Analysis of polyphenolic metabolites from in vitro gastrointestinal digested soft fruit extracts identify malvidin-3-glucoside as an inhibitor of PTP1B

Acknowledgements We are grateful to the Scottish Government Rural and Environment Science and Analytical Services (RESAS), the University of Aberdeen and Nutricia Research Foundation for funding. We thank Graham Horgan from Biomathematics and Statistics Scotland for their assistance with the statistical analysis. We thank Gordon J. McDougall and Rex M. Brennan, from The James Hutton Institute for supplying the soft fruits.Peer reviewedPostprin

Binding-Site Assessment by Virtual Fragment Screening

The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules) would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock ∼11000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors

Computational Insight into Protein Tyrosine Phosphatase 1B Inhibition: A Case Study of the Combined Ligand- and Structure-Based Approach

Protein tyrosine phosphatase 1B (PTP1B) is an attractive target for treating cancer, obesity, and type 2 diabetes. In our work, the way of combined ligand- and structure-based approach was applied to analyze the characteristics of PTP1B enzyme and its interaction with competitive inhibitors. Firstly, the pharmacophore model of PTP1B inhibitors was built based on the common feature of sixteen compounds. It was found that the pharmacophore model consisted of five chemical features: one aromatic ring (R) region, two hydrophobic (H) groups, and two hydrogen bond acceptors (A). To further elucidate the binding modes of these inhibitors with PTP1B active sites, four docking programs (AutoDock 4.0, AutoDock Vina 1.0, standard precision (SP) Glide 9.7, and extra precision (XP) Glide 9.7) were used. The characteristics of the active sites were then described by the conformations of the docking results. In conclusion, a combination of various pharmacophore features and the integration information of structure activity relationship (SAR) can be used to design novel potent PTP1B inhibitors

Computational and experimental druggability assessment of human DNA glycosylases

Due to a polar or even charged binding interface, DNA-binding proteins are considered extraordinarily difficult targets for development of small-molecule ligands and only a handful of proteins have been targeted successfully to date. Recently, however, it has been shown that development of selective and efficient inhibitors of 8-oxoguanine DNA glycosylase is possible. Here, we describe the initial druggability assessment of DNA glycosylases in a computational setting and experimentally investigate several methods to target endonuclease VIII-like 1 (NEIL1) with small-molecule inhibitors. We find that DNA glycosylases exhibit good predicted druggability in both DNA-bound and -unbound states. Furthermore, we find catalytic sites to be highly flexible, allowing for a range of interactions and binding partners. One flexible catalytic site was rationalized for NEIL1 and further investigated experimentally using both a biochemical assay in the presence of DNA and a thermal shift assay in the absence of DNA

TIES 20: Relative Binding Free Energy with a Flexible Superimposition Algorithm and Partial Ring Morphing

The TIES (Thermodynamic Integration with Enhanced Sampling) protocol is a formally exact alchemical approach in computational chemistry to the calculation of relative binding free energies. The validity of TIES relies on the correctness of matching atoms across compared pairs of ligands, laying the foundation for the transformation along an alchemical pathway. We implement a flexible topology superimposition algorithm which uses an exhaustive joint-traversal for computing the largest common component(s). The algorithm is employed to enable matching and morphing of partial rings in the TIES protocol along with a validation study using 55 transformations and five different proteins from our previous work. We find that TIES 20 with the RESP charge system, using the new superimposition algorithm, reproduces the previous results with mean unsigned error of 0.75 kcal/mol with respect to the experimental data. Enabling the morphing of partial rings decreases the size of the alchemical region in the dual-topology transformations resulting in a significant improvement in the prediction precision. We find that increasing the ensemble size from 5 to 20 replicas per λ window only has a minimal impact on the accuracy. However, the non-normal nature of the relative free energy distributions underscores the importance of ensemble simulation. We further compare the results with the AM1-BCC charge system and show that it improves agreement with the experimental data by slightly over 10%. This improvement is partly due to AM1-BCC affecting only the charges of the atoms local to the mutation, which translates to even fewer morphed atoms, consequently reducing issues with sampling and therefore ensemble averaging. TIES 20, in conjunction with the enablement of ring morphing, reduces the size of the alchemical region and significantly improves the precision of the predicted free energies

An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering

Abstract: Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here, we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small- molecule fragment soaks. New modeling approaches reveal ’hidden’ low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function

Applications of nuclear magnetic resonance spectroscopy: from drug discovery to protein structure and dynamics.

