ABSTRACT
Nesprins comprise a family of multi-isomeric scaffolding proteins, forming the linker of nucleoskeleton-and-cytoskeleton complex with lamin A/C, emerin and SUN1/2 at the nuclear envelope. Mutations in nesprin-1/-2 are associated with Emery-Dreifuss muscular dystrophy (EDMD) with conduction defects and dilated cardiomyopathy (DCM). We have previously observed sarcomeric staining of nesprin-1/-2 in cardiac and skeletal muscle, but nesprin function in this compartment remains unknown. In this study, we show that specific nesprin-2 isoforms are highly expressed in cardiac muscle and localize to the Z-disc and I band of the sarcomere. Expression of GFP-tagged nesprin-2 giant spectrin repeats 52 to 53, localized to the sarcomere of neonatal rat cardiomyocytes. Yeast two-hybrid screening of a cardiac muscle cDNA library identified telethonin and four-and-half LIM domain (FHL)-2 as potential nesprin-2 binding partners. GST pull-down and immunoprecipitation confirmed the individual interactions between nesprin-2/telethonin and nesprin-2/FHL-2, and showed that nesprin-2 and telethonin binding was dependent on telethonin phosphorylation status. Importantly, the interactions between these binding partners were impaired by mutations in nesprin-2, telethonin, and FHL-2 identified in EDMD with DCM and hypertrophic cardiomyopathy patients. These data suggest that nesprin-2 is a novel sarcomeric scaffold protein that may potentially participate in the maintenance and/or regulation of sarcomeric organization and function.
Subject(s)
Connectin , LIM Domain Proteins , Muscle Proteins , Myocytes, Cardiac , Nerve Tissue Proteins , Nuclear Proteins , Sarcomeres , Animals , Humans , Mice , Rats , Connectin/metabolism , Connectin/genetics , Cytoskeletal Proteins/metabolism , Cytoskeletal Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , LIM Domain Proteins/metabolism , LIM Domain Proteins/genetics , LIM-Homeodomain Proteins , Microfilament Proteins/metabolism , Microfilament Proteins/genetics , Muscle Proteins/metabolism , Muscle Proteins/genetics , Myocytes, Cardiac/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Protein Binding , Sarcomeres/metabolism , Transcription FactorsABSTRACT
Emery-Dreifuss muscular dystrophy (EDMD) is a genetically and clinically variable disorder. Previous attempts to use gene expression changes to find its pathomechanism were unavailing, so we engaged a functional pathway analysis. RNA-Seq was performed on cells from 10 patients diagnosed with an EDMD spectrum disease with different mutations in seven genes. Upon comparing to controls, the pathway analysis revealed that multiple genes involved in fibrosis, metabolism, myogenic signaling and splicing were affected in all patients. Splice variant analysis revealed alterations of muscle-specific variants for several important muscle genes. Deeper analysis of metabolic pathways revealed a reduction in glycolytic and oxidative metabolism and reduced numbers of mitochondria across a larger set of 14 EDMD spectrum patients and 7 controls. Intriguingly, the gene expression signatures segregated the patients into three subgroups whose distinctions could potentially relate to differences in clinical presentation. Finally, differential expression analysis of miRNAs changing in the patients similarly highlighted fibrosis, metabolism and myogenic signaling pathways. This pathway approach revealed a transcriptome profile that can both be used as a template for establishing a biomarker panel for EDMD and direct further investigation into its pathomechanism. Furthermore, the segregation of specific gene changes into distinct groups that appear to correlate with clinical presentation may template development of prognostic biomarkers, though this will first require their testing in a wider set of patients with more clinical information.
