The biased apelin receptor agonist, MM07, reverses Sugen/hypoxia-induced pulmonary arterial hypertension as effectively as the endothelin antagonist macitentan.

Introduction: Pulmonary arterial hypertension (PAH) is characterised by endothelial dysfunction and pathological vascular remodelling, resulting in the occlusion of pulmonary arteries and arterioles, right ventricular hypertrophy, and eventually fatal heart failure. Targeting the apelin receptor with the novel, G protein-biased peptide agonist, MM07, is hypothesised to reverse the developed symptoms of elevated right ventricular systolic pressure and right ventricular hypertrophy. Here, the effects of MM07 were compared with the clinical standard-of-care endothelin receptor antagonist macitentan. Methods: Male Sprague-Dawley rats were randomised and treated with either normoxia/saline, or Sugen/hypoxia (SuHx) to induce an established model of PAH, before subsequent treatment with either saline, macitentan (30 mg/kg), or MM07 (10 mg/kg). Rats were then anaesthetised and catheterised for haemodynamic measurements, and tissues collected for histopathological assessment. Results: The SuHx/saline group presented with significant increases in right ventricular hypertrophy, right ventricular systolic pressure, and muscularization of pulmonary arteries compared to normoxic/saline controls. Critically, MM07 was as at least as effective as macitentan in significantly reversing detrimental structural and haemodynamic changes after 4 weeks of treatment. Discussion: These results support the development of G protein-biased apelin receptor agonists with improved pharmacokinetic profiles for use in human disease.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D-proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR-based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

Understanding and Promoting Preventive Health Service Use Among Black Men: Community-Driven and Informed Insights.

Black men experience high rates of adverse health that can be prevented or mitigated by the regular use of preventive health services. Efforts are urgently needed to promote this type of health service use among Black men. The U.S. Preventive Services Task Force and the Institute of Medicine indicate that such efforts must align with Black men's values, perspectives, and preferences. However, little guidance exists on how to align these efforts for Black men. The present qualitative study was developed to understand factors associated with preventive health service use among Black men and community-informed strategies to promote preventive health service use among these men. An approach rooted in community-based participatory research and ecological theory was used. A core leadership team consisting of five Black men from the area guided the project's development, implementation, and evaluation. The core leadership team conducted 22 interviews with Black men from their communities. Four themes emerged from these interviews: (1) holistic well-being challenges faced by Black men: interaction of mental, physical, and societal forces; (2) the interplay of financial, informational, and gendered barriers/facilitators to using preventative health services among Black men; (3) the importance of shared identity in peer health education about preventive health service use; and (4) the need for community-centered initiatives to improve preventive health service use among Black men that prioritize accessibility and information. Findings of the present study can be used to tailor preventive health service use efforts for Black men. Such efforts have the potential to promote health and mitigate health disparities.

Expanding the apelin receptor pharmacological toolbox using novel fluorescent ligands.

Introduction: The apelin receptor binds two distinct endogenous peptides, apelin and ELA, which act in an autocrine/paracrine manner to regulate the human cardiovascular system. As a class A GPCR, targeting the apelin receptor is an attractive therapeutic strategy. With improvements in imaging techniques, and the stability and brightness of dyes, fluorescent ligands are becoming increasingly useful in studying protein targets. Here, we describe the design and validation of four novel fluorescent ligands; two based on [Pyr1]apelin-13 (apelin488 and apelin647), and two based on ELA-14 (ELA488 and ELA647). Methods: Fluorescent ligands were pharmacologically assessed using radioligand and functional in vitro assays. Apelin647 was validated in high content imaging and internalisation studies, and in a clinically relevant human embryonic stem cell-derived cardiomyocyte model. Apelin488 and ELA488 were used to visualise apelin receptor binding in human renal tissue. Results: All four fluorescent ligands retained the ability to bind and activate the apelin receptor and, crucially, triggered receptor internalisation. In high content imaging studies, apelin647 bound specifically to CHO-K1 cells stably expressing apelin receptor, providing proof-of-principle for a platform that could screen novel hits targeting this GPCR. The ligand also bound specifically to endogenous apelin receptor in stem cell-derived cardiomyocytes. Apelin488 and ELA488 bound specifically to apelin receptor, localising to blood vessels and tubules of the renal cortex. Discussion: Our data indicate that the described novel fluorescent ligands expand the pharmacological toolbox for studying the apelin receptor across multiple platforms to facilitate drug discovery.

