Role of ALDH2 in nitroglycerin bioactivation

Nitroglycerin (glyceryl trinitrate, GTN) has been used since more than 130 years for the therapy of coronary artery disease and other cardiovascular disorders. The clinical benefit of GTN and related organic nitrates results from dilation of large blood vessels, in particular coronary arteries and large veins, leading to improved blood supply to cardiac muscle and reduction of preload, respectively. GTN is a prodrug that becomes enzymatically bioactivated in vascular smooth muscle to yield nitric oxide (NO) which triggers cGMP-mediated vasodilation through activation of soluble guanylate cyclase (sGC). The key enzyme of GTN bioactivation is thought to be mitochondrial aldehyde dehydrogenase (ALDH2). Continuous exposure of ALDH2 to GTN results in oxidative inactivation of the enzyme, presumably explaining the development of nitrate tolerance, i.e. the loss of therapeutic effect of GTN upon long-term application. It is well documented that ALDH2 denitrates GTN to yield 1,2-glyceryl dinitrate and inorganic nitrite but the link between this clearance-based metabolism of GTN and bioactivation resulting in sGC activation is not well understood. Recently, we discovered that ALDH2 is able to reduce GTN directly to NO and identified a mutant of the enzyme (E268Q) which exhibited hyperactivity with respect to GTN bioactivation (sGC activation), even though it had lost all of its classical activities, including clearance-based denitration of GTN to 1,2-GDN and nitrite. These data suggest that ALDH2 catalyzes GTN biotransformation by two independent reactions: clearance- based 2-electron reduction yielding nitrite, and 3-electron reduction resulting in formation of NO. It is the aim of the present proposal to elucidate the molecular mechanisms underlying the two independent reactions of GTN biotransformation catalyzed by ALDH2 and to settle the in vivo relevance of NO formation. We will solve the 3-dimensional structures of wildtype ALDH2 and selected mutants of the protein with bound GTN to identify essential amino acid residues in the active site, test for a potential interference of classical GTN denitration with NO formation to explain the observed hyperactivity of the E268Q mutant, and study the molecular consequences of enzyme inactivation by GTN as well as possible pathways of reactivation. The in vivo relevance of NO formation will be addressed by measuring GTN reactivity and nitrate tolerance in ALDH2-deficient mice with vascular smooth muscle-specific overexpression of the hyperactive E268Q mutant. The proposed studies will provide new insights into the molecular mechanisms underlying GTN bioactivation and the development of vascular nitrate tolerance.

Nitroglycerin (glyceryl trinitrate, GTN) has been used since more than 130 years for the therapy of coronary artery disease and other cardiovascular disorders. The clinical benefit of GTN and related organic nitrates results from dilation of large blood vessels, in particular coronary arteries and large veins, leading to improved blood supply to cardiac muscle and reduction of preload, respectively. GTN is a prodrug that becomes enzymatically bioactivated in vascular smooth muscle to yield nitric oxide (NO) which triggers cGMP-mediated vasodilation through activation of soluble guanylate cyclase (sGC). The key enzyme of GTN bioactivation is thought to be mitochondrial aldehyde dehydrogenase (ALDH2). Continuous exposure of ALDH2 to GTN results in oxidative inactivation of the enzyme, presumably explaining the development of nitrate tolerance, i.e. the loss of therapeutic effect of GTN upon long-term application. It is well documented that ALDH2 denitrates GTN to yield 1,2-glyceryl dinitrate and inorganic nitrite but the link between this clearance-based metabolism of GTN and bioactivation resulting in sGC activation is not well understood.In this project we characterized several ALDH2 mutants which are able to reduce GTN directly to NO, one of which exhibited exhibited hyperactivity with respect to GTN bioactivation (sGC activation), even though it had lost all of its classical activities, including clearance-based denitration of GTN ("NO-mutant"). These data suggested that ALDH2 catalyzes GTN biotransformation by two independent reactions: clearance-based 2-electron reduction yielding nitrite, and 3-electron reduction resulting in formation of NO. In collaboration with Karl Gruber (Department of Molecular Biosciences, University of Graz) and Leslie Poole (Wake Forest University, NC, USA) we determined the 3D structure of the ALDH2 mutant in complex with GTN and identified intermediates of the GTN/ALDH2 reaction by mass spectrometry. To clarify whether ALDH2-catalyzed NO formation explains GTN-induced vasorelaxation, we established a method for overexpression of ALDH2 in ALDH2-deficient blood vessels isolated from knockout mice. Although expression of the NO-mutant has not yet been successul, overexpression of the wildtype enzyme unexpectedly showed that cytosolic localization of the enzyme is essential for GTN-induced relaxation, qestioning the current view that vascular bioactivation of nitroglycerin takes place in mitochondria.The results provide new insights into the cellular and molecular mechanisms of nitroglycerin bioactivation in blood vessels and pave the way for the development of new drugs, which could be useful for the treatment of coronary artery disease without causing nitrate tolerance.