Peptides Derived from Histone 3 and Modified at Position 18 Inhibit Histone Demethylase KDM6 Enzymes.

The KDM6 subfamily of histone lysine demethylases has recently been implicated as a putative target in the treatment of a number of diseases; this makes the availability of potent and selective inhibitors important. Due to high sequence similarity of the catalytic domain of Jumonji C histone demethylases, the development of small-molecule, family-specific inhibitors has, however, proven challenging. One approach to achieve the selective inhibition of these enzymes is the use of peptides derived from the substrate, the histone 3 C terminus. Here we used computational methods to optimize such inhibitors of the KDM6 family. Through natural amino acid substitution, it is shown that a K18I variant of a histone H3 derived peptide significantly increases affinity towards the KDM6 enzymes. The crystal structure of KDM6B in complex with a histone 3 derived K18I peptide reveals a tighter fit of the isoleucine side chain, compared with that of the arginine. As a consequence, the peptide R17 residue also has increased hydrophilic interactions. These interactions of the optimized peptide are likely to be responsible for the increased affinity to the KDM6 enzymes.

Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen-Deuterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation.

Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helices 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the impact of the new disulfide bond on the conformational dynamics of this new hGH variant. Compared to wild type hGH, the variant exhibits reduced loop dynamics, indicating a stabilizing effect of the introduced disulfide bond. Furthermore, the disulfide bond exhibits longer ranging effects, stabilizing a short α-helix quite distant from the mutation sites, but also rendering a part of the α-helical hGH core slightly more dynamic. In the regions where the hGH variant exhibits a different deuterium uptake than the wild type protein, electron transfer dissociation (ETD) fragmentation has been used to pinpoint the residues responsible for the observed differences (HDX-ETD). Finally, by use of surface plasmon resonance (SPR) measurements, we show that the new disulfide bond does not compromise receptor affinity. Our work highlight the analytical potential of HDX-ETD combined with functional assays to guide protein engineering.

Mechanism of the N-hydroxylation of primary and secondary amines by cytochrome P450.

Cytochrome P450 enzymes (CYPs) metabolize alkyl- and arylamines, generating several different products. For the primary and secondary amines, some of these reactions result in hydroxylated amines, which may be toxic. Thus, when designing new drugs containing amine groups, it is important to be able to predict if a given compound will be a substrate for CYPs, in order to avoid toxic metabolites, and hence to understand the mechanism that is utilized by CYPs. Two possible mechanisms, for the N-hydroxylation of primary and secondary amines mediated by CYPs, are studied by density functional theory (DFT) for four different amines (aniline, N-methylaniline, propan-2-amine, and dimethylamine). The hydrogen abstraction and rebound mechanism is found to be preferred over a direct oxygen transfer mechanism for all four amines. However, in contrast to the same mechanism for the hydroxylation of aliphatic carbon atoms, the rebound step is shown to be rate-limiting in most cases.