In-silico guided chemical exploration of KDM4A fragments hits.

BACKGROUND: Lysine demethylase enzymes (KDMs) are an emerging class of therapeutic targets, that catalyse the removal of methyl marks from histone lysine residues regulating chromatin structure and gene expression. KDM4A isoform plays an important role in the epigenetic dysregulation in various cancers and is linked to aggressive disease and poor clinical outcomes. Despite several efforts, the KDM4 family lacks successful specific molecular inhibitors. RESULTS: Herein, starting from a structure-based fragments virtual screening campaign we developed a synergic framework as a guide to rationally design efficient KDM4A inhibitors. Commercial libraries were used to create a fragments collection and perform a virtual screening campaign combining docking and pharmacophore approaches. The most promising compounds were tested in-vitro by a Homogeneous Time-Resolved Fluorescence-based assay developed for identifying selective substrate-competitive inhibitors by means of inhibition of H3K9me3 peptide demethylation. 2-(methylcarbamoyl)isonicotinic acid was identified as a preliminary active fragment, displaying inhibition of KDM4A enzymatic activity. Its chemical exploration was deeply investigated by computational and experimental approaches which allowed a rational fragment growing process. The in-silico studies guided the development of derivatives designed as expansion of the primary fragment hit and provided further knowledge on the structure-activity relationship. CONCLUSIONS: Our study describes useful insights into key ligand-KDM4A protein interaction and provides structural features for the development of successful selective KDM4A inhibitors.

Drug repositioning as a therapeutic strategy for neurodegenerations associated with OPA1 mutations.

OPA1 mutations are the major cause of dominant optic atrophy (DOA) and the syndromic form DOA plus, pathologies for which there is no established cure. We used a 'drug repurposing' approach to identify FDA-approved molecules able to rescue the mitochondrial dysfunctions induced by OPA1 mutations. We screened two different chemical libraries by using two yeast strains carrying the mgm1I322M and the chim3P646L mutations, identifying 26 drugs able to rescue their oxidative growth phenotype. Six of them, able to reduce the mitochondrial DNA instability in yeast, have been then tested in Opa1 deleted mouse embryonic fibroblasts expressing the human OPA1 isoform 1 bearing the R445H and D603H mutations. Some of these molecules were able to ameliorate the energetic functions and/or the mitochondrial network morphology, depending on the type of OPA1 mutation. The final validation has been performed in patients' fibroblasts, allowing to select the most effective molecules. Our current results are instrumental to rapidly translating the findings of this drug repurposing approach into clinical trial for DOA and other neurodegenerations caused by OPA1 mutations.

Metabolomics hallmarks OPA1 variants correlating with their in vitro phenotype and predicting clinical severity.

Interpretation of variants of uncertain significance is an actual major challenge. We addressed this question on a set of OPA1 missense variants responsible for variable severity of neurological impairments. We used targeted metabolomics to explore the different signatures of OPA1 variants expressed in Opa1 deleted mouse embryonic fibroblasts (Opa1-/- MEFs), grown under selective conditions. Multivariate analyses of data discriminated Opa1+/+ from Opa1-/- MEFs metabolic signatures and classified OPA1 variants according to their in vitro severity. Indeed, the mild p.I382M hypomorphic variant was segregating close to the wild-type allele, while the most severe p.R445H variant was close to Opa1-/- MEFs, and the p.D603H and p.G439V alleles, responsible for isolated and syndromic presentations, respectively, were intermediary between the p.I382M and the p.R445H variants. The most discriminant metabolic features were hydroxyproline, the spermine/spermidine ratio, amino acid pool and several phospholipids, emphasizing proteostasis, endoplasmic reticulum (ER) stress and phospholipid remodeling as the main mechanisms ranking OPA1 allele impacts on metabolism. These results demonstrate the high resolving power of metabolomics in hierarchizing OPA1 missense mutations by their in vitro severity, fitting clinical expressivity. This suggests that our methodological approach can be used to discriminate the pathological significance of variants in genes responsible for other rare metabolic diseases and may be instrumental to select possible compounds eligible for supplementation treatment.

Deciphering OPA1 mutations pathogenicity by combined analysis of human, mouse and yeast cell models.

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA "plus". Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two "ad hoc" generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1-/- MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA "plus" phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions.

Eight human OPA1 isoforms, long and short: What are they for?

OPA1 is a dynamin-related GTPase that controls mitochondrial dynamics, cristae integrity, energetics and mtDNA maintenance. The exceptional complexity of this protein is determined by the presence, in humans, of eight different isoforms that, in turn, are proteolytically cleaved into combinations of membrane-anchored long forms and soluble short forms. Recent advances highlight how each OPA1 isoform is able to fulfill "essential" mitochondrial functions, whereas only some variants carry out "specialized" features. Long forms determine fusion, long or short forms alone build cristae, whereas long and short forms together tune mitochondrial morphology. These findings offer novel challenging therapeutic potential to gene therapy.

OPA1: How much do we know to approach therapy?

OPA1 is a GTPase that controls several functions, such as mitochondrial dynamics and energetics, mtDNA maintenance and cristae integrity. In the last years, there have been described other cellular pathways and mechanisms involving OPA1 directly or through its interaction. All this new information, by implementing our knowledge on OPA1 is instrumental to elucidating the pathogenic mechanisms of OPA1 mutations. Indeed, these are associated with dominant optic atrophy (DOA), one of the most common inherited optic neuropathies, and with an increasing number of heterogeneous neurodegenerative disorders. In this review, we overview all recent findings on OPA1 protein functions, on its dysfunction and related clinical phenotypes, focusing on the current therapeutic options and future perspectives to treat DOA and the other associated neurological disorders due to OPA1 mutations.

OPA1 Isoforms in the Hierarchical Organization of Mitochondrial Functions.

OPA1 is a GTPase that controls mitochondrial fusion, cristae integrity, and mtDNA maintenance. In humans, eight isoforms are expressed as combinations of long and short forms, but it is unclear whether OPA1 functions are associated with specific isoforms and/or domains. To address this, we expressed each of the eight isoforms or different constructs of isoform 1 in Opa1-/- MEFs. We observed that any isoform could restore cristae structure, mtDNA abundance, and energetic efficiency independently of mitochondrial network morphology. Long forms supported mitochondrial fusion; short forms were better able to restore energetic efficiency. The complete rescue of mitochondrial network morphology required a balance of long and short forms of at least two isoforms, as shown by combinatorial isoform silencing and co-expression experiments. Thus, multiple OPA1 isoforms are required for mitochondrial dynamics, while any single isoform can support all other functions. These findings will be useful in designing gene therapies for patients with OPA1 haploinsufficiency.