Inducible apelin receptor knockdown reduces differentiation efficiency and contractility of hESC-derived cardiomyocytes.

AIMS: The apelin receptor, a G protein-coupled receptor, has emerged as a key regulator of cardiovascular development, physiology, and disease. However, there is a lack of suitable human in vitro models to investigate the apelinergic system in cardiovascular cell types. For the first time we have used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and a novel inducible knockdown system to examine the role of the apelin receptor in both cardiomyocyte development and to determine the consequences of loss of apelin receptor function as a model of disease. METHODS AND RESULTS: Expression of the apelin receptor and its ligands in hESCs and hESC-CMs was determined. hESCs carrying a tetracycline-inducible short hairpin RNA targeting the apelin receptor were generated using the sOPTiKD system. Phenotypic assays characterized the consequences of either apelin receptor knockdown before hESC-CM differentiation (early knockdown) or in 3D engineered heart tissues as a disease model (late knockdown). hESC-CMs expressed the apelin signalling system at a similar level to the adult heart. Early apelin receptor knockdown decreased cardiomyocyte differentiation efficiency and prolonged voltage sensing, associated with asynchronous contraction. Late apelin receptor knockdown had detrimental consequences on 3D engineered heart tissue contractile properties, decreasing contractility and increasing stiffness. CONCLUSIONS: We have successfully knocked down the apelin receptor, using an inducible system, to demonstrate a key role in hESC-CM differentiation. Knockdown in 3D engineered heart tissues recapitulated the phenotype of apelin receptor down-regulation in a failing heart, providing a potential platform for modelling heart failure and testing novel therapeutic strategies.

FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2.

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.

Machine Learning-Based Rapid Detection of Volatile Organic Compounds in a Graphene Electronic Nose.

Rapid detection of volatile organic compounds (VOCs) is growing in importance in many sectors. Noninvasive medical diagnoses may be based upon particular combinations of VOCs in human breath; detecting VOCs emitted from environmental hazards such as fungal growth could prevent illness; and waste could be reduced through monitoring of gases produced during food storage. Electronic noses have been applied to such problems, however, a common limitation is in improving selectivity. Graphene is an adaptable material that can be functionalized with many chemical receptors. Here, we use this versatility to demonstrate selective and rapid detection of multiple VOCs at varying concentrations with graphene-based variable capacitor (varactor) arrays. Each array contains 108 sensors functionalized with 36 chemical receptors for cross-selectivity. Multiplexer data acquisition from 108 sensors is accomplished in tens of seconds. While this rapid measurement reduces the signal magnitude, classification using supervised machine learning (Bootstrap Aggregated Random Forest) shows excellent results of 98% accuracy between 5 analytes (ethanol, hexanal, methyl ethyl ketone, toluene, and octane) at 4 concentrations each. With the addition of 1-octene, an analyte highly similar in structure to octane, an accuracy of 89% is achieved. These results demonstrate the important role of the choice of analysis method, particularly in the presence of noisy data. This is an important step toward fully utilizing graphene-based sensor arrays for rapid gas sensing applications from environmental monitoring to disease detection in human breath.

Apelin is expressed throughout the human kidney, is elevated in chronic kidney disease & associates independently with decline in kidney function.

