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Recent genome-wide association studies have identified a missense variant p.A165T in mitochondrial amidoxime-reducing component 1 (mARC1) that is strongly associated with protection from all-cause cirrhosis and improved prognosis in nonalcoholic steatohepatitis. The precise mechanism of this protective effect is unknown. Substitution of alanine 165 with threonine is predicted to affect mARC1 protein stability and to have deleterious effects on its function. To investigate the mechanism, we have generated a knock-in mutant mARC1 A165T and a catalytically dead mutant C273A (as a control) in human hepatoma HepG2 cells, enabling characterization of protein subcellular distribution, stability, and biochemical functions of the mARC1 mutant protein expressed from its endogenous locus. Compared to WT mARC1, we found that the A165T mutant exhibits significant mislocalization outside of its traditional location anchored in the mitochondrial outer membrane and reduces protein stability, resulting in lower basal levels. We evaluated the involvement of the ubiquitin proteasome system in mARC1 A165T degradation and observed increased ubiquitination and faster degradation of the A165T variant. In addition, we have shown that HepG2 cells carrying the MTARC1 p.A165T variant exhibit lower N-reductive activity on exogenously added amidoxime substrates in vitro. The data from these biochemical and functional assays suggest a mechanism by which the MTARC1 p.A165T variant abrogates enzyme function which may contribute to its protective effect in liver disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A series of diarylurea inhibitors of the cardiac-specific kinase TNNI3K were developed to elucidate the biological function of TNNI3K and evaluate TNNI3K as a therapeutic target for the treatment of cardiovascular diseases. Utilizing a structure-based design, enhancements in kinase selectivity were engineered into the series, capitalizing on the established X-ray crystal structures of TNNI3K, VEGFR2, p38α, and B-Raf. Our efforts culminated in the discovery of an in vivo tool compound 47 (GSK329), which exhibited desirable TNNI3K potency and rat pharmacokinetic properties as well as promising kinase selectivity against VEGFR2 (40-fold), p38α (80-fold), and B-Raf (>200-fold). Compound 47 demonstrated positive cardioprotective outcomes in a mouse model of ischemia/reperfusion cardiac injury, indicating that optimized exemplars from this series, such as 47 , are favorable leads for discovering novel medicines for cardiac diseases.

Increased fibroblast growth factor 23 (FGF23) levels are an independent predictor for adverse cardiac events suggesting a role as a link that drives cardiomyopathic changes in cardiorenal syndrome. The search for the underlying mechanism driving this interaction has led to the hypothesis that FGF23 causes pathogenic changes in the heart. Increased serum FGF23 has been independently shown to cause increased cardiac morbidity, mortality, and hypertrophy by signalling through FGF receptor 4. This mechanistic concept was based on preclinical studies demonstrating inhibition of FGF23 signaling through FGF4, which led to suppression of left ventricular hypertrophy and fibrosis in a 2-week rat 5/6 nephrectomy study and a 12-week (2%) high-phosphate diet mouse model in which FGF23 levels were markedly elevated. In this report, renal dysfunction was observed in the 5/6 nephrectomy model, and FGF23 levels were significantly elevated, whereas no changes in left ventricular hypertrophy were observed at 2 or 4 weeks postnephrectomy. Mice placed on a high-phosphate diet that did not cause significant renal dysfunction resulted in significantly elevated FGF23 but no changes in left ventricular hypertrophy. The in vivo studies reported here, which were performed to recapitulate the observations of FGF23 as a driver of cardiac hypertrophy, did not lend support to the FGF23-driven cardiac remodelling hypothesis., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Structure-guided progression of a purine-derived series of TNNI3K inhibitors directed design efforts that produced a novel series of 4,6-diaminopyrimidine inhibitors, an emerging kinase binding motif. Herein, we report a detailed understanding of the intrinsic conformational preferences of the scaffold, which impart high specificity for TNNI3K. Further manipulation of the template based on the conformational analysis and additional structure-activity relationship studies provided enhancements in kinase selectivity and pharmacokinetics that furnished an advanced series of potent inhibitors. The optimized compounds (e.g., GSK854) are suitable leads for identifying new cardiac medicines and have been employed as in vivo tools in investigational studies aimed at defining the role of TNNI3K within heart failure.

