Intracellular deposits of α-synuclein in the form of Lewy bodies are major hallmarks of Parkinson's disease (PD) and a range of related neurodegenerative disorders. Post-translational modifications (PTMs) of α-synuclein are increasingly thought to be major modulators of its structure, function, degradation and toxicity. Among these PTMs, phosphorylation near the C-terminus at S129 has emerged as a dominant pathogenic modification as it is consistently observed to occur within the brain and cerebrospinal fluid (CSF) of post-mortem PD patients, and its level appears to correlate with disease progression. Phosphorylation at the neighboring tyrosine residue Y125 has also been shown to protect against α-synuclein toxicity in a Drosophila model of PD. In the present study we address the potential roles of C-terminal phosphorylation in modulating the interaction of α-synuclein with other protein partners, using a single domain antibody fragment (NbSyn87) that binds to the C-terminal region of α-synuclein with nanomolar affinity. The results reveal that phosphorylation at S129 has negligible effect on the binding affinity of NbSyn87 to α-synuclein while phosphorylation at Y125, only four residues away, decreases the binding affinity by a factor of 400. These findings show that, despite the fact that α-synuclein is intrinsically disordered in solution, selective phosphorylation can modulate significantly its interactions with other molecules and suggest how this particular form of modification could play a key role in regulating the normal and aberrant function of α-synuclein.

a b s t r a c tMacrophage migration inhibitory factor (MIF) is a major proinflammatory cytokine that has been increasingly implicated in the pathogenesis of several inflammatory, autoimmune, infectious and oncogenic diseases. Accumulating evidence suggests that the tautomerase activity of MIF plays a role in modulating some of its intra-and extra-cellular activities. Therefore, the identification and development of smallmolecule inhibitors targeting the catalytic activity of MIF has emerged as an attractive and viable therapeutic strategy to attenuate its function in health and disease. Herein we report a novel virtual screening protocol for the discovery of new inhibitors of MIF's tautomerase activity. Our protocol takes into account the flexibility and dynamics of the catalytic site by coupling molecular dynamics (MD) simulations aimed at modeling the protein's flexibility in solution to (i) docking with FlexX, or (ii) docking with FlexX and pharmacophoric filtering with Unity. In addition, we applied in parallel a standalone docking using the new version of Surflex software. The three approaches were used to screen the ChemBridge chemical library and the inhibitory activity of the top-ranked 333 compound obtained from each approach (1000 compound in total) was assessed in vitro using the tautomerase assay. This biochemical validation process resulted in the identification of 12 novel MIF inhibitors corresponding to a 1.2% hit rate. Six of these hits came from Surflex docking; two from FlexX docking with MD simulations and four hits were identified with MDS and pharmacophore filtering with minimal overlap between the hits from each approach. Six hits were identified with IC 50 values lower than 10 lM (three hits with IC 50 lower than 1 lM); four were shown to be suicide inhibitors and act via covalent modification of the N-terminal catalytic residues Pro1. One additional inhibitor, N-phenyl-N-1,3,4-thiadiazol-2-yl-thiourea, (IC 50 = 300 nM) was obtained from FlexX docking combined to pharmacophoric filtering on one of the eight MD structures. These results demonstrate the power of integrative in silico approaches in the discovery of new modulator of MIF's tautomerase activity. The chemical diversity and mode of action of these compounds suggest that they could be used as molecular probes to elucidate the functions and biology of MIF and as lead candidates in drug developments of anti-MIF drugs.

Background Mucopolysaccharidoses (MPS) are a group of inherited metabolic diseases characterized by chronic, progressive multi-system manifestations with varying degrees of severity. Disease-modifying therapies exist to treat some types of MPS; however, they are not curative, underscoring the need to identify and evaluate co-interventions that optimize functioning, participation in preferred activities, and quality of life. A Canadian pediatric MPS registry is under development and may serve as a platform to launch randomized controlled trials to evaluate such interventions. To promote the standardized collection of patient/family-reported and clinical outcomes considered important to patients/families, health care providers (HCPs), and policymakers, the choice of outcomes to include in the registry will be informed by a core outcome set (COS). We aim to establish a patient-oriented COS for pediatric MPS using a multi-stakeholder approach. Methods In step 1 of the six-step process to develop the COS, we will identify relevant outcomes through a rapid literature review and candidate outcomes survey. A two-phase screening approach will be implemented to identify eligible publications, followed by extraction of outcomes and other pre-specified data elements. Simultaneously, we will conduct a candidate outcomes survey with children with MPS and their families to identify outcomes most important to them. In step 2, HCPs experienced in treating patients with MPS will be invited to review the list of outcomes generated in step 1 and identify additional clinically relevant outcomes. We will then ask patients/families, HCPs, and policymakers to rate the outcomes in a set of Delphi Surveys (step 3), and to participate in a subsequent consensus meeting to finalize the COS (step 4). Step 5 involves establishing a set of outcome measurement instruments for the COS. Finally, we will disseminate the COS to knowledge users (step 6). Discussion The proposed COS will inform the choice of outcomes to include in the MPS registry and, more broadly, promote the standardized collection of patient-oriented outcomes for pediatric MPS research. By involving patients/families from the earliest stage of the research, we will ensure that the COS will be relevant to those who will ultimately benefit from the research. Trial registration PROSPERO CRD42021267531, COMET

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