Inhibition of O‐GlcNAcase in perfused rat hearts by NAG‐thiazolines at the time of reperfusion is cardioprotective in an O‐GlcNAc dependent manner

Abstract

Increased O‐linked‐N‐acetylglucosamine (O‐GlcNAc) levels of cardiac proteins afford protection against ischemia‐reperfusion (I/R) injury. One strategy to increase cellular O‐GlcNAc levels is inhibition of O‐GlcNAcase, which catalyzes O‐GlcNAc removal. Here we tested the cardioprotective efficacy of two novel and highly selective O‐GlcNAcase inhibitors, NAG‐Bt (Bt) and NAG‐Ae (Ae). Isolated perfused rat hearts were subjected to 20 min no‐flow ischemia followed by 60 min reperfusion. At the time of reperfusion hearts were assigned to 4 groups 1) untreated control (CON); 2) 50µM Bt; 3) 100µM Bt or 50µM Ae. All treatment groups significantly increased O‐GlcNAc levels and improved functional recovery compared to CON group. O‐GlcNAc increased in a dose dependent manner with Bt and was highest in Ae group. There was a strong correlation between O‐GlcNAc, improved functional recovery and reduced troponin I release. Immunohistochemistry of non‐ischemic hearts showed higher O‐GlcNAc intensity at the Z‐lines and intense nuclear staining. There was a marked loss of nuclear O‐GlcNAc and disruption of the striated Z‐line structures following I/R. Bt treated hearts exhibited less structural disruption and higher overall O‐GlcNAc. These data further support the notion that increasing cardiac O‐GlcNAc by inhibition of O‐GlcNAcase may be a clinically relevant approach for ischemic cardioprotection. (NIH HL079364)

Protein O‐GlcNAcylation in the heart

“…7,32 Increasing O-GlcNAc levels in isolated rat hearts with glucosamine or OGA inhibitors such as O-(2-acetamido-2deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 2'-methyl-2-acetamido-2-deoxy-αd-glu copyranosyl-[2,1d]-∆2'-thiazoline (NAG-thiazoline) derivatives: NAG-Bt, and NAG-Ae prior to the induction of ischaemia or during ischaemia-reperfusion improves left ventricular developed pressure (LVDP) and reduces the release of cardiac troponin I, after 20 minutes of ischaemia followed by 60 minutes of reperfusion injury. 26,33,34 Conversely, inhibition of OGT with alloxan or the HBP with azaserine completely abolishes these cardioprotective effects. 34,35 These in vitro and ex vivo findings show that acute increases in cardiac protein O-GlcNAcylation exert beneficial effects against ischaemia-reperfusion injury.…”

“…In neonatal rat ventricular myocytes, increased O‐GlcNAc levels via overexpression of OGT or increased HBP flux enhance cell viability, and attenuate necrosis and apoptosis following ischaemia‐reperfusion injury with a significant correlation between protein O‐GlcNAcylation and cell survival 7,32 . Increasing O‐GlcNAc levels in isolated rat hearts with glucosamine or OGA inhibitors such as O‐(2‐acetamido‐2‐deoxy‐D‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc) and 2'‐methyl‐2‐acetamido‐2‐deoxy‐α‐ d ‐glucopyranosyl‐[2,1‐ d ]‐∆2'‐thiazoline (NAG‐thiazoline) derivatives: NAG‐Bt, and NAG‐Ae prior to the induction of ischaemia or during ischaemia‐reperfusion improves left ventricular developed pressure (LVDP) and reduces the release of cardiac troponin I, after 20 minutes of ischaemia followed by 60 minutes of reperfusion injury 26,33,34 . Conversely, inhibition of OGT with alloxan or the HBP with azaserine completely abolishes these cardioprotective effects 34,35 .…”

“…Adenoviral‐mediated overexpression of OGT increases O‐GlcNAc in cardiomyocytes and pharmacological OGT inhibition reduces the O‐GlcNAc level 25 . Conversely, OGA overexpression reduces O‐GlcNAc levels whereas inhibition of OGA in isolated perfused rat hearts with NAG‐thiazoline derivatives, including NAG‐Bt and NAG‐Ae, elevates O‐GlcNAc levels 26,27 . Consequently, the relatively simple two enzyme‐based system makes this pathway attractive for research aimed at controlling the level of protein O‐GlcNAcylation in the heart.…”

Computational Analysis of Human OGA Structure in Complex with PUGNAc and NAG-Thiazoline Derivatives

“…Several PUGNAc and NGT derivatives (Table ) are characterized as potent inhibitors of hOGA and human Hexosaminidases . These inhibitors have been used to identify the function of O- GlcNAc at the cellular level. ,, …”

O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry

“…35 With respect to cardiovascular disease, inhibition of OGA using NAG thiazolines and NButGT was shown to be protective and attenuate tissue necrosis after ischemia/reperfusion injury. 36,37 Furthermore, genetic studies have suggested that deletion of oga is perinatally lethal, although it is not yet clear whether this essentiality is limited to early development. 3,4 While potent and selective, the existing inhibitors of OGA possess a carbohydrate scaffold, which has poor drug-like properties with respect to Lipinski's rules.…”