Biology subjects

Smith, N. J.

Publications and source records attributed to Smith, N. J..

9 recordsLinked to original sources

bioRxiv2026

Pharmacological proximities in the GPCR family discovered using contact-informed amino-acid and binding pocket similarities

Understanding protein proximities in the theoretical ligand space is essential for developing therapeutics with desirable polypharmacology, predicting off-targets, and discovering surrogate ligands for poorly characterized proteins. This is especially important for G protein-coupled receptors (GPCRs) - a major class of drug targets, many of which still lack known ligands. Circumventing this limitation, we present GPCR-CoINPocket v2, a contact-informed metric for detecting GPCR pharmacological similarities from amino-acid sequences alone. We first establish a "gold standard" of pharmacological relatedness using ChEMBL-derived ligand sets. We then replace traditional evolutionary amino acid similarity matrices with a chemically-informed matrix derived from protein:ligand interaction patterns across 3,306 structures, significantly improving early detection of shared pharmacology between distantly homologous receptors. An additional unconstrained, contact-informed matrix further enhances predictive performance. Pilot application of the method revealed previously unrecognized similarities between the {beta}2 adrenoceptor and three Class A peptide GPCRs, which we confirmed experimentally by demonstrating the binding of select ligands of these receptors to the {beta}2. Dimensionality reduction of similarity scores recapitulates known receptor relationships and predicts neighbors of orphan GPCRs later confirmed experimentally. Overall, GPCR-CoINPocket v2 provides a powerful sequence-based framework to prioritize ligand space, predict polypharmacology, and accelerate GPCR drug discovery and deorphanization.

bioinformatics↗

bioRxiv2025

Exercise remodels hippocampal extracellular matrix to alleviate chondroitin-4-sulphate-induced memory impairment.

Research utilising exercise therapeutically to enhance neuroplasticity mainly reports enhanced expression of neurotrophic (neural growth) factors which are associated with increased neurogenesis, structural changes, and improved memory. However, these neural changes are tightly regulated by both promotors and inhibitors of neuroplasticity, and the effect of exercise on inhibitory pathways is currently understudied. We show that exercise also modulates inhibitors of neuroplasticity. Six weeks of treadmill training reduced the gene expression of aggrecan, a chondroitin sulphate proteoglycan (CSPG), and altered the composition of CSPG containing structures, perineuronal nets, in the rodent hippocampus. Genetically manipulating chondroitin-4-sulphation of CSPGs by overexpressing hippocampal Chst11 impaired memory performance, which was mitigated by treadmill training. We provide evidence that hippocampal CSPGs are involved in object recognition memory, and that there is a link between exercise, modulating hippocampal inhibitors of neuroplasticity, and memory performance.

neuroscience↗

bioRxiv2024

Inhibition of CELA1 Improves Septation in the Mouse Hyperoxia Model of Impaired Alveolar Development

A key feature of bronchopulmonary dysplasia (BPD) is impaired alveolar septation. In later live, BPD survivors are more susceptible to childhood respiratory problems and have reduced respiratory function as adults. Chymotrypsin-like elastase 1 (CELA1) is a serine protease expressed in AT2 cells that mediates emphysema progression in adult mouse models. CELA1 binds and cleaves tropoelastin in response to strain. Its expression is developmentally regulated. Using the mouse hyperoxia model of impaired alveolar development we hypothesized a role for CELA1 in impaired alveolar development (IAD). In C57BL6 mouse pup lungs exposed to 80% oxygen for 14 days Cela1 mRNA increased 1.9-fold (p<0.05) and protein 2.6-fold (p<0.01). Protein levels normalized after 14 days in room air. Analysis of an existing single cell mRNA-seq dataset showed Cela1 mRNA in AT2 cells, alveolar macrophages and interstitial macrophages. The fraction of cells with Cela1 mRNA increased with hyperoxia. By flow cytometry the only Cela1-specific difference in immune cell populations was a 2-fold increase in lung eosinophils in room air (p<0.05). After 14 days of exposure to 80% oxygen Cela1-/- mice had better alveolarization with an average mean linear intercept of 80 m compared to 111m (p<0.001). Treatment of hyperoxia-exposed pups with subcutaneous anti-Cela1 KF4 antibody offered similar protection compared to IgG (59 m vs. 67 m, p<0.001).Human BPD specimens demonstrated CELA1 in AT2 cells and myeloid cells. These data indicate that hyperoxia-induced increases in CELA1 are partially responsible for IAD and suggest a potential role in premature neonates exposed to high FiO2.

