Deborah K. Shoemark

The Rho GTPase RhoJ is a key regulator of tumour angiogenesis and has been identified as a target for anti-angiogenic drug design. Its high sequence conservation with the related Rho GTPase Cdc42 complicates selective drug discovery. RhoJ has a unique N-terminal extension (Asn2-Lys19) that is absent in Cdc42 and other Rho GTPases. Here, we use molecular dynamics simulations to identify a novel allosteric pocket formed by the RhoJ N-terminus and show that this pocket contributes to interactions with key downstream signalling partners. Structure-based virtual screening of small molecules targeting this pocket identified the antifungal terconazole as a potential binder. Computational docking revealed that terconazole occupies the N-terminal pocket, sharing critical contacts with the interface between RhoJ and PAK3, a key interacting partner. To validate this, we developed an optimized bioluminescence resonance energy transfer (eBRET ² )-based biosensor to monitor the interaction between RhoJ and PAK3 in live cells and saw that terconazole disrupted the RhoJ:PAK3 interaction in a dose-dependent manner. Together, our MD results show that the RhoJ N-terminal extension assumes structure on binding to PAK3 and contributes to that interaction, while our cellular biosensor data support targeting this pocket to modulate the RhoJ:PAK3 interaction. The ability of terconazole to bind to the N-terminal pocket and disrupt PAK3 binding provides proof of concept for the specific targeting of RhoJ in the development of novel anti-angiogenic drugs. Graphical

Cystic Fibrosis (CF), one of the most common life-shortening autosomal recessive diseases, arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and leads to severe respiratory and digestive dysfunction. CFTR-targeting therapies such as ivacaftor have transformed CF care by directly modulating CFTR function. However, the precise mechanism by which ivacaftor alters CFTR’s conformational dynamics remains incompletely understood. In this study, we refined and employed a CFTR model derived from the phosphorylated, ATP- and ivacaftor-bound cryo-EM structure (PDB ID 6O2P). Through 32.5 [ ]s of Molecular Dynamics (MD) simulations, we investigated the structural and dynamic effects of the common F508del mutation and the gating mutations G551D, G1349D and S549N. Our results show that mutations within the ATP-binding regions cause local rearrangements at the nucleotide-binding sites, affecting the interface between the Nucleotide-Binding Domains (NBDs) relative to wild-type CFTR, rather than abolishing ATP binding entirely. We further indicate that, for the NBDs to approach one another productively, protonation of the aspartate side chains in G551D and G1349D CFTR is necessary to mitigate charge repulsion. The F508del mutation induces increased rigidity within the protein core, impeding the rearrangement of transmembrane helices required for channel opening. Ivacaftor does not directly perturb the ATP-binding sites, consistent with emerging evidence that it exerts its effects allosterically on regions distal to the nucleotide-binding sites. Collectively, our findings enhance our understanding of CFTR dynamics and provide a versatile framework for exploring the molecular effects of disease-causing mutations and evaluating potential therapeutic agents.

Cystic fibrosis (CF), one of the most common life-shortening autosomal recessive diseases, arises from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and leads to severe respiratory and digestive dysfunction. CFTR-targeting therapies such as ivacaftor have transformed CF care by directly modulating CFTR function. However, the precise mechanism by which ivacaftor alters CFTR’s conformational dynamics remains incompletely understood. In this study, we refined and employed a CFTR model derived from the phosphorylated, ATP- and ivacaftor-bound cryo-EM structure (PDB ID 6O2P). Through 32.5 μs of molecular dynamics simulations, we investigated the structural and dynamic effects of the common F508del mutation and the gating mutations G551D, G1349D and S549N. Our results show that mutations within the ATP-binding regions cause local rearrangements at the nucleotide-binding sites, affecting the interface between the nucleotide-binding domains (NBDs) relative to wild-type CFTR, rather than abolishing ATP binding entirely. We further indicate that, for the NBDs to approach one another productively, protonation of the aspartate side chains in G551D and G1349D CFTR is necessary to mitigate charge repulsion. The F508del mutation induces increased rigidity within the protein core, impeding the rearrangement of transmembrane helices required for channel opening. Ivacaftor does not directly perturb the ATP-binding sites, consistent with emerging evidence that it exerts its effects allosterically on regions distal to the nucleotide-binding sites. Collectively, our findings enhance our understanding of CFTR dynamics and provide a versatile framework for exploring the molecular effects of disease-causing mutations and evaluating potential therapeutic agents.

The regulation of reactive oxygen species (ROS) in red blood cells (RBCs) is crucial for maintaining functionality and lifespan. Indeed, dysregulated ROS occurs in haematological diseases such as sickle cell disease and β-thalassaemia. In order to combat this, RBCs possess high levels of protective antioxidant enzymes. We aimed to further boost RBC antioxidant capacity by overexpressing peroxiredoxin (Prxs) and glutathione peroxidase (GPxs) enzymes. Multiple antioxidant enzyme cDNAs were individually overexpressed in expanding immortalised erythroblasts using lentivirus, including Prx isoforms 1, 2, and 6 and GPx isoforms 1 and 4. Enhancing Prx protein expression proved straightforward, but GPx overexpression required modifications. For GPx4, these modifications included adding a SECIS element in the 3’UTR, the removal of a mitochondrial-targeting sequence, and removing putative ubiquitination sites. Culture-derived reticulocytes exhibiting enhanced levels of Prx and GPx antioxidant proteins were successfully engineered, demonstrating a novel approach to improve RBC resilience to oxidative stress. Further work is needed to explore the activity of these proteins and their impact on RBC metabolism, but this strategy shows promise for improving RBC function in physiological and pathological contexts and during storage for transfusion. Enhancing the antioxidant capacity of reticulocytes has exciting promise for developing culture-derived RBCs with enhanced resistance to oxidative damage and offers new therapeutic interventions in diseases with elevated oxidative stress.

