SIRT6. As predicted based on SIRT6's biological functions, the identified new SIRT6 inhibitors increase histone H3 lysine 9 acetylation, reduce TNF-α production and increase glucose uptake in cultured cells. In addition, these compounds exacerbate DNA damage and cell death in response to the PARP inhibitor olaparib in BRCA2-deficient Capan-1 cells and cooperate with gemcitabine to the killing of pancreatic cancer cells. In conclusion, new SIRT6 inhibitors with a quinazolinedione-based structure have been identified which are active in cells and could potentially find applications in cancer treatment.

Selectivity hot-spots of sirtuin catalytic cores.

Sirtuins are NAD(+)-dependent deacetylases with several biological roles in DNA regulation, genomic stability, metabolism, longevity and immune cell functions. Numerous disease conditions are linked to sirtuins including metabolic disorders, inflammatory and autoimmune processes and cancer. Although few specific small molecule modulators have been reported to date, the need to identify selective ligands would be crucial not only for the development of active pharmaceutical ingredients for new targeted therapies but also as a tool for dissecting the biological roles of sirtuin family members. Herein, we report a comprehensive study aimed to classify and identify the selectivity hot-spots for targeting the catalytic cores of human sirtuins using small molecule modulators. Our selectivity analysis suggests that catalytic cores can be divided into different clusters that can constitute the basis for the development of selective ligands. The ensemble of hot-spot information is expected to be helpful to devise new selective chemicals targeting sirtuin family members.

Discovery of novel and selective SIRT6 inhibitors.

SIRT6 is an NAD(+)-dependent deacetylase with a role in the transcriptional control of metabolism and aging but also in genome stability and inflammation. Broad therapeutic applications are foreseen for SIRT6 inhibitors, including uses in diabetes, immune-mediated disorders, and cancer. Here we report on the identification of the first selective SIRT6 inhibitors by in silico screening. The most promising leads show micromolar IC50s, have significant selectivity for SIRT6 versus SIRT1 and SIRT2, and are active in cells, as shown by increased acetylation at SIRT6 target lysines on histone 3, reduced TNF-α secretion, GLUT-1 upregulation, and increased glucose uptake. Taken together, these results show the value of these compounds as starting leads for the development of new SIRT6-targeting therapeutic agents.

Rejuvenating sirtuins: the rise of a new family of cancer drug targets.

Sirtuins are a family of NAD+-dependent enzymes that was proposed to control organismal life span about a decade ago. While such role of sirtuins is now debated, mounting evidence involves these enzymes in numerous physiological processes and disease conditions, including metabolism, nutritional behavior, circadian rhythm, but also inflammation and cancer. SIRT1, SIRT2, SIRT3, SIRT6, and SIRT7 have all been linked to carcinogenesis either as tumor suppressor or as cancer promoting proteins. Here, we review the biological rationale for the search of sirtuin inhibitors and activators for treating cancer and the experimental approaches to their identification.

Immunoproteasome in cancer and neuropathologies: a new therapeutic target?

Immunoproteasome is an emerging biological target that constitutes a key element not only in antigen presentation but also in T cell and cytokine regulation as well as cellular homeostasis. Respect to standard proteasome, the inducible expression and different sensitivity towards activity modulators of immunoproteasome render it a potential therapeutic target for tumours and central nervous system diseases. In this review we report the cutting edge studies for understanding when immunoproteasome expression is induced and how it regulates pivotal pathways involved in tumours and neuropathologies, including apoptosis and inflammation. We emphasize its role as a new pharmacological target by describing the recent medicinal chemistry efforts aimed at design selective small-molecule modulators of both standard- and immuno-proteasome forms. Finally, we also present an in silico model of the human immunoproteasome structure by the major molecular differences with the 20S standard proteasome and discuss the perspective for the design of novel specific smallmolecule modulators for the different proteasome isoforms.