Subject(s)
Muscular Dystrophy, Emery-Dreifuss , Humans , Muscular Dystrophy, Emery-Dreifuss/genetics , Mutation , Fibrosis , BiomarkersABSTRACT
Tuberculosis remains a leading cause of death from a single infection worldwide. Drug resistance to existing and even new antimycobacterials calls for research into novel targets and unexplored mechanisms of action. Recently we reported on the development of tight-binding inhibitors of Mycobacterium tuberculosis (Mtb) lipoamide dehydrogenase (Lpd), which selectively inhibit the bacterial but not the human enzyme based on a differential modality of inhibitor interaction with these targets. Here we report on the striking improvement in inhibitor residence time on the Mtb enzyme associated with scaffold progression from an indazole to 2-cyanoindole. Cryo-EM of Lpd with the bound 2-cyanoindole inhibitor 19 confirmed displacement of the buried water molecule deep in the binding channel with a cyano group. The ensuing hours-long improvement in on-target residence time is associated with enhanced antibacterial activity in axenic culture and in primary mouse macrophages. Resistance to 2-cyanoindole inhibitors involves mutations within the inhibitor binding site that have little effect on inhibitor affinity but change the modality of inhibitor-target interaction, resulting in fast dissociation from Lpd. These findings underscore that on-target residence time is a major determinant of antibacterial activity and in vivo efficacy.
ABSTRACT
Late-onset Pompe disease (LOPD) is a rare genetic disorder produced by mutations in the GAA gene and is characterized by progressive muscle weakness. LOPD muscle biopsies show accumulation of glycogen along with the autophagic vacuoles associated with atrophic muscle fibers. The expression of molecules related to muscle fiber atrophy in muscle biopsies of LOPD patients was studied using immunofluorescence and real-time PCR. BCL2 and adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a well-known atrogene, was identified as a potential mediator of muscle fiber atrophy in LOPD muscle biopsies. Vacuolated fibers in LOPD patient muscle biopsies were smaller than nonvacuolated fibers and expressed BNIP3. The current data suggested that BNIP3 expression is regulated by inhibition of the AKT-mammalian target of rapamycin pathway, leading to phosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1) at Ser317 by AMP-activated protein kinase. Myoblasts and myotubes obtained from LOPD patients and age-matched controls were studied to confirm these results using different molecular techniques. Myotubes derived from LOPD patients were likewise smaller and expressed BNIP3. Conclusively, transfection of BNIP3 into control myotubes led to myotube atrophy. These findings suggest a cascade that starts with the inhibition of the AKT-mammalian target of rapamycin pathway and activation of BNIP3 expression, leading to progressive muscle fiber atrophy. These results open the door to potential new treatments targeting BNIP3 to reduce its deleterious effects on muscle fiber atrophy in Pompe disease.
Subject(s)
Glycogen Storage Disease Type II , Atrophy/pathology , Glycogen Storage Disease Type II/genetics , Glycogen Storage Disease Type II/pathology , Humans , Membrane Proteins/genetics , Muscle Fibers, Skeletal/metabolism , Proto-Oncogene Proteins , Proto-Oncogene Proteins c-akt , TOR Serine-Threonine Kinases/metabolismABSTRACT
Free energy perturbation is a computational technique that can be used to predict how small changes to an inhibitor structure will affect the binding free energy to its target. In this paper, we describe the utility of free energy perturbation with FEP+ in the hit-to-lead stage of a drug discovery project targeting soluble adenyl cyclase. The project was structurally enabled by X-ray crystallography throughout. We employed free energy perturbation to first scaffold hop to a preferable chemotype and then optimize the binding affinity to sub-nanomolar levels while retaining druglike properties. The results illustrate that effective use of free energy perturbation can enable a drug discovery campaign to progress rapidly from hit to lead, facilitating proof-of-concept studies that enable target validation.