AIMS: Chronic kidney disease (CKD) is common and cardiovascular disease (CVD) is its commonest complication. The apelin system is a potential therapeutic target for CVD but data relating to apelin in CKD are limited. We examined expression of the apelin system in human kidney, and investigated apelin and Elabela/Toddler (ELA), the endogenous ligands for the apelin receptor, in patients with CKD. METHODS: Using autoradiography, immunohistochemistry and enzyme-linked immunosorbent assay, we assessed expression of apelin, ELA and the apelin receptor in healthy human kidney, and measured plasma apelin and ELA in 155 subjects (128 patients with CKD, 27 matched controls) followed up for 5 years. Cardiovascular assessments included blood pressure, arterial stiffness (pulse wave velocity) and brachial artery flow-mediated dilation. Surrogate markers of endothelial function (plasma asymmetric dimethylarginine and endothelin-1) and inflammation (C-reactive protein and interleukin-6) were measured. RESULTS: The apelin system was expressed in healthy human kidney, throughout the nephron. Plasma apelin concentrations were 60% higher in women than men (6.48 [3.62-9.89] vs. 3.95 [2.02-5.85] pg/mL; P < .0001), and increased as glomerular filtration rate declined (R = -0.41, P < .0001), and albuminuria rose (R = 0.52, P < .0001). Plasma apelin and ELA were associated with vascular dysfunction. Plasma apelin associated independently with a 50% decline in glomerular filtration rate at 5 years. CONCLUSION: We show for the first time that the apelin system is expressed in healthy human kidney. Plasma apelin is elevated in CKD and may be a potential biomarker of risk of decline in kidney function. Clinical studies exploring the therapeutic potential of apelin agonism in CKD are warranted.

Differential expression in humans of the viral entry receptor ACE2 compared with the short deltaACE2 isoform lacking SARS-CoV-2 binding sites.

ACE2 is a membrane protein that regulates the cardiovascular system. Additionally, ACE2 acts as a receptor for host cell infection by human coronaviruses, including SARS-CoV-2 that emerged as the cause of the on-going COVID-19 pandemic and has brought unprecedented burden to economy and health. ACE2 binds the spike protein of SARS-CoV-2 with high affinity and shows little variation in amino acid sequence meaning natural resistance is rare. The discovery of a novel short ACE2 isoform (deltaACE2) provides evidence for inter-individual differences in SARS-CoV-2 susceptibility and severity, and likelihood of developing subsequent 'Long COVID'. Critically, deltaACE2 loses SARS-CoV-2 spike protein binding sites in the extracellular domain, and is predicted to confer reduced susceptibility to viral infection. We aimed to assess the differential expression of full-length ACE2 versus deltaACE2 in a panel of human tissues (kidney, heart, lung, and liver) that are implicated in COVID-19, and confirm ACE2 protein in these tissues. Using dual antibody staining, we show that deltaACE2 localises, and is enriched, in lung airway epithelia and bile duct epithelia in the liver. Finally, we also confirm that a fluorescently tagged SARS-CoV-2 spike protein monomer shows low binding at lung and bile duct epithelia where dACE2 is enriched.

Selection for constrained peptides that bind to a single target protein.

Peptide secondary metabolites are common in nature and have diverse pharmacologically-relevant functions, from antibiotics to cross-kingdom signaling. Here, we present a method to design large libraries of modified peptides in Escherichia coli and screen them in vivo to identify those that bind to a single target-of-interest. Constrained peptide scaffolds were produced using modified enzymes gleaned from microbial RiPP (ribosomally synthesized and post-translationally modified peptide) pathways and diversified to build large libraries. The binding of a RiPP to a protein target leads to the intein-catalyzed release of an RNA polymerase σ factor, which drives the expression of selectable markers. As a proof-of-concept, a selection was performed for binding to the SARS-CoV-2 Spike receptor binding domain. A 1625 Da constrained peptide (AMK-1057) was found that binds with similar affinity (990 ± 5 nM) as an ACE2-derived peptide. This demonstrates a generalizable method to identify constrained peptides that adhere to a single protein target, as a step towards "molecular glues" for therapeutics and diagnostics.

Human embryonic stem cell-derived cardiomyocyte platform screens inhibitors of SARS-CoV-2 infection.

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated.

Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency.

Genetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

Enabling protein-hosted organocatalytic transformations.

In this review, the development of organocatalytic artificial enzymes will be discussed. This area of protein engineering research has underlying importance, as it enhances the biocompatibility of organocatalysis for applications in chemical and synthetic biology research whilst expanding the catalytic repertoire of enzymes. The approaches towards the preparation of organocatalytic artificial enzymes, techniques used to improve their performance (selectivity and reactivity) as well as examples of their applications are presented. Challenges and opportunities are also discussed.