Modulation of epigenetic targets for anticancer therapy: clinicopathological relevance, structural data and drug discovery perspectives.

Research on cancer epigenetics has flourished in the last decade. Nevertheless growing evidence point on the importance to understand the mechanisms by which epigenetic changes regulate the genesis and progression of cancer growth. Several epigenetic targets have been discovered and are currently under validation for new anticancer therapies. Drug discovery approaches aiming to target these epigenetic enzymes with small-molecules inhibitors have produced the first pre-clinical and clinical outcomes and many other compounds are now entering the pipeline as new candidate epidrugs. The most studied targets can be ascribed to histone deacetylases and DNA methyltransferases, although several other classes of enzymes are able to operate post-translational modifications to histone tails are also likely to represent new frontiers for therapeutic interventions. By acknowledging that the field of cancer epigenetics is evolving with an impressive rate of new findings, with this review we aim to provide a current overview of pre-clinical applications of smallmolecules for cancer pathologies, combining them with the current knowledge of epigenetic targets in terms of available structural data and drug design perspectives.

Advances and applications of binding affinity prediction methods in drug discovery.

Nowadays, the improvement of R&D productivity is the primary commitment in pharmaceutical research, both in big pharma and smaller biotech companies. To reduce costs, to speed up the discovery process and to increase the chance of success, advanced methods of rational drug design are very helpful, as demonstrated by several successful applications. Among these, computational methods able to predict the binding affinity of small molecules to specific biological targets are of special interest because they can accelerate the discovery of new hit compounds. Here we provide an overview of the most widely used methods in the field of binding affinity prediction, as well as of our own work in developing BEAR, an innovative methodology specifically devised to overtake some limitations in existing approaches. The BEAR method was successfully validated against different biological targets, and proved its efficacy in retrieving active compounds from virtual screening campaigns. The results obtained so far indicate that BEAR may become a leading tool in the drug discovery pipeline. We primarily discuss advantages and drawbacks of each technique and show relevant examples and applications in drug discovery.

BEAR, a novel virtual screening methodology for drug discovery.

BEAR (binding estimation after refinement) is a new virtual screening technology based on the conformational refinement of docking poses through molecular dynamics and prediction of binding free energies using accurate scoring functions. Here, the authors report the results of an extensive benchmark of the BEAR performance in identifying a smaller subset of known inhibitors seeded in a large (1.5 million) database of compounds. BEAR performance proved strikingly better if compared with standard docking screening methods. The validations performed so far showed that BEAR is a reliable tool for drug discovery. It is fast, modular, and automated, and it can be applied to virtual screenings against any biological target with known structure and any database of compounds.

A computational workflow for the design of irreversible inhibitors of protein kinases.

Design of irreversible inhibitors is an emerging and relatively less explored strategy for the design of protein kinase inhibitors. In this paper, we present a computational workflow that was specifically conceived to assist such design. The workflow takes the form of a multi-step procedure that includes: the creation of a database of already known reversible inhibitors of protein kinases, the selection of the most promising scaffolds that bind one or more desired kinase templates, the modification of the scaffolds by introduction of chemically reactive groups (suitable cysteine traps) and the final evaluation of the reversible and irreversible protein-ligand complexes with molecular dynamics simulations and binding free energy predictions. Most of these steps were automated. In order to prove that this is viable, the workflow was tested on a database of known inhibitors of ERK2, a protein kinase possessing a cysteine in the ATP site. The modeled ERK2-ligand complexes and the values of the estimated binding free energies of the putative ligands provide useful indicators of their aptitude to bind reversibly and irreversibly to the protein kinase. Moreover, the computational data are used to rank the ligands according to their computed binding free energies and their ability to bind specific protein residues in the reversible and irreversible complexes, thereby providing a useful decision-making tool for each step of the design. In this work we present the overall procedure and the first proof of concept results.

