The association of bound aldehyde content with bioprosthetic tissue calcification.

The rapid progression of mineralization seen in glutaraldehyde-treated valves has prompted a wide variety of secondary treatments aimed at mitigating dystrophic calcification. We tested the hypothesis that aldehyde residuals bound to bioprosthetic tissue is a significant promoter of calcification. We developed a novel assay to measure residual aldehyde functional groups and assessed aldehyde content in three different groups: glutaraldehyde-fixed tissue (Glut-only), Edwards ThermaFix™ treated tissue and Edwards RESILIA™ tissue. The amount of tissue calcification in these same groups was assessed in vivo using a well-established rabbit model, in which tissue samples were implanted intramuscularly for 60 days. The aldehyde content of the Glut-only, ThermaFix™ treated and RESILIA™ tissues were 225.7 ± 31.5, 101.9 ± 79.7 and 32.5 ± 48.4 nmol/g, respectively. The differences among all three groups were highly significant (p < 0.001, Student's unpaired t test). The median (interquartile range) calcium content of the Glut-only, ThermaFix™ treated and RESILIA™ tissues were 227.4 (221.8-243.6), 101.0 (23.05-169.6), and 10.1 (0.28-51.7) µg/mg. The differences among all three groups were highly significant (p < 0.001, Mann-Whitney U test). The results indicated that our novel assay was able to reliably measure aldehyde content in bovine pericardial tissue. Furthermore, there appeared to be a close association between aldehyde content and tissue calcium content. The processing of bioprosthetic valves to reduce their aldehyde content may offer a significant advantage in terms of reducing the potential for long-term calcification in human implants.

Inhibition of prenylated KRAS in a lipid environment.

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.