Apelin peptides linked to anti-serum albumin domain antibodies retain affinity in vitro and are efficacious receptor agonists in vivo.

The apelin receptor is a potential target in the treatment of heart failure and pulmonary arterial hypertension where levels of endogenous apelin peptides are reduced but significant receptor levels remain. Our aim was to characterise the pharmacology of a modified peptide agonist, MM202, designed to have high affinity for the apelin receptor and resistance to peptidase degradation and linked to an anti-serum albumin domain antibody (AlbudAb) to extend half-life in the blood. In competition, binding experiments in human heart MM202-AlbudAb (pKi  = 9.39 ± 0.09) bound with similar high affinity as the endogenous peptides [Pyr1 ]apelin-13 (pKi  = 8.83 ± 0.06) and apelin-17 (pKi  = 9.57 ± 0.08). [Pyr1 ]apelin-13 was tenfold more potent in the cAMP (pD2  = 9.52 ± 0.05) compared to the ß-arrestin (pD2  = 8.53 ± 0.03) assay, whereas apelin-17 (pD2  = 10.31 ± 0.28; pD2  = 10.15 ± 0.13, respectively) and MM202-AlbudAb (pD2  = 9.15 ± 0.12; pD2  = 9.26 ± 0.03, respectively) were equipotent in both assays, with MM202-AlbudAb tenfold less potent than apelin-17. MM202-AlbudAb bound to immobilised human serum albumin with high affinity (pKD  = 9.02). In anaesthetised, male Sprague Dawley rats, MM202-AlbudAb (5 nmol, n = 15) significantly reduced left ventricular systolic pressure by 6.61 ± 1.46 mm Hg and systolic arterial pressure by 14.12 ± 3.35 mm Hg and significantly increased cardiac contractility by 533 ± 170 mm Hg/s, cardiac output by 1277 ± 190 RVU/min, stroke volume by 3.09 ± 0.47 RVU and heart rate by 4.64 ± 2.24 bpm. This study demonstrates that conjugating an apelin mimetic peptide to the AlbudAb structure retains receptor and in vivo activity and may be a new strategy for development of apelin peptides as therapeutic agents.

The G Protein Biased Small Molecule Apelin Agonist CMF-019 is Disease Modifying in Endothelial Cell Apoptosis In Vitro and Induces Vasodilatation Without Desensitisation In Vivo.

Signaling through the apelin receptor is beneficial for a number of diseases including pulmonary arterial hypertension. The endogenous small peptides, apelin and elabela/toddler, are downregulated in pulmonary arterial hypertension but are not suitable for exogenous administration owing to a lack of bioavailability, proteolytic instability and susceptibility to renal clearance. CMF-019, a small molecule apelin agonist that displays strong bias towards G protein signaling over ß-arrestin (∼400 fold), may be more suitable. This study demonstrates that in addition to being a positive inotrope, CMF-019 caused dose-dependent vasodilatation in vivo (50 nmol 4.16 ± 1.18 mmHg, **p < 0.01; 500 nmol 6.62 ± 1.85 mmHg, **p < 0.01), without receptor desensitization. Furthermore, CMF-019 rescues human pulmonary artery endothelial cells from apoptosis induced by tumor necrosis factor α and cycloheximide (5.66 ± 0.97%, **p < 0.01) by approximately 50% of that observable with rhVEGF (11.59 ± 1.85%, **p < 0.01), suggesting it has disease-modifying potential in vitro. CMF-019 displays remarkable bias at the apelin receptor for a small molecule and importantly recapitulates all aspects of the cardiovascular responses to the endogenous ligand, [Pyr1]apelin-13, in vivo. Additionally, it is able to protect human pulmonary artery endothelial cells from apoptosis, suggesting that the beneficial effects observed with apelin agonists extend beyond hemodynamic alleviation and address disease etiology itself. These findings support CMF-019 as a G protein biased small molecule apelin agonist in vitro and in vivo that could form the basis for the design of novel therapeutic agents in chronic diseases, such as, pulmonary arterial hypertension.