Three-dimensional quantitative structure-activity relationship analysis of a set of Plasmodium falciparum dihydrofolate reductase inhibitors using a pharmacophore generation approach.

A 3D pharmacophore model able to quantitatively predict inhibition constants was derived for a series of inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR), a validated target for antimalarial therapy. The data set included 52 inhibitors, with 23 of these comprising the training set and 29 an external test set. The activity range, expressed as Ki, of the training set molecules was from 0.3 to 11 300 nM. The 3D pharmacophore, generated with the HypoGen module of Catalyst 4.7, consisted of two hydrogen bond donors, one positive ionizable feature, one hydrophobic aliphatic feature, and one hydrophobic aromatic feature and provided a 3D-QSAR model with a correlation coefficient of 0.954. Importantly, the type and spatial location of the chemical features encoded in the pharmacophore were in full agreement with the key binding interactions of PfDHFR inhibitors as previously established by molecular modeling and crystallography of enzyme-inhibitor complexes. The model was validated using several techniques, namely, Fisher's randomization test using CatScramble, leave-one-out test to ensure that the QSAR model is not strictly dependent on one particular compound of the training set, and activity prediction in an external test set of compounds. In addition, the pharmacophore was able to correctly classify as active and inactive the dihydrofolate reductase and aldose reductase inhibitors extracted from the MDDR database, respectively. This test was performed in order to challenge the predictive ability of the pharmacophore with two classes of inhibitors that target very different binding sites. Molecular diversity of the data sets was finally estimated by means of the Tanimoto approach. The results obtained provide confidence for the utility of the pharmacophore in the virtual screening of libraries and databases of compounds to discover novel PfDHFR inhibitors.

Structure of Plasmodium vivax dihydrofolate reductase determined by homology modeling and molecular dynamics refinement.

The structure of Plasmodium vivax dihydrofolate reductase (PvDHFR), a potentially important target for antimalarial chemotherapy, was determined by means of homology modeling and molecular dynamics refinement. The structure proved to be consistent with DHFRs of known crystal structure. The comparison of the complexes of the antifolate inhibitor pyrimethamine bound at the active sites of PvDHFR and PfDHFR, the related enzyme from Plasmodium falciparum, prospected the possibility of using structure-based drug design to develop inhibitors that are effective against both malarial enzymes. This study constitutes a first step toward understanding of the antifolate-PvDHFR molecular interactions and possible rationalization of resistance in vivax malaria.

Docking and database screening reveal new classes of Plasmodium falciparum dihydrofolate reductase inhibitors.

Plasmodium falciparum dihydrofolate reductase (PfDHFR) is an important target for antimalarial chemotherapy. Unfortunately, the emergence of resistant parasites has significantly reduced the efficiency of classical antifolate drugs such as cycloguanil and pyrimethamine. In this study, an approach toward molecular docking of the structures contained in the Available Chemicals Directory (ACD) database to search for novel inhibitors of PfDHFR is described. Instead of docking the whole ACD database, specific 3D pharmacophores were used to reduce the number of molecules in the database by excluding a priori molecules lacking essential requisites for the interaction with the enzyme and potentially unable to bind to resistant mutant PfDHFRs. The molecules in the resulting "focused" database were then evaluated with regard to their fit into the PfDHFR active site. Twelve new compounds whose structures are completely unrelated to known antifolates were identified and found to inhibit, at the micromolar level, the wild-type and resistant mutant PfDHFRs harboring A16V, S108T, A16V + S108T, C59R + S108N + I164L, and N51I + C59R + S108N + I164L mutations. Depending on the functional groups interacting with key active site residues of the enzyme, these inhibitors were classified as N-hydroxyamidine, hydrazine, urea, and thiourea derivatives. The structures of the complexes of the most active inhibitors, as refined by molecular mechanics and molecular dynamics, provided insight into how these inhibitors bind to the enzyme and suggested prospects for these novel derivatives as potential leads for antimalarial development.