Non-innocuous Consequences of Metabolic Oxidation of Alkyls on Arenes.

Reactive metabolite (RM) formation is widely accepted as playing a pivotal role in causing adverse idiosyncratic drug reactions, with most attention paid to drug-induced liver injury. Mechanisms of RM formation are determined by the drug's properties in relation to human enzymes transforming the drug. This Perspective focuses on enzymatic oxidation of alkyl groups on aromatics leading to quinone methides and benzylic alcohol sulfates as RMs, a topic that has not received very much attention. Unlike previous overviews, we will include in our Perspective several fulvene-like methides such as 3-methyleneindole. We also speculate that a few older drugs may form non-reported methides of this class. In addition, we report a few guiding DFT calculations of changes in free energy on going from a benzylic alcohol to the corresponding methide. Particularly facile reactions of 2-aminothiazole-5-methanol and 4-aminobenzyl alcohol are noted.

Discovery of USP7 small-molecule allosteric inhibitors.

Ubiquitin specific protease-7 (USP7) is considered an attractive target for cancer therapy by promoting degradation of the tumor suppressor p53 and negatively affecting the immune response to tumors. However, the development of selective non-covalent USP7 inhibitors has proven challenging. In this work we report the NMR characterization of a weak binder from SPR screening of an in-house fragment library which reveals that it binds to the allosteric palm site of the catalytic domain. Molecular modeling combined with 1HNMR saturation transfer difference and NOESY experiments enabled structure-based design of additional compounds showing IC50 values in the low-micromolar range with good selectivity over the closest homolog USP47. The most potent analogue represents a promising starting point for the development of novel, selective USP7 inhibitors.

The optimal DFT approach in DP4 NMR structure analysis - pushing the limits of relative configuration elucidation.

What computational methods should be used to achieve the most reliable result in computational structure elucidation? A study on the effect of quality and quantity of geometries on computational NMR structure elucidation performance is reported. Semi-empirical, HF and DFT methods were explored, and B3LYP optimized geometries in combination with mPW1PW91 shifts and M06-2X conformer energies was found to be best. The required number of conformers considered has also been investigated, as well as several methods for the reduction of this number. Clear guidelines for the best computational NMR structure elucidation methods for different levels of available computing power are provided.

Confident application of a global human liver microsomal activity QSAR.

Metabolic stability is an important property of drug candidates and pharmaceutical companies often have human liver microsomal (HLM) data for a large number of molecules, enabling development of global quantitative structure-activity relationship models. RESULTS: This study describes a strategy for building a global HLM quantitative structure-activity relationship model, applicable also to datasets of limited size. By using external congeneric test sets, a realistic description of the performance in the future applied setting and a reliable prediction confidence method is obtained. CONCLUSION: The limited ability of the HLM model to generalize in chemical space to show the importance of internal model development and continuous updating of global HLM models, as well as the importance of a validated prediction confidence method.

Expanding DP4: application to drug compounds and automation.

The DP4 parameter, which provides a confidence level for NMR assignment, has been widely used to help assign the structures of many stereochemically-rich molecules. We present an improved version of the procedure, which can be downloaded as Python script instead of running within a web-browser, and which analyses output from open-source molecular modelling programs (TINKER and NWChem) in addition to being able to use output from commercial packages (Schrodinger's Macromodel and Jaguar; Gaussian). The new open-source workflow incorporates a method for the automatic generation of diastereomers using InChI strings and has been tested on a range of new structures. This improved workflow permits the rapid and convenient computational elucidation of structure and relative stereochemistry.

Design and synthesis of HIV-1 protease inhibitors for a long-acting injectable drug application.

The design and synthesis of novel HIV-1 protease inhibitors (PIs) (1-22), which display high potency against HIV-1 wild-type and multi-PI-resistant HIV-mutant clinical isolates, is described. Lead optimization was initiated from compound 1, a Phe-Phe hydroxyethylene peptidomimetic PI, and was directed towards the discovery of new PIs suitable for a long-acting (LA) injectable drug application. Introducing a heterocyclic 6-methoxy-3-pyridinyl or a 6-(dimethylamino)-3-pyridinyl moiety (R(3)) at the para-position of the P1' benzyl fragment generated compounds with antiviral potency in the low single digit nanomolar range. Halogenation or alkylation of the metabolic hot spots on the various aromatic rings resulted in PIs with high stability against degradation in human liver microsomes and low plasma clearance in rats. Replacing the chromanolamine moiety (R(1)) in the P2 protease binding site by a cyclopentanolamine or a cyclohexanolamine derivative provided a series of high clearance PIs (16-22) with EC(50)s on wild-type HIV-1 in the range of 0.8-1.8 nM. PIs 18 and 22, formulated as nanosuspensions, showed gradual but sustained and complete release from the injection site over two months in rats, and were therefore identified as interesting candidates for a LA injectable drug application for treating HIV/AIDS.

