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As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.

In 2007, a genome wide association study identified a SNP in intron one of the gene encoding human FTO that was associated with increased body mass index. Homozygous risk allele carriers are on average three kg heavier than those homozygous for the protective allele. FTO is a DNA/RNA demethylase, however, how this function affects body weight, if at all, is unknown. Here we aimed to pharmacologically inhibit FTO to examine the effect of its demethylase function in vitro and in vivo as a first step in evaluating the therapeutic potential of FTO. We showed that IOX3, a known inhibitor of the HIF prolyl hydroxylases, decreased protein expression of FTO (in C2C12 cells) and reduced maximal respiration rate in vitro. However, FTO protein levels were not significantly altered by treatment of mice with IOX3 at 60 mg/kg every two days. This treatment did not affect body weight, or RER, but did significantly reduce bone mineral density and content and alter adipose tissue distribution. Future compounds designed to selectively inhibit FTO's demethylase activity could be therapeutically useful for the treatment of obesity.

In the last decade, dynamic combinatorial mass spectrometry (DCMS) with protein targets has emerged as a promising method for the identification of enzyme-inhibitors. 2-Oxoglutarate (2OG) oxygenases are involved in important biological processes related to many diseases; several human 2OG oxygenases are targeted for pharmaceutical intervention. This thesis describes inhibition studies on three 2OG oxygenases using DCMS and structure activity relation (SAR) studies. Disulphide based DCMS was used for the identification of N-oxalyl based lead inhibitors for the 2OG oxygenase AlkB from Escherichia coli. Crystallographic analyses of AlkB with a lead inhibitor assisted in the design of a second generation of inhibitors using N-oxalyl, pyridyl and quinolinyl scaffolds. Crystallographic and kinetic data of three potent and selective AlkB inhibitors validates the DCMS approach for the development of 2OG oxygenase inhibitors. The hypoxia inducible factor hydroxylase, prolyl hydroxylase domain 2 (PHD2), was then used as the model enzyme for the development of a novel DCMS approach employing the reversible reaction of boronic acids with diols to form boronate esters. The ‘boronate’ DCMS method was used to identify pyridyl- substituted lead compounds. Further modification of the pyridine scaffold, based on structural analyses, led to the development of highly potent and selective PHD2 inhibitors. To identify inhibitors for the fat mass and obesity associated protein (FTO), another 2OG oxygenase, an inhibition assay was developed. The inhibition assay was used in conjunction with a differential scanning fluorimetry (DSF) binding assay to identify isoquinolinyl and pyridyl inhibitor scaffolds, related to those used in the DCMS studies. FTO complexed structures of these compounds, and with a natural product anthraquinone, enabled the design and synthesis of new inhibitors that are both co-substrate and substrate competitors of FTO. One such compound proved to be a potent FTO inhibitor with improved selectivity over other 2OG oxygenases. Overall, the work validates the use of DCMS methods for the development of potent and selective inhibitors for 2OG oxygenases, and by implication of other enzyme families.

As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIF alpha in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.

Background The early management of polytrauma patients with traumatic spinal cord injury (tSCI) is a major challenge. Sparse data is available to provide optimal care in this scenario and worldwide variability in clinical practice has been documented in recent studies. Methods A multidisciplinary consensus panel of physicians selected for their established clinical and scientific expertise in the acute management of tSCI polytrauma patients with different specializations was established. The World Society of Emergency Surgery (WSES) and the European Association of Neurosurgical Societies (EANS) endorsed the consensus, and a modified Delphi approach was adopted. Results A total of 17 statements were proposed and discussed. A consensus was reached generating 17 recommendations (16 strong and 1 weak). Conclusions This consensus provides practical recommendations to support a clinician’s decision making in the management of tSCI polytrauma patients.

Background The early management of polytrauma patients with traumatic spinal cord injury (tSCI) is a major challenge. Sparse data is available to provide optimal care in this scenario and worldwide variability in clinical practice has been documented in recent studies. Methods A multidisciplinary consensus panel of physicians selected for their established clinical and scientific expertise in the acute management of tSCI polytrauma patients with different specializations was established. The World Society of Emergency Surgery (WSES) and the European Association of Neurosurgical Societies (EANS) endorsed the consensus, and a modified Delphi approach was adopted. Results A total of 17 statements were proposed and discussed. A consensus was reached generating 17 recommendations (16 strong and 1 weak). Conclusions This consensus provides practical recommendations to support a clinician's decision making in the management of tSCI polytrauma patients. Keywords: Traumatic spinal cord injury, Polytrauma, Management

BACKGROUND: The early management of polytrauma patients with traumatic spinal cord injury (tSCI) is a major challenge. Sparse data is available to provide optimal care in this scenario and worldwide variability in clinical practice has been documented in recent studies. METHODS: A multidisciplinary consensus panel of physicians selected for their established clinical and scientific expertise in the acute management of tSCI polytrauma patients with different specializations was established. The World Society of Emergency Surgery (WSES) and the European Association of Neurosurgical Societies (EANS) endorsed the consensus, and a modified Delphi approach was adopted. RESULTS: A total of 17 statements were proposed and discussed. A consensus was reached generating 17 recommendations (16 strong and 1 weak). CONCLUSIONS: This consensus provides practical recommendations to support a clinician's decision making in the management of tSCI polytrauma patients.

Lysosomes are multifunctional organelles that play important roles in cellular recycling, signaling, and homeostasis, relying on precise trafficking and activation of lysosomal enzymes. While the Golgi apparatus plays a central role in lysosomal enzyme sorting, the mechanisms linking Golgi function to lysosomal activity remain incompletely understood. Here, we identify the Golgi-resident protein GRASP55, but not its paralog GRASP65, as a key regulator of lysosome function. More specifically, we demonstrate that loss of GRASP55 expression leads to missorting and secretion of lysosomal enzymes, lysosomal dysfunction and bloating. GRASP55 deficiency also disrupts lysosomal mTORC1 signaling, reducing the phosphorylation of its lysosomal substrates, TFEB and TFE3, while sparing its non-lysosomal targets. Mechanistically, GRASP55 interacts with GNPTAB, a critical enzyme required for mannose 6-phosphate (M6P) tagging of lysosomal enzymes, and is necessary for its correct trafficking and stability. These findings reveal an essential role for GRASP55 in Golgi-lysosome communication and lysosomal enzyme trafficking, and suggest that GRASP55/GORASP2 may act as a susceptibility gene for lysosomal storage disorder (LSD)-like conditions. Overall, this work underscores the importance of Golgi-mediated protein sorting in lysosome function and lysosomal mTORC1 signaling, and provides insights into the molecular basis of LSD-related pathologies.Competing Interest StatementThe authors have declared no competing interest.