(020) Allosteric Modulation of Transglutaminase 2 for Targeting Vascular and Fibrotic Mechanisms in Diabetes-Induced Female Sexual Dysfunction

Introduction Women with diabetes are several times more likely to experience sexual dysfunction (SD) than non-diabetic women. Diabetes-associated vasculopathy and neuropathy impair genital perfusion/innervation, driving diabetes-induced SD (DISD). Chronic hyperglycaemia triggers oxidative stress and TGF-β1 activation, leading to irreversible genital tissue fibrosis. Current treatments show limited efficacy, significant side effects, and fail to address vascular or fibrotic mechanisms underlying female SD, representing an unmet need. Transglutaminase 2 (TG2), an enzyme existing in open (pro-fibrotic) and closed (vasodilatory) conformations, may play a central role in DISD. Objective To evaluate whether promoting TG2’s closed conformation with the allosteric inhibitor LDN27219 can limit genital tissue fibrosis and improve vascular function, thereby supporting TG2 as a potential therapeutic target in women with DISD. Methods Human primary uterine fibroblasts (HUFs) were seeded (20,000 cells/well) on collagen I/III–coated 96-well plates. A pro-fibrotic milieu was induced with high glucose (25 mM) plus connective tissue growth factor (CTGF, 100 ng/mL) or TGF-β (10 ng/mL) and calcium (1 mM) for 48 h. The effects of Z-DON (40 μM; locks TG2 open conformation) and LDN27219 (10 μM) on TG2 activity were assessed. Transamidase activity was quantified via incorporation of a substrate 5-(biotinamido)pentylamine (BPA, 0.5 mM, 4 h), detected with streptavidin–Alexa Fluor 488 and read fluorometrically. Pudendal arteries (PAs)-the primary arterial supply to the genital tissues and a critical regulator of genital perfusion-were isolated from female Sprague–Dawley rats and mounted on wire myographs. After phenylephrine (Phe) pre-contraction, concentration–response curves (10 nM–100 μM) for vehicle, LDN27219, or Z-DON were obtained in the presence/absence of L-NAME (nitric oxide synthase inhibitor, 100 μM, 30 min). KCl (20–120 mM), Phe (1 nM–100 μM), and acetylcholine (ACh, 1 nM–100 μM) responses were obtained in the presence/absence of vehicle, LDN27219 (3 μM), or Z-DON (40 μM). The relevant national authority approved all procedures on animals. Statistical significance was determined using one- and two-way ANOVA followed by Tukey’s post hoc test. Results In HUFs, high glucose+CTGF (P<0.001) and high glucose+TGF-β (P<0.01) significantly increased TG2 transamidase activity (n=6), which was inhibited by LDN27219 (P<0.05) and Z-DON (P<0.001) (n=6). In PAs, LDN27219 produced concentration-dependent relaxation compared to vehicle (EC50≈1.6 μM; P<0.001; n=6), which was attenuated in the presence of L-NAME (P<0.01; n=6). Z-DON showed no relaxant effect in pre-contracted PAs but induced contraction at 40 μM at resting tone compared to vehicle (P<0.05, n=6). LDN27219 enhanced ACh-induced relaxations (P<0.05, n=3) and reduced contractions to KCl (P<0.05, n=6) and Phe (P<0.01, n=6), whereas Z-DON did not alter these responses (n=3–6). Conclusions Allosteric TG2 inhibition enhanced endothelium-dependent vascular relaxation and suppressed pro-fibrotic stimulus-induced TG2 activity, supporting the closed conformation as a therapeutic target for DISD and genital tissue fibrosis. Future in vivo studies should test the therapeutic benefit of TG2 modulation in DISD. Acknowledgments: This project is funded by the European Union (MSCA Postdoctoral Fellowship; Grant No. 101203332). Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Initiator Pharma A/S