Different crystal structures of both HOs were

Different crystal structures of both HOs were reported and showed that the three-dimensional structures of proteins (HO-1: PDB code 1N45, HO-2: PDB code 2QPP) are predominantly in an α-helical conformation, with the heme packed between two helices (distal and proximal), and present a remarkable structural conservation [24,25]. Several glycines are present in both isoforms in the distal helix that provides flexibility to accommodate the substrate and release the product. The active site of the apoenzyme usually is more open than the holoenzyme. In the holoenzyme, the heme iron is coordinated by a histidine (His25 in HO-1 and His45 in HO-2) and by a water molecule serving as the sixth ligand [26]. The distal and proximal helices are closer together to form a closed conformation in the holoenzyme, but the distal helix has an inherent flexibility that allows crystallizing the protein in both open and closed conformations. In both apo and holoenzymes, a hydrogen-bond network involving Asn210, Arg136, Tyr58, and Tyr114 stabilizes the catalytically critical Asp140 residue of HO-1 (Asp160 in HO-2). In the crystal structure of the HO-1 holoenzyme [24], it was revealed that the distal and proximal helices “clamp down” to tighten the structure and allow coordination of the heme moiety with the His25. On the other hand, the tightened structure also blocks water FMK receptor (catalytically relevant) in the distal cavity, which form part of a well-ordered hydrogen-bond network involving Asp140. This network may serve as a proton shuttle to anchor the catalytically critical distal water ligand of heme required for oxygen activation [24]. The major point of sequence divergence between HO-1 and HO-2 is in the C-terminal region, as well as the presence of three heme regulatory motifs (HRMs) in HO-2 [25,27]. Owing to its regulatory capacity, HO-1 induction has widely been acknowledged as an adaptive cellular response able to counteract oxidative stress [28]. Therefore, the induction of the HO system has been studied in order to develop a potential therapeutic strategy in some diseases. These cytoprotective properties can be exploited during inflammatory processes or different pathological conditions, such as diabetes, hypertension, heart diseases and neurological disorders [1,[28], [29], [30], [31], [32]]. On the other hand, elevated expression of this enzyme under unfavorable conditions is related to tissues and organs damage. In fact, high levels of HO-1 have been reported in several diseases, such as neonatal jaundice, Alzheimer disease, and cancers (for example leukemias, multiple myeloma, neuroblastoma, melanoma, breast, prostate, pancreatic, lung tumors, etc.) [33,34]. Numerous studies confirmed that HO-1 up-regulation or induction contributes to the development and maintenance of these pathological conditions, through different mechanisms. The involvement of HO-1 in tumors is related to its anti-apoptotic and pro-angiogenic properties sustaining cancer proliferation and metastasis. Also, HO-1 overexpression has been evidenced at the onset of resistance towards commonly used cancer therapies [33]. These findings suggest that HO-1 inhibition could be a therapeutic approach in anticancer therapy. Therefore, it is conceivable that HO-1 inhibition can be therapeutically useful in all those pathological conditions associated with aberrant HO-1 overexpression and HO-1 selective inhibitors can be regarded as potential cancer treatment enhancers under regimens of chemotherapy, radiotherapy, or photodynamic therapy [35,36]. Moreover, the identification of selective/specific HO-1 inhibitors lacking undesired side effects to the constitutive HO-2 isoform may support a more in-depth knowledge of the role of HO-1 in human malignancies and show the way forward to future clinical applications. This review focuses on the development of heme oxygenase (HO) inhibitors with a major focus on HO-1 and their potential application in several diseases, according to discoveries achieved during the last six years (2013–2018), as previous comprehensive reviews concern the most significant works published until 2013 [37,38].