Autoantibodies to glutamate decarboxylase (GADA) are widely used in the prediction and classification of type 1 diabetes. GADA radiobinding assays (RBAs) using N-terminally truncated antigens offer improved specificity but radioisotopes limit the high-throughput potential for population screening. Luciferase-based Immunoprecipitation System (LIPS) assays are sensitive and specific alternatives to RBAs with the potential to improve risk stratification. The performance of assays using the Luciferase (Nluc-) conjugated GAD ₆₅ constructs, Nluc-GAD65(96-585) and full length Nluc-GAD65(1-585) were evaluated in 434 well-characterised sera from recent-onset type 1 diabetes patients and first-degree relatives. Non-radioactive, high-throughput LIPS assays are quicker and require less serum than RBAs. Of 171 relatives previously tested single autoantibody positive for autoantibodies to full-length GAD ₆₅ by RBA but had not progressed to diabetes, fewer retested positive by LIPS using either truncated (n=72) or full-length (n=111) antigen. The Nluc-GAD65(96-585) truncation demonstrated the highest specificity in LIPS assays overall but in contrast to RBA, N-terminus truncations did not result in a significant increase in disease-specificity compared with the full-length antigen. This suggests that binding of non-specific antibodies is affected by the conformational changes resulting from addition of the Nluc antigen. Nluc-GAD65(96-585) LIPS assays offer low blood volume, high specificity GADA tests for screening and diagnostics.

The LIM homeodomain transcription factors LMX1A and LMX1B are essential mediators of midbrain dopaminergic neuronal (mDAN) differentiation and survival. Here we show that LMX1A and LMX1B are autophagy transcription factors that provide cellular stress protection. Their suppression dampens the autophagy response, lowers mitochondrial respiration, and elevates mitochondrial ROS, and their inducible overexpression protects against rotenone toxicity in human iPSC-derived mDANs in vitro. Significantly, we show that LMX1A and LMX1B stability is in part regulated by autophagy, and that these transcription factors bind to multiple ATG8 proteins. Binding is dependent on subcellular localization and nutrient status, with LMX1B interacting with LC3B in the nucleus under basal conditions and associating with both cytosolic and nuclear LC3B during nutrient starvation. Crucially, ATG8 binding stimulates LMX1B-mediated transcription for efficient autophagy and cell stress protection, thereby establishing a novel LMX1B-autophagy regulatory axis that contributes to mDAN maintenance and survival in the adult brain.

The SARS-CoV-2 spike protein contains a functionally important fatty acid (FA) binding site, which is also found in some other coronaviruses, e.g. SARS-CoV and MERS-CoV. The occupancy of the FA site by linoleic acid (LA) reduces infectivity by ‘locking’ the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the allosteric responses of spike variants to LA removal. D-NEMD simulations show that the FA site is coupled to other functional regions of the protein, e.g. the receptor-binding motif (RBM), N-terminal domain (NTD), furin cleavage site, and regions surrounding the fusion peptide. D-NEMD simulations also identify the allosteric networks connecting the FA site to these functional regions. The comparison between the wild-type spike and four variants (Alpha, Delta, Delta plus, and Omicron BA.1) shows that the variants differ significantly in their responses to LA removal. The allosteric connections to the FA site on Alpha are generally similar to those on the wild-type protein, with the exception of the RBM and the S71–R78 region, which show a weaker link to the FA site. In contrast, Omicron is the most different variant, exhibiting significant differences in the RBM, NTD, V622–L629, and furin cleavage site. These differences in the allosteric modulation may be of functional relevance, potentially affecting transmissibility and virulence. Experimental comparison of the effects of LA on SARS-CoV-2 variants, including emerging variants, is warranted.

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed “cargoes”) from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans -Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

Targeting the colchicine binding site of α/β tubulin microtubules can lead to suppression of microtubule dynamics, cell cycle arrest and apoptosis. Therefore, the development of microtubule (MT) inhibitors is considered a promising route to anticancer agents. Our approach to identify novel scaffolds as MT inhibitors depends on a 3D-structure-based pharmacophore approach and docking using three programs MOE, Autodock and BUDE (Bristol University Docking Engine) to screen a library of virtual compounds. From this work we identified the compound 7-(3-hydroxy-4-methoxy-phenyl)-3-(3-trifluoromethyl-phenyl)-6,7-dihydro-3H-imidazo[4,5-b]pyridin-5-ol (6) as a novel inhibitor scaffold. This compound inhibited several types of cancer cell proliferation at low micromolar concentrations with low toxicity. Compound 6 caused cell cycle arrest in the G2/M phase and blocked tubulin polymerization at low micromolar concentration (IC₅₀ = 6.1 ±0.1 μM), inducing apoptosis via activation of caspase 9, increasing the level of the pro-apoptotic protein Bax and decreasing the level of the anti-apoptotic protein Bcl2. In summary, our approach identified a lead compound with potential antimitotic and antiproliferative activity.