Connectivity and binding-site recognition: applications relevant to drug design.

Here, we describe a family of methods based on residue-residue connectivity for characterizing binding sites and apply variants of the method to various types of protein-ligand complexes including proteases, allosteric-binding sites, correctly and incorrectly docked poses, and inhibitors of protein-protein interactions. Residues within ligand-binding sites have about 25% more contact neighbors than surface residues in general; high-connectivity residues are found in contact with the ligand in 84% of all complexes studied. In addition, a k-means algorithm was developed that may be useful for identifying potential binding sites with no obvious geometric or connectivity features. The analysis was primarily carried out on 61 protein-ligand structures from the MEROPS protease database, 250 protein-ligand structures from the PDBSelect (25%), and 30 protein-protein complexes. Analysis of four proteases with crystal structures for multiple bound ligands has shown that residues with high connectivity tend to have less variable side-chain conformation. The relevance to drug design is discussed in terms of identifying allosteric-binding sites, distinguishing between alternative docked poses and designing protein interface inhibitors. Taken together, this data indicate that residue-residue connectivity is highly relevant to medicinal chemistry.

Closed loop folding units from structural alignments: experimental foldons revisited.

Nonoverlapping closed loops of around 25-35 amino acids formed via nonlocal interactions at the loop ends have been proposed as an important unit of protein structure. This hypothesis is significant as such short loops can fold quickly and so would not be bound by the Leventhal paradox, giving insight into the possible nature of the funnel in protein folding. Previously, these closed loops have been identified either by sequence analysis (conservation and autocorrelation) or studies of the geometry of individual proteins. Given the potential significance of the closed loop hypothesis, we have explored a new strategy for determining closed loops from the insertions identified by the structural alignment of proteins sharing the same overall fold. We determined the locations of the closed loops in 37 pairs of proteins and obtained excellent agreement with previously published closed loops. The relevance of NMR structures to closed loop determination is briefly discussed. For cytochrome c, cytochrome b(562) and triosephophate isomerase, independent folding units have been determined on the basis of hydrogen exchange experiments and misincorporation proton-alkyl exchange experiments. The correspondence between these experimentally derived foldons and the theoretically derived closed loops indicates that the closed loop hypothesis may provide a useful framework for analyzing such experimental data.

Design and synthesis of novel P2 substituents in diol-based HIV protease inhibitors.

The synthesis and SAR of HIV-1 protease inhibitors containing novel P2 structural elements are presented. The inhibitors were designed having hydrogen bond accepting P2 substituents to probe potential favorable interactions to Asp-29/Asp-30 of the HIV-1 protease backbone utilizing inhibitor 3 as a model template. Several inhibitors were synthesized from an L-Val methyl amide P2 motif by appending hydrogen bonding moieties from either the isopropyl side-chain or from the methyl amide portion. The most promising inhibitors 4a and 4e displayed Ki values of 1.0 nM and 0.7 nM respectively and EC50 values in the MT4 cell-based assay of 0.17 microM and 0.33 microM respectively, a slight loss in potency compared to lead inhibitor 3. These inhibitors were also tested against an HIV protease inhibitor resistant strain carrying the M46I, V82F, and I84V mutations. Inhibitors 4a and 4e displayed a 3 and 4 fold change respectively compared with HIV wild type, whereas lead inhibitor 3 showed a higher 9 fold change. This study further demonstrate the chemical tractability of the approach where various P2 substituents can be introduced in just one chemical step from lactone 21 enabling facile modifications of the overall properties in this inhibitor class.

Assessing the role of polarization in docking.

We describe a strategy for including ligand and protein polarization in docking that is based on the conversion of induced dipoles to induced charges. Induced charges have a distinct advantage in that they are readily implemented into a number of different computer programs, including many docking programs and hybrid QM/MM programs; induced charges are also more readily interpreted. In this study, the ligand was treated quantum mechanically to avoid parametrization issues and was polarized by the target protein, which was treated as a set of point charges. The induced dipole at a given target atom, due to polarization by the ligand and neighboring residues, was reformulated as induced charges at the given atom and its bonded neighbors, and these were allowed to repolarize the ligand in an iterative manner. The final set of polarized charges was evaluated in docking using AutoDock 4.0 on 12 protein-ligand systems against the default empirical Gasteiger charges, and against nonpolarized and partially polarized potential-derived charges. One advantage of AutoDock is that the best rmsd structure can be identified not only from the lowest energy pose but also from the largest cluster of poses. Inclusion of polarization does not always lead to the lowest energy pose having a lower rmsd, because docking is designed by necessity to be rapid rather than accurate. However, whenever an improvement in methodology, corresponding to a more thorough treatment of polarization, resulted in an increased cluster size, then there was also a corresponding decrease in the rmsd. The options for implementing polarization within a purely classical docking framework are discussed.

