Cellular Active N-Hydroxyurea FEN1 Inhibitors Block Substrate Entry to the Active Site

The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the first crystal structure of inhibitor-bound hFEN1 and show a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein– substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the unpairing of substrate DNA necessary for reaction. Other compounds were more competitive with substrate. Cellular thermal shift data showed engagement of both inhibitor types with hFEN1 in cells with activation of the DNA damage response evident upon treatment. However, cellular EC50s were significantly higher than in vitro inhibition constants and the implications of this for exploitation of hFEN1 as a drug target are discussed

Determination of the association constant of strongly bonded host-guest systems by multivariate regression of infrared spectroscopic data

A new method applying infrared spectroscopy in combination with multivariate regression for the determination of association constants has been developed to overcome the sensitivity problems arising when strongly associating complexes are investigated by NMR or UV–VIS methods. A concentration profile for the complex is derived by correction of multivariate regression data. Subsequent iterative fitting of the corrected data yields the association constant.The regression part is not integrated in the process of the association constant determination itself. Separating data treatment from the actual fitting procedure offers a means of evaluating the quality of the data set and the order of association before the actual calculation of the association constant. Furthermore, the method of regression can be varied and the applicability of the selected method can be assessed from the data-regression output.From simulated data, it is estimated that an association constant range of 102–106M–1 can be determined when measuring at millimolar concentration levels.Examples are shown for halide complexation of a thiourea compound and a urea-substituted resorcinarene cavitand. \u

An infrared study of host-guest association in solution by substituted resorcinarene cavitands. Part II. Comparison of halide complexation by tetrathiourea cavitands and a simple thiourea

Binding of halide has been studied for two thioureido-substituted resorcin[4]arenes and a model compound, with special attention to the NH stretching region and the bonding behaviour. Mathematical techniques such as curve fitting, Fourier self-deconvolution and Partial Least Squares analysis have been applied to investigate the characteristics of bonding, and the results are correlated with information obtained from a steric analysis.It is shown that association of the halide guest takes place via hydrogen bonding between halide ions and thioureido moieties solely for all ligands. Compared to a simple thiourea compound, pre-organisation of the thioureide side-chains on the upper rim by intramolecular bonding enhances the capability of association by a factor of 10 to 100.An analogy is found between complexation of a halide anion in the cavitands and complexation of an iodide anion by the model compound. This suggests that within the cavitand, chloride, bromide and iodide are all complexed by at least two thioureido moieties, whereas a simple thiourea binds chloride and bromide in a 1-to-1 fashion, and iodide in a 2-to-1 fashion. \u

An infrared study of host-guest association in solution by substituted resorcinarene cavitands. Part I. Structural aspects of halide complexation by a tetraurea cavitand

Complexation of chloride, bromide and iodide ion was investigated for a resorcinarene cavitand substituted on its upper rim with four N-(o-nitrophenoxy-n-octyl)ureido groups. Association in chloroform takes place solely via hydrogen bonding by the urea moieties, and is monitored well by infrared spectroscopy. Association constants are high, about 104 M–1, and a small preference for chloride over bromide and iodide is observed.Upon binding of the anion, the array of weak intramolecular bonds is disrupted and replaced by hydrogen bonds towards the halide lone pairs. No significant reorganisation of the urea o-nitrophenoxy-n-octyl substituents was found, which suggests a re-ordering of the urea groups only upon binding. Co-operation of the bonding moieties is observed in the complexation of halide ions as a result of pre-organisation of the urea groups on the upper rim of the resorcinarene cavitand by weak hydrogen bonding interactions

Does a machine-learned potential perform better than an optimally tuned traditional force field?: a case study on fluorohydrins

We present a comparative study that evaluates the performance of a machine learning potential (ANI-2x), a conventional force field (GAFF), and an optimally tuned GAFF-like force field in the modeling of a set of 10 γ-fluorohydrins that exhibit a complex interplay between intra- and intermolecular interactions in determining conformer stability. To benchmark the performance of each molecular model, we evaluated their energetic, geometric, and sampling accuracies relative to quantum-mechanical data. This benchmark involved conformational analysis both in the gas phase and chloroform solution. We also assessed the performance of the aforementioned molecular models in estimating nuclear spin–spin coupling constants by comparing their predictions to experimental data available in chloroform. The results and discussion presented in this study demonstrate that ANI-2x tends to predict stronger-than-expected hydrogen bonding and overstabilize global minima and shows problems related to inadequate description of dispersion interactions. Furthermore, while ANI-2x is a viable model for modeling in the gas phase, conventional force fields still play an important role, especially for condensed-phase simulations. Overall, this study highlights the strengths and weaknesses of each model, providing guidelines for the use and future development of force fields and machine learning potentials

MTH1 Substrate Recognition--An Example of Specific Promiscuity.

MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1