Background: The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti-inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl-tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure., Methods and Results: Consistent with its ability to inhibit prolyl-tRNA synthetase, halofuginone elicited a general control nonderepressible 2-dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l-proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2-dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell-derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin-1-mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α-dependent manner., Conclusions: Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress., (© 2017 The Authors and GlaxoSmithKline. Published on behalf of the American Heart Association, Inc., by Wiley.)

Investigation of troponin I-interacting kinase (TNNI3K) as a potential target for the treatment of heart failure has produced a series of substituted N-methyl-3-(pyrimidin-4-ylamino)benzenesulfonamide inhibitors that display excellent potency and selectivity against a broad spectrum of protein kinases. Crystal structures of prototypical members bound to the ATP-binding site of TNNI3K reveal two anchoring hydrogen bond contacts: (1) from the hinge region amide N-H to the pyrimidine nitrogen and (2) from the sulfonamide N-H to the gatekeeper threonine. Evaluation of various para-substituted benzenesulfonamides defined a substituent effect on binding affinity resulting from modulation of the sulfonamide H-bond donor strength. An opposite electronic effect emerged for the hinge NH-pyrimidine H-bond interaction, which is further illuminated in the correlation of calculated H-bond acceptor strength and TNNI3K affinity for a variety of hinge binding heterocycles. These fundamental correlations on drug-receptor H-bond interactions may be generally useful tools for the optimization of potency and selectivity in the design of kinase inhibitors.

A series of selective TNNI3K inhibitors were developed by modifying the hinge-binding heterocycle of a previously reported dual TNNI3K/B-Raf inhibitor. The resulting quinazoline-containing compounds exhibit a large preference (up to 250-fold) for binding to TNNI3K versus B-Raf, are useful probes for elucidating the biological pathways associated with TNNI3K, and are leads for discovering novel cardiac medicines. GSK114 emerged as a leading inhibitor, displaying significant bias (40-fold) for TNNI3K over B-Raf, exceptional broad spectrum kinase selectivity, and adequate oral exposure to enable its use in cellular and in vivo studies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A series of cardiac troponin I-interacting kinase (TNNI3K) inhibitors arising from 3-((9H-purin-6-yl)amino)-N-methyl-benzenesulfonamide (1) is disclosed along with fundamental structure-function relationships that delineate the role of each element of 1 for TNNI3K recognition. An X-ray structure of 1 bound to TNNI3K confirmed its Type I binding mode and is used to rationalize the structure-activity relationship and employed to design potent, selective, and orally bioavailable TNNI3K inhibitors. Identification of the 7-deazapurine heterocycle as a superior template (vs purine) and its elaboration by introduction of C4-benzenesulfonamide and C7- and C8-7-deazapurine substituents produced compounds with substantial improvements in potency (>1000-fold), general kinase selectivity (10-fold improvement), and pharmacokinetic properties (>10-fold increase in poDNAUC). Optimal members of the series have properties suitable for use in in vitro and in vivo experiments aimed at elucidating the role of TNNI3K in cardiac biology and serve as leads for developing novel heart failure medicines.

Percutaneous coronary intervention is first-line therapy for acute coronary syndromes (ACS) but can promote cardiomyocyte death and cardiac dysfunction via reperfusion injury, a phenomenon driven in large part by oxidative stress. Therapies to limit this progression have proven elusive, with no major classes of new agents since the development of anti-platelets/anti-thrombotics. We report that cardiac troponin I-interacting kinase (TNNI3K), a cardiomyocyte-specific kinase, promotes ischemia/reperfusion injury, oxidative stress, and myocyte death. TNNI3K-mediated injury occurs through increased mitochondrial superoxide production and impaired mitochondrial function and is largely dependent on p38 mitogen-activated protein kinase (MAPK) activation. We developed a series of small-molecule TNNI3K inhibitors that reduce mitochondrial-derived superoxide generation, p38 activation, and infarct size when delivered at reperfusion to mimic clinical intervention. TNNI3K inhibition also preserves cardiac function and limits chronic adverse remodeling. Our findings demonstrate that TNNI3K modulates reperfusion injury in the ischemic heart and is a tractable therapeutic target for ACS. Pharmacologic TNNI3K inhibition would be cardiac-selective, preventing potential adverse effects of systemic kinase inhibition.