Subject(s)
Adenylyl Cyclases , Drug Discovery , Thermodynamics , EntropyABSTRACT
BACKGROUND: Glucocorticoids play a significant role in metabolic processes and pathways that impact muscle size, mass, and function. The expression of 11-beta-hydroxysteroid dehydrogenase type 1 (HSD11B1) has been previously described as a major regulator of skeletal muscle function in glucocorticoid-induced muscle atrophy and aging humans. Our study aimed to investigate glucocorticoid metabolism, including the expression of HSD11B1 in skeletal muscle, in patients with sarcopenia. METHODS: Muscle biopsies were taken from the vastus lateralis muscle of thirty-three patients over 60 years of age with hip fractures. Sarcopenia status was assessed according to the criteria of the European Working Group on Sarcopenia in Older People 2. Skeletal muscle mass was measured by bioelectrical impedance analysis. Cortisol and cortisone concentrations were measured in serum. Gene expression analysis of HSD11B1, NR3C1, FBXO32, and TRIM63 in muscle biopsies was performed. Serial cross sections of skeletal muscle were labeled with myosin heavy chain slow (fiber type-1) and fast (fiber type-2) antibodies. RESULTS: The study included 33 patients (21 women) with a mean age of 82.5 ± 6.3 years, 17 patients revealed sarcopenic (n = 16 non-sarcopenic). Serum cortisone concentrations were negatively correlated with muscle mass (ß = - 0.425; p = 0.034) and type-2 fiber diameter (ß = - 0.591; p = 0.003). Gene expression of HSD11B1 (ß = - 0.673; p = 0.008) showed a negative correlation with muscle mass in the sarcopenic group. A significant correlation was found for the non-sarcopenic group for NR3C1 (ß = 0.548; p = 0.028) and muscle mass. CONCLUSION: These findings suggest a pathogenetic role of HSD11B1 in sarcopenic muscle.
Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 1 , Cortisone , Sarcopenia , Aged , Aged, 80 and over , Female , Humans , Middle Aged , 11-beta-Hydroxysteroid Dehydrogenase Type 1/genetics , 11-beta-Hydroxysteroid Dehydrogenase Type 1/metabolism , Cortisone/metabolism , Gene Expression , Glucocorticoids/metabolism , Muscle, Skeletal , Sarcopenia/geneticsABSTRACT
A drug accelerator that partners the creative power of academic scientists with drug discovery professionals to consistently advance groundbreaking biological discoveries would be transformational. One such model, the Tri-Institutional Therapeutics Discovery Institute, evolved a series of best practices for identifying, selecting, executing, and completing academic-initiated drug discovery projects, is described.
Subject(s)
Academies and Institutes , Drug DiscoveryABSTRACT
Novel bacterial topoisomerase inhibitors (NBTIs) are the newest members of gyrase inhibitor broad-spectrum antibacterial agents, represented by the most advanced member, gepotidacin, a 4-amino-piperidine linked NBTI, which is undergoing phase III clinical trials for treatment of urinary tract infections (UTI). We have extensively reported studies on oxabicyclooctane linked NBTIs, including AM-8722. The present study summarizes structure activity relationship (SAR) of AM-8722 leading to identification of 7-fluoro-1-cyanomethyl-1,5-naphthyridin-2-one based NBTI (16, AM-8888) with improved potency and spectrum (MIC values of 0.016-4 µg/mL), with Pseudomonas aeruginosa being the least sensitive strain (MIC 4 µg/mL).
Subject(s)
Anti-Bacterial Agents , Topoisomerase Inhibitors , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , DNA Gyrase/metabolism , DNA Topoisomerase IV , Microbial Sensitivity Tests , Staphylococcus aureus/metabolism , Structure-Activity Relationship , Thioinosine/analogs & derivatives , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacology , Topoisomerase Inhibitors/chemistry , Topoisomerase Inhibitors/pharmacologyABSTRACT
BACKGROUND: Hereditary angioedema (HAE) is a potentially fatal disorder resulting in recurrent attacks of severe swelling. It may be associated with a genetic deficiency of functional C1 inhibitor or with normal C1 inhibitor (HAEnCI). In families with HAEnCI, HAE-linked mutations in the F12, PLG, KNG1, ANGPT1, or MYOF genes have been identified. In many families with HAEnCI the genetic cause of the disease is currently unknown. OBJECTIVE: The aim of this study was to identify a novel disease-linked mutation for HAEnCI. METHODS: The study methods comprised whole exome sequencing, Sanger sequencing analysis, pedigree analysis, bioinformatic analysis of the mutation, and biochemical analysis of parameters of the kallikrein-kinin (contact) system. RESULTS: By performing whole exome sequencing on a multigenerational family with HAEnCI we were able to identify the heparan sulfate (HS)-glucosamine 3-O-sulfotransferase 6 (HS3ST6) mutation c.430A>T (p.Thr144Ser) in all 3 affected family members who were sequenced. This gene encodes HS-glucosamine 3-O-sulfotransferase 6 (3-OST-6), which is involved in the last step of HS biosynthesis. The p.Thr144Ser mutation is likely to affect the interaction between 2 ß-sheets stabilizing the active center of the 3-OST-6 protein. CONCLUSIONS: We conclude that mutant 3-OST-6 fails to transfer sulfo groups to the 3-OH position of HS, resulting in incomplete HS biosynthesis. This likely affects cell surface interactions of key players in angioedema formation and is a novel mechanism for disease development.