developmental biology↗

bioRxiv2024

KF4 anti-CELA1 Antibody and Purified α1-Antitrypsin Have Similar but Not Additive Efficacy in Preventing Emphysema in Murine α1-Antitrypsin Deficiency

Alpha-1 antitrypsin (AAT) deficiency is the most common genetic cause of emphysema. Chymotrypsin-like Elastase 1 (CELA1) is a serine protease neutralized by AAT and is important in emphysema progression. Cela1-deficiency is protective in a murine models of AAT-deficient emphysema. KF4 anti-CELA1 antibody prevented emphysema in PPE and cigarette smoke models in wild type mice. We evaluated potential toxicities of KF4 and its ability to prevent emphysema in AAT deficiency. We found Cela1 protein expression in mouse lung, pancreas, small intestine, and spleen. In toxicity studies, mice treated with KF4 25 mg/kg weekly for four weeks showed an elevation in blood urea nitrogen and slower weight gain compared to lower doses or equivalent dose IgG. In histologic grading of tissue injury of the lung, kidney, liver, and heart, there was some evidence of liver injury with KF4 25 mg/kg, but in all tissues, injury was less than in control mice subjected to cecal ligation and puncture. In efficacy studies, KF4 doses as low as 0.5 mg/kg reduced the lung elastase activity of AAT-/-mice treated with 0.2 units of PPE. In this injury model, AAT-/-mice treated with KF4 1 mg/kg weekly, human purified AAT 60 mg/kg weekly, and combined KF4 and AAT treatment had less emphysema than mice treated with IgG 1 mg/kg weekly. However, the efficacy of KF4, AAT, or KF4 & AAT was similar. While KF4 might be an alternative to AAT replacement, combined KF4 and AAT replacement does not confer additional benefit.

pathology↗

bioRxiv2022

Diversity-oriented synthesis encoded by deoxyoligonucleotides

Diversity-oriented synthesis (DOS)is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors, derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

biochemistry↗

bioRxiv2021

A standardized method to purify cardiomyocytes from individual mouse hearts of any age

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different agedependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2x106 cells/heart) and viability (~70-100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell-type specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR<0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rodshaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.

cell biology↗

bioRxiv2020

Orphan G protein-coupled receptor, GPR37L1: pharmacological toolbox empty once again

Orphan G protein-coupled receptors (GPCRs) are largely intractable therapeutic targets, owing to the lack of chemical tools for exploring their pharmacology. The discovery of such tools, however, is hampered by a number of unknowns, such as effector coupling and appropriate positive controls. In our 2017 Nature Chemical Biology paper1, we developed a computational chemical tool discovery approach called GPCR Contact-Informed Neighboring Pocket (GPCR-CoINPocket). This method predicted pharmacological similarity of GPCRs in a ligand- and structure-independent manner, to enable the discovery of off-target activities of known compounds at orphan GPCRs and hence the identification of so-called surrogate ligands. Our orphan GPCR target for prospective surrogate ligand discovery efforts was GPR37L1, a brain-specific receptor linked to cerebellar development2 and seizures3. We had previously demonstrated that GPR37L1 constitutively coupled to Gs and generated ligand-independent increases in intracellular cAMP4[§]. Thus, the inverse agonist activities of computationally predicted surrogates were tested in the cAMP response element luciferase (CRE-luc) reporter gene assay in human embryonic kidney (HEK293) cells expressing either vector control or what we thought was untagged GPR37L1 in pcDNA3.1. However, we recently discovered that the GPR37L1 construct used in that study was incorrect: instead of pcDNA3.1, it carried the receptor inserted backwards into a yeast p426GPD vector (hereafter referred to as p426-r37L1). Here, we correct the cloning error and describe our subsequent unsuccessful efforts to re-test the computationally predicted GPR37L1 ligands (triggering an author-initiated retraction of1). NoteWe, the authors, are working with the Nature Chemical Biology Editors to retract our 2017 paper Orphan receptor ligand discovery by pickpocketing pharmacological neighbors1. The present manuscript is under review at Nature Chemical Biology as a Matters Arising accompaniment to the anticipated author-initiated retraction. We initiated the steps towards the retraction upon discovering a regrettable cloning error that put into question the in vitro findings reported in1. This action was unanimously agreed upon by all authors. The computational aspects of the original manuscript1 are unaffected by this error.

