A novel family of small molecule HIF-1 alpha stabilizers for the treatment of diabetic wounds; an integrated in silico, in vitro, and in vivo strategy

Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a crucial regulator of wound healing, which includes epithelialization, angiogenesis, granulation, tissue development, and wound contraction. Even though diabetic wounds are hypoxic, HIF-1 alpha levels are decreased during healing. Diabetic wound healing necessitates the modulation of hypoxia-induced responses by VHL-HIF-1 alpha protein-protein inhibition. Our proposed hypothesis is to increase HIF-1 alpha levels by inhibiting VHL and HIF-1 alpha interactions by novel small bioactive molecules, accelerating diabetic wound healing. A three features (two aromatic rings and one hydrogen bond acceptor) pharmacophore hypothesis was generated from the existing HIF-1 alpha modulators. Virtual screening was done based on the generated pharmacophore, and a library consisting of the top 20 out of 3728 compounds was selected using ZINCPharmer. Of the top 20 molecules, the pyrazole moiety was identified as the top "HIT". Five analogues of pyrazole were designed, and Scifinder ascertained the novelty. The designed compounds were synthesized and characterized by IR, Mass, and NMR. Preliminarily, we have carried out a scratch wound assay using 3T3L1 cell lines. All the synthesized compounds showed significant wound healing activity. Further, to validate the in vitro assay, the compound CI, which showed effective in vitro results was used for in vivo study. Using the diabetes mouse model, comprising streptozotocin-induced (STZ) diabetic mice and scratch wound assay, we demonstrated that inhibiting the VHL and HIF-1 alpha connection is a promising strategy for treating diabetic ulcers. Molecules CI and CP were found to have substantial in silico, in vitro, and in vivo outcomes.