Subject(s)
Angioedemas, Hereditary/genetics , Sulfotransferases/genetics , Adult , Aged, 80 and over , Female , Humans , Middle Aged , Mutation , Exome SequencingABSTRACT
Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkalinization, beat frequency and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and human sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in women.
Subject(s)
Adenylyl Cyclase Inhibitors/pharmacology , Fertilization/drug effects , Spermatozoa/drug effects , Adenylyl Cyclases/genetics , Adenylyl Cyclases/physiology , Animals , Cells, Cultured , Female , Fertilization/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Knockout , Pregnancy , Spermatozoa/physiologyABSTRACT
BACKGROUND: Sarcopenia is the age-related loss of muscle mass and strength. Undiagnosed late-onset neuromuscular disorders need to be considered in the differential diagnosis of sarcopenia. AIM: Based on emblematic case reports and current neuromuscular diagnostic guidelines for three common late-onset neuromuscular disorders, a differential diagnostic approach for geriatric patients presenting with a sarcopenic phenotype is given. METHODS: Patients over 65 years of age with sarcopenia, amyotrophic lateral sclerosis, inclusion body myositis and myotonic dystrophy type 2 were recruited. All patients were assessed for sarcopenia based on the revised European consensus definition. Patients with neuromuscular diseases were diagnosed according to the revised El Escorial criteria and the European neuromuscular centre criteria. Phenotypes and diagnostic criteria for all patients were summarized including their specific histopathological findings. RESULTS: All patients with neuromuscular diseases were positively screened for sarcopenia and classified as severe sarcopenic by means of assessment. The clinical phenotype, the evolution pattern of weakness and muscle atrophy combined with laboratory finding including electromyography could unquestionably distinguish the diseases. DISCUSSION: Neuromuscular disorders can manifest beyond the age of 65 years and misdiagnosed as sarcopenia. The most common diseases are inclusion body myositis, amyotrophic lateral sclerosis and myotonic dystrophy type 2. A diagnostic work-up for neuromuscular diseases ensures their correct diagnosis by clinical-, electrophysiological, histopathological, and genetic work-up. CONCLUSIONS: In geriatric patients with a focal or asymmetrical muscular weakness and atrophy, sarcopenia assessment should be extended with patient's history of disease course. Furthermore, concomitant diseases, analysis of serum creatine kinase, electrophysiological examination, and in selected patients muscle biopsy and gene analysis is needed to rule out a late-onset neuromuscular disorder.