pharmacology and toxicology↗

bioRxiv2020

The N-terminus of GPR37L1 is proteolytically processed by matrix metalloproteases

GPR37L1 is an orphan G protein-coupled receptor expressed exclusively in the brain and linked to seizures, neuroprotection and cardiovascular disease. Based upon the observation that fragments of the GPR37L1 N-terminus are found in human cerebrospinal fluid, we hypothesized that GPR37L1 was subject to post-translational modification. Heterologous expression of GPR37L1-eYFP in either HEK293 or U87 glioblastoma cells yielded two cell surface species of approximately equivalent abundance, the larger of which is N-glycosylated at Asn105. The smaller species is produced by matrix metalloprotease/ADAM-mediated proteolysis (shown by the use of pharmacological inhibitors) and has a molecular weight identical to that of a mutant lacking the entire N-terminus, Δ122 GPR37L1. Serial truncation of the N-terminus prevented GPR37L1 expression except when the entire N-terminus was removed, narrowing the predicted site of N-terminal proteolysis to residues 105-122. Using yeast expressing different G protein chimeras, we found that wild type GPR37L1, but not Δ122 GPR37L1, coupled constitutively to Gpa1/Gαs and Gpa1/Gα16 chimeras, in contrast to previous studies. We tested the peptides identified in cerebrospinal fluid as well as their putative newly-generated N-terminal ‘tethered’ counterparts in both wild type and Δ122 GPR37L1 Gpa1/Gαs strains but saw no effect, suggesting that GPR37L1 does not signal in a manner akin to the protease-activated receptor family. We also saw no evidence of receptor activation or regulation by the reported GPR37L1 ligand, prosaptide/TX14A. Finally, the proteolytically processed species predominated both in vivo and ex vivo in organotypic cerebellar slice preparations, suggesting that GPR37L1 is rapidly processed to a signaling-inactive form. Our data indicate that the function of GPR37L1 in vivo is tightly regulated by metalloprotease-dependent N-terminal cleavage.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2019

A mutation in Hnrnph1 that decreases methamphetamine-induced reinforcement, reward, and dopamine release and increases synaptosomal hnRNP H and mitochondrial proteins

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, mice (both male and female) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1+/-) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1+/- mice showed a robust decrease in methamphetamine-induced dopamine release in the nucleus accumbens with no change in baseline extracellular dopamine, striatal whole tissue dopamine, dopamine transporter protein, or dopamine uptake. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for tyrosine hydroxylase did not reveal any major changes in staining intensity, cell number, or in the number of forebrain puncta. Surprisingly, there was a two-fold increase in hnRNP H protein in the striatal synaptosome of H1+/- mice with no change in whole tissue levels. To gain insight into the molecular mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min post-methamphetamine administration in H1+/- mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in wild-type mice showed a rapid increase in response to methamphetamine. We conclude that H1+/- decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.\n\nSIGNIFICANCE STATEMENTMethamphetamine dependence is a significant public health concern with no FDA-approved treatment. We discovered a role for the RNA binding protein hnRNP H in methamphetamine reward and reinforcement. Hnrnph1 mutation also blunted methamphetamine-induced dopamine release in the nucleus accumbens - a key neurochemical event contributing to methamphetamine addiction liability. Finally, Hnrnph1 mutants showed a marked increase in basal level of synaptosomal hnRNP H and mitochondrial proteins that decreased in response to methamphetamine whereas wild-type mice showed a methamphetamine-induced increase in synaptosomal mitochondrial proteins. Thus, we identified a potential role for hnRNP H in basal and dynamic mitochondrial function that informs methamphetamine-induced cellular adaptations associated with reduced addiction liability.

neuroscience↗