The versatility of nuclear magnetic resonance (NMR) spectroscopy is apparent when presented with diverse applications to which it can contribute. Here, NMR is used i) as a screening/ validation tool for a drug discovery program targeting the Phosphatase of Regenerating Liver 3 (PRL3), ii) to characterize the conformational heterogeneity of p53 regulator, Murine Double Minute X (MDMX), and iii) to characterize the solution dynamics of guanosine monophosphate kinase (GMPK). Mounting evidence suggesting roles for PRL3 in oncogenesis and metastasis has catapulted it into prominence as a cancer drug target. Yet, despite significant efforts, there are no PRL3 small molecule inhibitors currently in clinical trials. This work combines screening of an FDA-approved drug panel and the identification of binders by protein-observed NMR. FDA-approved drugs salirasib and candesartan were identified as potent inhibitors in in vitro inhibition and migration assays while a weak inhibitor, olsalazine, was identified by NMR as the first small molecule inhibitor to directly bind PRL3. NMR was also used to validate the binding of additional compounds identified as experimental PRL3 inhibitors. Thienopyridone, a potent experimental inhibitor, did not show direct binding to PRL3 but instead inhibited phosphatase activity via redox mechanism. NMR also revealed that other experimental inhibitors did not engage PRL3. Thus, there remains a need to identify potent PRL3-directed inhibitors. Meanwhile, molecular modeling revealed a putative druggable site that has not been thoroughly explored before. The current study provides some scaffolds such as candesartan and particularly, olsalazine, the only binder identified, that could be the starting point of further drug discovery efforts, as well as a putative site that can be targeted in silico. MDMX, a negative regulator of p53, is another important therapeutic target in cancer, along with the homologous protein, MDM2. Inhibitors that block the MDM2-p53 interaction have been identified and despite similarities in the binding site of these homologous proteins, these inhibitors are ineffective against MDMX. It is hypothesized that the flexibility of MDMX contributes to this significant difference in response to inhibitors, despite comparable affinity to their endogenous target, p53. Examination of available inhibitor-bound structures of MDMX reveal a conserved pharmacophore but the structures adopt distinct conformations away from the binding site. This implies that global motions of the protein might contribute to molecular recognition. The conformational heterogeneity in MDMX was further confirmed by collecting residual dipolar couplings (RDCs). Further investigations on both MDMX and MDM2 are necessary to uncover whether the flexibility of MDMX contributes to the differential binding to inhibitors. Finally, NMR relaxation methods and state-of-the-art high-power Carr-Purcell-Meiboom Gill (CPMG) relaxation dispersion measurements, the first documented application on an enzyme, were used to characterize the solution dynamics of GMPK and the changes in dynamics upon GMP binding. Substrate binding resulted in restricting the amplitudes of motion for backbone amide bonds within the picosecond-nanosecond timescale. Meanwhile, CPMG showed dispersion in both in the absence and presence of GMP, such that substrate binding did not quench dynamics within the microsecond-millisecond timescale. Interestingly, more residues are observed to have dispersion in the bound form, some near the C-terminal of helix 3, which has previously been proposed to be involved in product release. Current studies show that substrate binding affect different timescales of protein motion. Future work shall follow how motions within different timescales are affected as GMPK processes its substrates – such as, for instance, binding of ATP analogs within the ATP binding site or simultaneous occupancy of both substrate binding pockets. This paves the way for a complete picture of the relationship of function and dynamics in the conformational enzymatic cycle of a bi-substrate enzyme using GMPK as a model. The current work illustrates some of the diverse applications of NMR on three unique systems that are also drug targets. Information collected here can be leveraged on future structure and dynamics studies as well as drug discovery efforts targeting any of these proteins