Subject(s)
Neuromuscular Diseases/diagnosis , Sarcopenia/diagnosis , Aged , Aged, 80 and over , Amyotrophic Lateral Sclerosis/diagnosis , Diagnosis, Differential , Electromyography , Humans , Myotonic Dystrophy/diagnosisABSTRACT
BACKGROUND: Previous research has described a neuroprotective effect of IGF-I, supporting neuronal survival, axon growth and proliferation of muscle cells. Therefore, the association between IGF-I concentration, muscle histology and electrophysiological markers in a cohort of patients with sarcopenia dares investigation. METHODS: Measurement of serum concentrations of IGF-I and binding partners, electromyographic measurements with the MUNIX (Motor Unit Number Index) method and muscle biopsies were performed in 31 patients with acute hip fracture older age 60 years. Molecular markers for denervation (neural cell adhesion molecule NCAM) and proliferation markers (Ki67) were assessed by immunofluorescence staining of muscle biopsy tissue. Skeletal muscle mass by bioelectrical impedance analysis and hand-grip strength were measured to assess sarcopenia status according to EWGSOP2 criteria. RESULTS: Thirty-one patients (20 women) with a mean age of 80.6 ± 7.4 years were included. Concentrations of IGF-I and its binding partners were significantly associated with sarcopenia (ß = - 0.360; p = 0.047) and MUNIX (ß = 0.512; p = 0.005). Further, expression of NCAM (ß = 0.380; p = 0.039) and Ki67 (ß = 0.424; p = 0.022) showed significant associations to IGF-I concentrations. CONCLUSIONS: The findings suggest a pathogenetic role of IGF-I in sarcopenia based on muscle denervation.
Subject(s)
Sarcopenia , Aged , Aged, 80 and over , Female , Hand Strength , Humans , Insulin-Like Growth Factor I , Muscle, Skeletal/pathology , Regeneration , Sarcopenia/diagnosisABSTRACT
Myotonic dystrophy type 1 (DM1) is caused by CTG-repeat expansions leading to a complex pathology with a multisystemic phenotype that primarily affects the muscles and brain. Despite a multitude of information, especially on the alternative splicing of several genes involved in the pathology, information about additional factors contributing to the disease development is still lacking. We performed RNAseq and gene expression analyses on proliferating primary human myoblasts and differentiated myotubes. GO-term analysis indicates that in myoblasts and myotubes, different molecular pathologies are involved in the development of the muscular phenotype. Gene set enrichment for splicing reveals the likelihood of whole, differentiation stage specific, splicing complexes that are misregulated in DM1. These data add complexity to the alternative splicing phenotype and we predict that it will be of high importance for therapeutic interventions to target not only mature muscle, but also satellite cells.
Subject(s)
Myoblasts/metabolism , Myotonic Dystrophy/genetics , RNA Splicing , Transcriptome , Adult , Cell Differentiation , Cells, Cultured , Humans , Myoblasts/cytology , Myotonic Dystrophy/metabolismABSTRACT
Plasmodium falciparum proteasome (Pf20S) inhibitors are active against Plasmodium at multiple stages-erythrocytic, gametocyte, liver, and gamete activation stages-indicating that selective Pf20S inhibitors possess the potential to be therapeutic, prophylactic, and transmission-blocking antimalarials. Starting from a reported compound, we developed a noncovalent, macrocyclic peptide inhibitor of the malarial proteasome with high species selectivity and improved pharmacokinetic properties. The compound demonstrates specific, time-dependent inhibition of the ß5 subunit of the Pf20S, kills artemisinin-sensitive and artemisinin-resistant P.â falciparum isolates inâ vitro and reduces parasitemia in humanized, P.â falciparum-infected mice.
Subject(s)
Antimalarials/pharmacology , Drug Development , Malaria, Falciparum/drug therapy , Plasmodium falciparum/drug effects , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Animals , Antimalarials/chemical synthesis , Antimalarials/chemistry , Malaria, Falciparum/metabolism , Mice , Models, Molecular , Molecular Conformation , Parasitic Sensitivity Tests , Plasmodium falciparum/enzymology , Proteasome Inhibitors/chemical synthesis , Proteasome Inhibitors/chemistryABSTRACT
Efforts to develop new male or female nonhormonal, orally available contraceptives assume that to be effective and safe, targets must be (1) essential for fertility; (2) amenable to targeting by small-molecule inhibitors; and (3) restricted to the germline. In this perspective, we question the third assumption and propose that despite its wide expression, soluble adenylyl cyclase (sAC: ADCY10), which is essential for male fertility, is a valid target. We hypothesize that an acute-acting sAC inhibitor may provide orally available, on-demand, nonhormonal contraception for men without adverse, mechanism-based effects. To test this concept, we describe a collaboration between academia and the unique capabilities of a public-private drug discovery institute.