NADPH oxidase isoform-2 (NOX2) generates reactive oxygen species (ROS) that contribute to neurodegenerative and cardiovascular pathologies. However, validation of NOX2 as a pharmacotherapeutic target has been hampered by a lack of mechanistically-defined inhibitors. Using cellular and biochemical assays, we explored previously reported inhibitors of ROS production (perhexiline, suramin, VAS2870 and two Shionogi patent compounds) as direct NOX2 inhibitors. All but suramin, which presumably lacks cell penetrance, inhibit cellular ROS production. However, only perhexiline and suramin inhibit biochemical NOX2 activity. Indeed, our data suggest that NOX2 inhibition by perhexiline may contribute significantly to its demonstrated cardioprotective effects. Inhibition of protein kinase CβII explains the cellular activity of the Shionogi compounds, whereas VAS2870 inhibits by an as-yet unidentified mechanism unrelated to direct NOX2 function or subunit assembly. These data delineate the mechanisms of action of these compounds and highlight their strengths and limitations for use in future target validation studies.

Superoxide affects many normal and pathogenic cellular processes, and the detection of superoxide produced by cells is therefore of interest for potential therapeutic applications. To develop a high-throughput cell-based assay for the detection of extracellular superoxide production that could be run in a 384-well or 1536-well format, 2 luminescent reagents, Lucigenin and Diogenes, and one fluorescent reagent, Oxyburst Green BSA, were tested. HL-60 cells, which had been differentiated to a neutrophil-like phenotype with DMSO and frozen in large batches, were used in assays. All 3 superoxide detection reagents performed well statistically in terms of IC(50) reproducibility and met a desired Z' value requirement of >0.4. When tested against a 1408-compound test set at 5 or 10 microM compound concentration, a higher hit rate was obtained with the 2 luminescent reagents compared with that obtained with the fluorescent Oxyburst Green BSA reagent. The Oxyburst Green BSA assay was ultimately chosen for compound profiling and high-throughput screening activities. This 1536 superoxide detection assay using cryopreserved differentiated HL-60 cells represents a shifting paradigm toward the utilization of more therapeutically relevant cells in early drug development activities.

IroB is a C-glycosyltransferase encoded in the iroA cluster. C-Glucosylation of the bacterial siderophore enterobactin by IroB is a strategy some pathogenic bacteria use to evade the host's innate immunity mediated by lipocalin 2 (Lcn2). Without this modification, enterobactin can be tightly bound by host Lcn2, rendering it ineffective as a siderophore. Therefore, IroB inhibitors could be potential antibiotics against iroA-harboring pathogenic bacteria. We used enterobactin analogues to probe the properties of the active site of IroB and found that enterobactin analogues brominated at the C5 positions of the 2,3-dihydroxybenzoyl rings are potent inhibitors of IroB. This finding could lead to the discovery of effective antibiotics targeting iroA-containing bacteria.

Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner.

The diversity of distinct covalent forms of proteins (the proteome) greatly exceeds the number of proteins predicted by DNA coding capacities owing to directed posttranslational modifications. Enzymes dedicated to such protein modifications include 500 human protein kinases, 150 protein phosphatases, and 500 proteases. The major types of protein covalent modifications, such as phosphorylation, acetylation, glycosylation, methylation, and ubiquitylation, can be classified according to the type of amino acid side chain modified, the category of the modifying enzyme, and the extent of reversibility. Chemical events such as protein splicing, green fluorescent protein maturation, and proteasome autoactivations also represent posttranslational modifications. An understanding of the scope and pattern of the many posttranslational modifications in eukaryotic cells provides insight into the function and dynamics of proteome compositions.

During biosynthesis of the anthracycline antitumor agents daunomycin, adriamycin, and aclacinomycin, the polyketide-derived tetracyclic aglycone is enzymatically glycosylated at the C7-OH by dedicated glycosyltransferases (Gtfs) that transfer L-2,3,6-trideoxy-3-aminohexoses. In aclacinomycins, the first deoxyhexose is predicted to be transferred via AknS action, then subjected to further elongation to a trisaccharide by the subsequent Gtf, AknK. We report here that purified AknS has very low activity in the absence of the adjacently encoded AknT; however, at a 3:1 ratio, AknT stimulates AknS k(cat) by 40-fold up to 0.22 min(-1) for transfer of L-2-deoxyfucose (2-dF) to the aglycone aklavinone. It is likely that several other Gtfs that glycosylate polyketide aglycones also act as two-component catalytic systems. Incubations of purified AknS/AknT/AknK with two aglycones and two dTDP-2-deoxyhexoses produced previously uncharacterized anthracycline disaccharides.