Toward a consistent treatment of polarization in model QM/MM calculations.

The concept of model chemistries within hybrid QM/MM calculations has been addressed through analysis of the polarization energy determined by two distinct approaches based on (i) induced charges and (ii) induced dipoles. The quantum mechanical polarization energy for four configurations of the water dimer has been determined for a range of basis sets using Morokuma energy decomposition analysis. This benchmark value has been compared to the fully classical polarization energy determined using the induced dipole approach, and the molecular mechanics polarization energy calculated using induced charges within the MM region of hybrid QM/MM calculations. From the water dimer calculations, it is concluded that the induced charge approach is consistent with medium sized basis set calculations whereas the induced dipole approach is consistent with large basis set calculations. This result is highly relevant to the concept of QM/MM model chemistries.

Quantitative measurement of protease ligand conformation.

The tendency for protease ligands to bind in an extended conformation has been suggested as an important factor for the identification of compounds of medicinal importance. Here we present a novel graph-theoretical method giving a quantitative measure of ligand conformation, and through application of this method to a representative set of protease ligands in bound and unbound conformations, derive the result that protease ligands are more extended in conformation when in their bound state.

Criteria for confirming sequence periodicity identified by Fourier transform analysis: application to GCR2, a candidate plant GPCR?

Methods to determine periodicity in protein sequences are useful for inferring function. Fourier transformation is one approach but care is required to ensure the periodicity is genuine. Here we have shown that empirically-derived statistical tables can be used as a measure of significance. Genuine protein sequences data rather than randomly generated sequences were used as the statistical backdrop. The method has been applied to G-protein coupled receptor (GPCR) sequences, by Fourier transformation of hydrophobicity values, codon frequencies and the extent of over-representation of codon pairs; the latter being related to translational step times. Genuine periodicity was observed in the hydrophobicity whereas the apparent periodicity (as inferred from previously reported measures) in the translation step times was not validated statistically. GCR2 has recently been proposed as the plant GPCR receptor for the hormone abscisic acid. It has homology to the Lanthionine synthetase C-like family of proteins, an observation confirmed by fold recognition. Application of the Fourier transform algorithm to the GCR2 family revealed strongly predicted seven fold periodicity in hydrophobicity, suggesting why GCR2 has been reported to be a GPCR, despite negative indications in most transmembrane prediction algorithms. The underlying multiple sequence alignment, also required for the Fourier transform analysis of periodicity, indicated that the hydrophobic regions around the 7 GXXG motifs commence near the C-terminal end of each of the 7 inner helices of the alpha-toroid and continue to the N-terminal region of the helix. The results clearly explain why GCR2 has been understandably but erroneously predicted to be a GPCR.

Activity of the isolated HIV RNase H domain and specific inhibition by N-hydroxyimides.

This report describes a procedure to generate enzymatically active, isolated HIV RNase H domain. In contrast to previously described preparations, the RNA cleavage activity of the untagged RNase H domain was surprisingly similar to that of the full-length HIV-RT protein. Signature cleavages at 18 and 9 nucleotides downstream of a recessed RNA 5'-end were retained with the isolated RNase H domain. Activity was strongly decreased by deletion of 3 amino acids from the C-terminus, consistent with an important structural or functional role of the C-terminal alpha-helix. A prototype N-hydroxyimide (2-hydroxy-4H-isoquinoline-1,3-dione) was found to inhibit the activity of the isolated HIV RNase H domain as well as the RNase H activity of full-length HIV reverse transcriptase. In contrast, the compound did not significantly inhibit the structurally closely related Escherichia coli RNase HI. Specific binding of N-hydroxyimide compounds to the isolated RNase H domain was observed by protein fluorescence quenching.

Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors.

Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.

Use of a pharmacophore model to discover a new class of influenza endonuclease inhibitors.

Data from both our own and literature studies of the biochemistry and inhibition of influenza virus endonuclease was combined with data on the mechanism of action and the likely active site mechanism to propose a pharmacophore. The pharmacophore was used to design a novel structural class of inhibitors, some of which were found to have activities similar to that of known influenza endonuclease inhibitors and were also antiviral in cell culture.