Insulin Inhibits Aß42 Aggregation and Prevents Aß42-Induced Membrane Disruption.

Alzheimer's disease (AD) is associated with self-assembly of amyloid ß-protein (Aß) into soluble oligomers. Of the two predominant Aß alloforms, Aß40 and Aß42, the latter is particularly strongly linked to AD. Longitudinal studies revealed a correlation between AD and type 2 diabetes (T2D), characterized by abnormal insulin levels and insulin resistance. Although administration of intranasal insulin is explored as a therapy against AD, the extent to which insulin affects Aß dynamics and activity is unclear. We here investigate the effect of insulin on Aß42 self-assembly and characterize the capacity of insulin, Aß42, and Aß42 co-incubated with insulin to disrupt the integrity of biomimetic lipid vesicles. We demonstrate that quiescently incubated insulin, which does not form amyloid fibrils, over time develops membrane-disrupting capacity, which we propose to originate in misfolded insulin monomers. These hypothetically toxic misfolded monomers might contribute to the development of insulin resistance in early stages of T2D that are associated with abnormally high insulin levels. We show that in contrast to quiescent incubation, insulin incubated under agitated conditions readily forms amyloid fibrils, which protect against membrane permeation. Insulin quiescently incubated with Aß42 attenuates both Aß42 fibril formation and the ability of Aß42 to disrupt membranes in a concentration-dependent manner. Our findings offer insights into interactions between insulin and Aß42 that are relevant to understanding the molecular basis of intranasal insulin as a therapy against Aß-induced AD pathology, thereby elucidating a plausible mechanism underlying the observed correlations between AD and T2D.

International Union of Basic and Clinical Pharmacology. CVII. Structure and Pharmacology of the Apelin Receptor with a Recommendation that Elabela/Toddler Is a Second Endogenous Peptide Ligand.

The predicted protein encoded by the APJ gene discovered in 1993 was originally classified as a class A G protein-coupled orphan receptor but was subsequently paired with a novel peptide ligand, apelin-36 in 1998. Substantial research identified a family of shorter peptides activating the apelin receptor, including apelin-17, apelin-13, and [Pyr1]apelin-13, with the latter peptide predominating in human plasma and cardiovascular system. A range of pharmacological tools have been developed, including radiolabeled ligands, analogs with improved plasma stability, peptides, and small molecules including biased agonists and antagonists, leading to the recommendation that the APJ gene be renamed APLNR and encode the apelin receptor protein. Recently, a second endogenous ligand has been identified and called Elabela/Toddler, a 54-amino acid peptide originally identified in the genomes of fish and humans but misclassified as noncoding. This precursor is also able to be cleaved to shorter sequences (32, 21, and 11 amino acids), and all are able to activate the apelin receptor and are blocked by apelin receptor antagonists. This review summarizes the pharmacology of these ligands and the apelin receptor, highlights the emerging physiologic and pathophysiological roles in a number of diseases, and recommends that Elabela/Toddler is a second endogenous peptide ligand of the apelin receptor protein.

Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] peptides that improve diet induced obesity are G protein biased ligands at the apelin receptor.

BACKGROUND: Apelin signalling pathways have important cardiovascular and metabolic functions. Recently, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)], were reported to function independent of the apelin receptor in vivo to produce beneficial metabolic effects without modulating blood pressure. We aimed to show that these peptides bound to the apelin receptor and to further characterise their pharmacology in vitro at the human apelin receptor. METHODS: [Pyr1]apelin-13 saturation binding experiments and competition binding experiments were performed in rat and human heart homogenates using [125I]apelin-13 (0.1 nM), and/or increasing concentrations of apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] (50pM-100µM). Apelin-36 and its analogues apelin-36-[F36A], apelin-36-[L28A], apelin-36-[L28C(30kDa-PEG)], apelin-36-[A28 A13] and [40kDa-PEG]-apelin-36 were tested in forskolin-induced cAMP inhibition and ß-arrestin assays in CHO-K1 cells heterologously expressing the human apelin receptor. Bias signaling was quantified using the operational model for bias. RESULTS: In both species, [Pyr1]apelin-13 had comparable subnanomolar affinity and the apelin receptor density was similar. Apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] competed for binding of [125I]apelin-13 with nanomolar affinities. Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] inhibited forskolin-induced cAMP release, with nanomolar potencies but they were less potent compared to apelin-36 at recruiting ß-arrestin. Bias analysis suggested that these peptides were G protein biased. Additionally, [40kDa-PEG]-apelin-36 and apelin-36-[F36A] retained nanomolar potencies in both cAMP and ß-arrestin assays whilst apelin-36-[A13 A28] exhibited a similar profile to apelin-36-[L28C(30kDa-PEG)] in the ß-arrestin assay but was more potent in the cAMP assay. CONCLUSIONS: Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] are G protein biased ligands of the apelin receptor, suggesting that the apelin receptor is an important therapeutic target in metabolic diseases.