Rapamycin, FK506, and FK520 are potent immunosuppressant natural product macrocycles generated by hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) systems in streptomycetes. An important functional element within these molecules is an l-pipecolate moiety that is incorporated into the completed polyketide chain by the action of RapP/FkbP, a four-domain NRPS that also putatively serves to cyclize the chain after amino acid insertion. Here we report the expression and purification of recombinant FkbP from the FK520 biosynthetic pathway. Using a combination of radioassays and Fourier transform mass spectrometry, we demonstrate that once FkbP has been phosphopantetheinylated in vitro, its peptidyl carrier protein domain can be successfully loaded with l-pipecolic acid and, to a lesser extent, l-proline. The first condensation domain of FkbP is shown to be active through the successful acetylation of aminoacyl-S-FkbP using the appropriately loaded terminal acyl carrier protein from the PKS array, FkbA, as the chain donor. Site-directed mutagenesis confirmed that the N-terminal condensation domain catalyzes the transfer reaction. Acetylation of prolyl-S-FkbP was more rapid and occurred to a greater extent than that of pipecolyl-S-FkbP, a trend which was also observed with alternative acyl chain donors. These observations suggest that the adenylation domain of FkbP serves as the primary selectivity filter for pipecolate incorporation.

The majority of proteins targeted to the peroxisomal lumen contain a C-terminal peroxisomal targeting signal-1 (PTS1) that is bound by the peroxin Pex5p. The PTS1 is generally regarded as a C-terminal tripeptide that adheres to the consensus (S/A/C)(K/R/H)(L/M). Previously, we studied the binding affinity of peptides of the form YQX(-3)X(-2)X(-1) to the peptide-binding domain of human Pex5p (referred to as Pex5p-C). Optimal affinity was found for YQSKL, which bound with an affinity of 200 +/- 40 nM. To extend this work, we investigated the properties of a peptide containing the last 9 residues of acyl-CoA oxidase (RHYLKPLQSKL) and discovered that it binds to Pex5p-C with a dissociation constant of 1.4 +/- 0.4 nM, 180 times tighter than YQSKL. Further analysis revealed that the enhanced affinity is primarily due to the presence of leucine in the (-5) position. In addition, a peptide corresponding to the luciferase C-terminus (YKGGKSKL) was found to bind Pex5p-C about 20 times tighter than YQSKL. The majority of this effect results from having lysine in position (-4). Catalase contains a noncanonical PTS1 (-AREKANL). The affinity of YQANL was found to be 3600 +/- 400 nM. This relatively weak binding is consistent with previous unsuccessful attempts to direct chloramphenicol acetyltransferase to the peroxisome by fusing -ANL to its C-terminus (-GGA-ANL). The peptides YKANL, YEKANL, YREKANL, and YAREKANL all bound Pex5p-C with higher affinities than did YQANL, but the affinities are still lower than peptides that correspond to functional targeting signals in other contexts. Because both catalase and Pex5p are tetramers (as opposed to the monomeric Pex5p-C and the peptides used in our studies), multidentate effects on binding affinity between Pex5p and other oligomeric proteins should be considered. Our study provides direct thermodynamic data revealing that peptide binding to Pex5p-C binding is favored by lysine in the (-4) position and leucine in the (-5) position. Our results suggest that peptides or proteins with optimized residues in the (-4) and/or (-5) positions can bind to Pex5p with affinities that are at least two orders of magnitude greater than that of YQSKL, and that this stabilization can compensates for otherwise weakly binding PTS1s., (Copyright 2004 Wiley-Liss, Inc.)

The availability of complete genome sequences enables the statistical analysis of sequence features without significant database-imposed bias. The carboxyl termini of proteins often contain regions associated with protein targeting and enhanced translational termination. We analyzed the frequency of occurrence of C-terminal tripeptides in representative archaeal, bacterial, and eukaryotic genomes. The sequence distribution in prokaryotic genomes nearly matches that generated by the randomization of the observed tripeptide set. In contrast, eukaryotic genomes contain large numbers of overrepresented sequences. Some of these correspond to highly repeated sequences from either duplicated endogenous genes or transposon open reading frames. Gratifyingly, others represent previously known targeting signals or sequences associated with an increase in translational termination efficiency. However, a number of overrepresented tripeptides have not been previously noted and may represent novel functional sequences. For example, the sequence XSS may enhance translational termination efficiency in plants, whereas FWC may be a targeting or processing signal for certain amino acid permeases in yeast.