Subject(s)
Contraceptive Agents , Drug Discovery , Adenylyl Cyclases , Humans , LeadABSTRACT
Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The result of the GAA deficiency is a ubiquitous lysosomal and non-lysosomal accumulation of glycogen. The most affected tissues are heart, skeletal muscle, liver, and the nervous system. Replacement therapy with the currently approved enzyme relies on M6P-mediated endocytosis. However, therapeutic outcomes still leave room for improvement, especially with regard to skeletal muscles. We tested the uptake, activity, and effect on glucose metabolism of a non-phosphorylated recombinant human GAA produced in moss (moss-GAA). Three variants of moss-GAA differing in glycosylation pattern have been analyzed: two with terminal mannose residues in a paucimannosidic (Man3) or high-mannose (Man 5) configuration and one with terminal N-acetylglucosamine residues (GnGn). Compared to alglucosidase alfa the moss-GAA GnGn variant showed increased uptake in differentiated myotubes. Moreover, incubation of immortalized muscle cells of Gaa-/- mice with moss-GAA GnGn led to similarly efficient clearance of accumulated glycogen as with alglucosidase alfa. These initial data suggest that M6P-residues might not always be necessary for the cellular uptake in enzyme replacement therapy (ERT) and indicate the potential of moss-GAA GnGn as novel alternative drug for targeting skeletal muscle in Pompe patients.
Subject(s)
Enzyme Replacement Therapy , Glycogen Storage Disease Type II/metabolism , Muscle Cells/drug effects , Muscle Cells/metabolism , Recombinant Proteins/pharmacology , Animals , Biomarkers , Bryophyta/genetics , Cells, Cultured , Energy Metabolism/drug effects , Enzyme Replacement Therapy/methods , Glycogen Storage Disease Type II/drug therapy , Glycogen Storage Disease Type II/etiology , Humans , Mice , Myoblasts/drug effects , Myoblasts/metabolism , Recombinant Proteins/therapeutic use , alpha-Glucosidases/pharmacology , alpha-Glucosidases/therapeutic useABSTRACT
Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease-Hutchinson-Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long-term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT-immortalized cell lines.
Subject(s)
Abnormal Karyotype , Genomic Instability , Membrane Proteins/genetics , Microtubule-Associated Proteins/genetics , Nuclear Proteins/genetics , Progeria/genetics , Telomerase/genetics , Telomere Homeostasis , Cell Line , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Membrane Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Nuclear Proteins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Telomerase/metabolismABSTRACT
Cancer cells reprogram their metabolism to support growth and to mitigate cellular stressors. The serine synthesis pathway has been identified as a metabolic pathway frequently altered in cancers and there has been considerable interest in developing pharmacological agents to target this pathway. Here, we report a series of indole amides that inhibit human 3-phosphoglycerate dehydrogenase (PHGDH), the enzyme that catalyzes the first committed step of the serine synthesis pathway. Using X-ray crystallography, we show that the indole amides bind the NAD+ pocket of PHGDH. Through structure-based optimization we were able to develop compounds with low nanomolar affinities for PHGDH in an enzymatic IC50 assay. In cellular assays, the most potent compounds inhibited de novo serine synthesis with low micromolar to sub-micromolar activities and these compounds successfully abrogated the proliferation of cancer cells in serine free media. The indole amide series reported here represent an important improvement over previously published PHGDH inhibitors as they are markedly more potent and their mechanism of action is better defined.
Subject(s)
Amides/chemistry , Enzyme Inhibitors/chemistry , Indoles/chemistry , Phosphoglycerate Dehydrogenase/antagonists & inhibitors , Serine/biosynthesis , Amides/metabolism , Amides/pharmacology , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Molecular Dynamics Simulation , Phosphoglycerate Dehydrogenase/metabolism , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning.