Tautomerism of Warfarin: Combined Chemoinformatics, Quantum Chemical, and NMR Investigation

Warfarin, an important anticoagulant drug, can exist in solution in 40 distinct tautomeric forms through both prototropic tautomerism and ring–chain tautomerism. We have investigated all warfarin tautomers with computational and NMR approaches. Relative energies calculated at the B3LYP/6-311G++(d,p) level of theory indicate that the 4-hydroxycoumarin cyclic hemiketal tautomer is the most stable tautomer in aqueous solution, followed by the 4-hydroxycoumarin open-chain tautomer. This is in agreement with our NMR experiments where the spectral assignments indicate that warfarin exists mainly as a mixture of cyclic hemiketal diastereomers, with an open-chain tautomer as a minor component. We present a diagram of the interconversion of warfarin created taking into account the calculated equilibrium constants (p<i>K</i>_T) for all tautomeric reactions. These findings help with gaining further understanding of proton transfer and ring closure tautomerization processes. We also discuss the results in the context of chemoinformatics rules for handling tautomerism

Enumeration of Ring–Chain Tautomers Based on SMIRKS Rules

A compound exhibits (prototropic) tautomerism if it can be represented by two or more structures that are related by a formal intramolecular movement of a hydrogen atom from one heavy atom position to another. When the movement of the proton is accompanied by the opening or closing of a ring it is called ring–chain tautomerism. This type of tautomerism is well observed in carbohydrates, but it also occurs in other molecules such as warfarin. In this work, we present an approach that allows for the generation of all ring–chain tautomers of a given chemical structure. Based on Baldwin’s Rules estimating the likelihood of ring closure reactions to occur, we have defined a set of transform rules covering the majority of ring–chain tautomerism cases. The rules automatically detect substructures in a given compound that can undergo a ring–chain tautomeric transformation. Each transformation is encoded in SMIRKS line notation. All work was implemented in the chemoinformatics toolkit CACTVS. We report on the application of our ring–chain tautomerism rules to a large database of commercially available screening samples in order to identify ring–chain tautomers

Identification of PPARgamma Partial Agonists of Natural Origin (II): In Silico Prediction in Natural Extracts with Known Antidiabetic Activity

<div><h3>Background</h3><p>Natural extracts have played an important role in the prevention and treatment of diseases and are important sources for drug discovery. However, to be effectively used in these processes, natural extracts must be characterized through the identification of their active compounds and their modes of action.</p> <h3>Methodology/Principal Findings</h3><p>From an initial set of 29,779 natural products that are annotated with their natural source and using a previously developed virtual screening procedure (carefully validated experimentally), we have predicted as potential peroxisome proliferators-activated receptor gamma (PPARγ) partial agonists 12 molecules from 11 extracts known to have antidiabetic activity. Six of these molecules are similar to molecules with described antidiabetic activity but whose mechanism of action is unknown. Therefore, it is plausible that these 12 molecules could be the bioactive molecules responsible, at least in part, for the antidiabetic activity of the extracts containing them. In addition, we have also identified as potential PPARγ partial agonists 10 molecules from 16 plants with undescribed antidiabetic activity but that are related (<em>i.e.</em>, they are from the same genus) to plants with known antidiabetic properties. None of the 22 molecules that we predict as PPARγ partial agonists show chemical similarity with a group of 211 known PPARγ partial agonists obtained from the literature.</p> <h3>Conclusions/Significance</h3><p>Our results provide a new hypothesis about the active molecules of natural extracts with antidiabetic properties and their mode of action. We also suggest plants with undescribed antidiabetic activity that may contain PPARγ partial agonists. These plants represent a new source of potential antidiabetic extracts. Consequently, our work opens the door to the discovery of new antidiabetic extracts and molecules that can be of use, for instance, in the design of new antidiabetic drugs or functional foods focused towards the prevention/treatment of type 2 Diabetes Mellitus.</p> </div

Natural extracts with described antidiabetic activity that contain one molecule that is predicted to be a PPARγ partial agonist by our virtual screening protocol.

<p>The Table shows the natural extracts (<i>i.e.</i>, third column) and the VS hits that have been purified from them (identified by their common name, when available, and CAS number). The bibliographic references for each extract are split in three columns where (a) the fifth column reports papers that describe the purification of each molecule from the corresponding extract; (b) the sixth column reports papers that describe the antidiabetic activity of the corresponding extract; and (c) the seventh column reports papers that describe the antidiabetic activity of the corresponding molecule or similar molecules (when available). The second column represents the number of the cluster that each molecule belongs when they were compared with a group of 211 synthetic PPARγ partial agonists. The 2D structures of the molecules of this table can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055889#pone.0055889.s001" target="_blank">Figure S1</a>.</p

Natural extracts that contain one molecule predicted to be a PPARγ partial agonist by our VS protocol and that are related to natural extracts that are described to have antidiabetic activity.

<p>The Table shows the natural extracts (<i>i.e.</i>, third column) and the VS hits that have been purified from them (identified by their common name, when available, and CAS number) and that are the related to extracts with described antidiabetic activity (<i>i.e.</i>, sixth column). The bibliographic references for each extract are split in three columns where (a) the fifth column reports papers that describe the purification of each molecule from the corresponding extract; (b) the seventh column reports papers that describe the antidiabetic activity of the related extract (see sixth column); and (c) the eighth column reports papers that describe the antidiabetic activity of the corresponding or similar molecules (when available). The second column represents the number of the cluster to which each molecule belongs when they were compared with a group of 211 synthetic PPARγ partial agonists. The 2D structures of the molecules of this table can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055889#pone.0055889.s001" target="_blank">Figure S1</a>.</p

Characterizing the Covalent Targets of a Small Molecule Inhibitor of the Lysine Acetyltransferase P300

C646 inhibits the lysine acetyltransferases (KATs) p300 and CBP and represents the most potent and selective small molecule KAT inhibitor identified to date. To gain insights into the cellular activity of this epigenetic probe, we applied chemoproteomics to identify covalent targets of the C646 chemotype. Modeling and synthetic derivatization was used to develop a clickable analogue (C646-yne) that inhibits p300 similarly to the parent compound and enables enrichment of bound proteins. LC–MS/MS identified the major covalent targets of C646-yne as highly abundant cysteine-containing proteins, and follow-up studies found that C646 can inhibit tubulin polymerization in vitro. Finally, we provide evidence that thiol reactivity of C646 may limit its ability to antagonize acetylation in cells. These findings should enable a more precise interpretation of studies utilizing C646 as a chemical probe of KAT activity and suggest that an underappreciated liability of electrophile-containing inhibitors is a reduction in their cellular potency due to consumption by abundant protein and metabolite thiol sinks

Validation and application of the Virtual Screening (VS) workflow.

<p>A dataset of 211 known PPARγ partial agonists and 3,122 decoys extracted from the DUD database were used to validate our VS workflow. Once the VS was validated, it was applied to a dataset of 29,779 natural products (NPs). The numbers represent the number of compounds from each set that <i>survived</i> each step when applied sequentially.</p

Chemical comparison between molecules that we predict as PPARγ partial agonists and molecules with described antidiabetic activity.

<p>Each row represents the comparison of the 2D chemical structure between a molecule predicted as a PPARγ partial agonist through our VS workflow and a similar molecule that has been described to present antidiabetic activity.</p

Experimental and Chemoinformatics Study of Tautomerism in a Database of Commercially Available Screening Samples

We investigated how many cases of the same chemical sold as different products (at possibly different prices) occurred in a prototypical large aggregated database and simultaneously tested the tautomerism definitions in the chemoinformatics toolkit CACTVS. We applied the standard CACTVS tautomeric transforms plus a set of recently developed ring–chain transforms to the Aldrich Market Select (AMS) database of 6 million screening samples and building blocks. In 30 000 cases, two or more AMS products were found to be just different tautomeric forms of the same compound. We purchased and analyzed 166 such tautomer pairs and triplets by ¹H and ¹³C NMR to determine whether the CACTVS transforms accurately predicted what is the same “stuff in the bottle”. Essentially all prototropic transforms with examples in the AMS were confirmed. Some of the ring–chain transforms were found to be too “aggressive”, i.e. to equate structures with one another that were different compounds

Peroxisome Proliferator-Activated Receptor γ (PPARγ) and Ligand Choreography: Newcomers Take the Stage

Thiazolidinediones (TZDs), such as rosiglitazone and pioglitazone, are peroxisome proliferator-activated receptor γ (PPARγ) full agonists that have been widely used in the treatment of type 2 diabetes mellitus. Despite the demonstrated beneficial effect of reducing glucose levels in the plasma, TZDs also induce several adverse effects. Consequently, the search for new compounds with potent antidiabetic effects but fewer undesired effects is an active field of research. Interestingly, the novel proposed mechanisms for the antidiabetic activity of PPARγ agonists, consisting of PPARγ Ser273 phosphorylation inhibition, ligand and receptor mutual dynamics, and the presence of an alternate binding site, have recently changed the view regarding the optimal characteristics for the screening of novel PPARγ ligands. Furthermore, transcriptional genomics could bring essential information about the genome-wide effects of PPARγ ligands. Consequently, facing the new mechanistic scenario proposed for these compounds is essential for resolving the paradoxes among their agonistic function, antidiabetic activities, and side effects and should allow the rational development of better and safer PPARγ-mediated antidiabetic drugs