Many proteins that are destined to reside within the lumen of the peroxisome contain the peroxisomal targeting signal-1 (PTS1), a C-terminal tripeptide approximating the consensus sequence -Ser-Lys-Leu-COO(-). The PTS1 is recognized by the tetratricopeptide repeat (TPR) domains of PEX5, a cytosolic receptor that cycles between the cytoplasm and the peroxisome. To gain insight into the energetics of PTS1 binding specificity and to correlate these with features from the recently determined structure of a PEX5:PTS1 complex, we used a fluorescence-based binding assay that enables the quantitation of the dissociation constants for PTS1-containing peptide complexes with the TPR region of human PEX5. Through application of this assay to a collection of pentapeptides containing different C-terminal tripeptide sequences, including both natural and unnatural amino acids, the thermodynamic effects of sequence variation were examined. PTS1 variants that correspond to known functional targeting signals bind to the PEX5 fragment with a change in the standard binding free energy within 1.8 kcal mol(-1) of that corresponding to the peptide ending with -Ser-Lys-Leu-COO(-). The results suggest that a binding energy threshold may determine the functionality of PTS1 sequences.

Many proteins contain targeting signals within their sequences that specify their delivery to particular organelles. The peroxisomal targeting signal-1 (PTS1) is a C-terminal tripeptide that is sufficient to direct proteins into peroxisomes. The PTS1 sequence closely approximates Ser-Lys-Leu-COO-. PEX5, the receptor for PTS1, interacts with the signal via a series of tetratricopeptide repeats (TPRs) within its C-terminal half. Here we report the crystal structure of a fragment of human PEX5 that includes all seven predicted TPR motifs in complex with a pentapeptide containing a PTS1 sequence. Two clusters of three TPRs almost completely surround the peptide, while a hinge region, previously identified as TPR4, forms a distinct structure that enables the two sets of TPRs to form a single binding site. This structure reveals the molecular basis for PTS1 recognition and demonstrates a novel mode of TPR-peptide interaction.

The three-dimensional structure of a protein can greatly illuminate the relationship between its sequence and its function. However, in the absence of a set of experimentally derived coordinates, one often seeks a model of the protein of interest to guide future study. We describe the combined utilization of orthologous sequence information along with knowledge of the related structural fold to model the interaction between PEX5 and its ligand, the peroxisomal targeting signal-1 (PTS1). With this model, we are able to identify residues within PEX5 that appear to be important for peptide recognition, as well as explain some of the sequence requirements of the PTS1. Specifically, our model highlights four asparagine residues as important for ligand backbone atom recognition, which, along with previously observed examples, suggests this as a general mechanism for the binding of extended polypeptides.

Most biological information is contained within gene and genome sequences. However, current methods for analyzing these data are limited primarily to the prediction of coding regions and identification of sequence similarities. We have developed a computer algorithm, CoSMoS (for context sensitive motif searches), which adds context sensitivity to sequence motif searches. CoSMoS was challenged to identify genes encoding peroxisome-associated and oleate-induced genes in the yeast Saccharomyces cerevisiae. Specifically, we searched for genes capable of encoding proteins with a type 1 or type 2 peroxisomal targeting signal and for genes containing the oleate-response element, a cis-acting element common to fatty acid-regulated genes. CoSMoS successfully identified 7 of 8 known PTS-containing peroxisomal proteins and 13 of 14 known oleate-regulated genes. More importantly, CoSMoS identified an additional 18 candidate peroxisomal proteins and 300 candidate oleate-regulated genes. Preliminary localization studies suggest that these include at least 10 previously unknown peroxisomal proteins. Phenotypic studies of selected gene disruption mutants suggests that several of these new peroxisomal proteins play roles in growth on fatty acids, one is involved in peroxisome biogenesis and at least two are required for synthesis of lysine, a heretofore unrecognized role for peroxisomes. These results expand our understanding of peroxisome content and function, demonstrate the utility of CoSMoS for context-sensitive motif scanning, and point to the benefits of improved in silico genome analysis.