Crystal Structure and Biophysical Analysis of Furfural-Detoxifying Aldehyde Reductase from Clostridium beijerinckii.

Many aldehydes, such as furfural, are present in high quantities in lignocellulose lysates and are fermentation inhibitors, which makes biofuel production from this abundant carbon source extremely challenging. Cbei_3974 has recently been identified as an aldo-keto reductase responsible for partial furfural resistance in Clostridium beijerinckii Rational engineering of this enzyme could enhance the furfural tolerance of this organism, thereby improving biofuel yields. We report an extensive characterization of Cbei_3974 and a single-crystal X-ray structure of Cbei_3974 in complex with NADPH at a resolution of 1.75 Å. Docking studies identified residues involved in substrate binding, and an activity screen revealed the substrate tolerance of the enzyme. Hydride transfer, which is partially rate limiting under physiological conditions, occurs from the pro-R hydrogen of NADPH. Enzyme isotope labeling revealed a temperature-independent enzyme isotope effect of unity, indicating that the enzyme does not use dynamic coupling for catalysis and suggesting that the active site of the enzyme is optimally configured for catalysis with the substrate tested.IMPORTANCE Here we report the crystal structure and biophysical properties of an aldehyde reductase that can detoxify furfural, a common inhibitor of biofuel fermentation found in lignocellulose lysates. The data contained here will serve as a guide for protein engineers to develop improved enzyme variants that would impart furfural resistance to the microorganisms used in biofuel production and thus lead to enhanced biofuel yields from this sustainable resource.

Polymer Masked-Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin-Colistin Conjugates.

Polymer masked-unmasked protein therapy (PUMPT) uses conjugation of a biodegradable polymer, such as dextrin, hyaluronic acid, or poly(l-glutamic acid), to mask a protein or peptide's activity; subsequent locally triggered degradation of the polymer at the target site regenerates bioactivity in a controllable fashion. Although the concept of PUMPT is well established, the relationship between protein unmasking and reinstatement of bioactivity is unclear. Here, we used dextrin-colistin conjugates to study the relationship between the molecular structure (degree of unmasking) and biological activity. Size exclusion chromatography was employed to collect fractions of differentially degraded conjugates and ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) employed to characterize the corresponding structures. Antimicrobial activity was studied using a minimum inhibitory concentration (MIC) assay and confocal laser scanning microscopy of LIVE/DEAD-stained biofilms with COMSTAT analysis. In vitro toxicity of the degraded conjugate was assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. UPLC-MS revealed that the fully "unmasked" dextrin-colistin conjugate composed of colistin bound to at least one linker, whereas larger species were composed of colistin with varying lengths of glucose units attached. Increasing the degree of dextrin modification by succinoylation typically led to a greater number of linkers bound to colistin. Greater antimicrobial and antibiofilm activity were observed for the fully "unmasked" conjugate compared to the partially degraded species (MIC = 0.25 and 2-8 µg/mL, respectively), whereas dextrin conjugation reduced colistin's in vitro toxicity toward kidney cells, even after complete unmasking. This study highlights the importance of defining the structure-antimicrobial activity relationship for novel antibiotic derivatives and demonstrates the suitability of LC-MS to aid the design of biodegradable polymer-antibiotic conjugates.