RESUMEN
Parasitic cysteine proteases such as rhodesain (TbCatL) from Trypanosoma brucei rhodesiense are relevant targets for developing new potential drugs against parasitic diseases (e. g. Human African Trypanosomiasis). Designing selective inhibitors for parasitic cathepsins can be challenging as they share high structural similarities with human cathepsins. In this paper, we describe the development of novel peptidomimetic rhodesain inhibitors by applying a structure-based de novo design approach and molecular docking protocols. The inhibitors with a new scaffold in P2 and P3 position display high selectivity towards trypanosomal rhodesain over human cathepsins L and B and high antitrypanosomal activity. Vinylsulfonate 2a has emerged as a potent rhodesain inhibitor (k2nd = 883 ⢠103 M-1 s-1) with single-digit nanomolar binding affinity (Ki = 9 nM) and more than 150-fold selectivity towards human cathepsins and it thus constitutes an interesting starting compound for the further development of selective drugs against Human African Trypanosomiasis.
Asunto(s)
Peptidomiméticos , Tripanocidas , Trypanosoma brucei brucei , Tripanosomiasis Africana , Animales , Catepsinas , Cisteína Endopeptidasas , Inhibidores de Cisteína Proteinasa/química , Humanos , Simulación del Acoplamiento Molecular , Peptidomiméticos/farmacología , Peptidomiméticos/uso terapéutico , Relación Estructura-Actividad , Tripanocidas/farmacología , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/tratamiento farmacológicoRESUMEN
Ubiquitin (Ub)-mediated regulation of plasmalemmal ion channel activity canonically occurs via stimulation of endocytosis. Whether ubiquitination can modulate channel activity by alternative mechanisms remains unknown. Here, we show that the transient receptor potential vanilloid 4 (TRPV4) cation channel is multiubiquitinated within its cytosolic N-terminal and C-terminal intrinsically disordered regions (IDRs). Mutagenizing select lysine residues to block ubiquitination of the N-terminal but not C-terminal IDR resulted in a marked elevation of TRPV4-mediated intracellular calcium influx, without increasing cell surface expression levels. Conversely, enhancing TRPV4 ubiquitination via expression of an E3 Ub ligase reduced TRPV4 channel activity but did not decrease plasma membrane abundance. These results demonstrate Ub-dependent regulation of TRPV4 channel function independent of effects on plasma membrane localization. Consistent with ubiquitination playing a key negative modulatory role of the channel, gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy, whereas increasing mutant TRPV4 ubiquitination partially suppressed channel overactivity. Together, these data reveal a novel mechanism via which ubiquitination of an intracellular flexible IDR domain modulates ion channel function independently of endocytic trafficking and identify a contributory role for this pathway in the dysregulation of TRPV4 channel activity by neuropathy-causing mutations.
Asunto(s)
Canales Catiónicos TRPV , Ubiquitinación , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Drosophila/genética , Drosophila/metabolismo , Humanos , Ratones , Mutación , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Ubiquitina/metabolismoRESUMEN
Rhodesain is a major cysteine protease of Trypanosoma brucei rhodesiense, a pathogen causing Human African Trypanosomiasis, and a validated drug target. Recently, we reported the development of α-halovinylsulfones as a new class of covalent reversible cysteine protease inhibitors. Here, α-fluorovinylsulfones/-sulfonates were optimized for rhodesain based on molecular modeling approaches. 2d, the most potent and selective inhibitor in the series, shows a single-digit nanomolar affinity and high selectivity toward mammalian cathepsins B and L. Enzymatic dilution assays and MS experiments indicate that 2d is a slow-tight binder (Ki = 3 nM). Furthermore, the nonfluorinated 2d-(H) shows favorable metabolism and biodistribution by accumulation in mice brain tissue after intraperitoneal and oral administration. The highest antitrypanosomal activity was observed for inhibitors with an N-terminal 2,3-dihydrobenzo[b][1,4]dioxine group and a 4-Me-Phe residue in P2 (2e/4e) with nanomolar EC50 values (0.14/0.80 µM). The different mechanisms of reversible and irreversible inhibitors were explained using QM/MM calculations and MD simulations.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Sulfonas/farmacología , Ácidos Sulfónicos/farmacología , Tripanocidas/farmacología , Compuestos de Vinilo/farmacología , Animales , Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/metabolismo , Inhibidores de Cisteína Proteinasa/toxicidad , Pruebas de Enzimas , Femenino , Células HeLa , Humanos , Cinética , Masculino , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Unión Proteica , Relación Estructura-Actividad , Sulfonas/síntesis química , Sulfonas/metabolismo , Sulfonas/toxicidad , Ácidos Sulfónicos/síntesis química , Ácidos Sulfónicos/metabolismo , Ácidos Sulfónicos/toxicidad , Tripanocidas/síntesis química , Tripanocidas/metabolismo , Tripanocidas/toxicidad , Trypanosoma brucei brucei/efectos de los fármacos , Compuestos de Vinilo/síntesis química , Compuestos de Vinilo/metabolismo , Compuestos de Vinilo/toxicidadRESUMEN
TRPV4 is a cell surface-expressed calcium-permeable cation channel that mediates cell-specific effects on cellular morphology and function. Dominant missense mutations of TRPV4 cause distinct, tissue-specific diseases, but the pathogenic mechanisms are unknown. Mutations causing peripheral neuropathy localize to the intracellular N-terminal domain whereas skeletal dysplasia mutations are in multiple domains. Using an unbiased screen, we identified the cytoskeletal remodeling GTPase RhoA as a TRPV4 interactor. TRPV4-RhoA binding occurs via the TRPV4 N-terminal domain, resulting in suppression of TRPV4 channel activity, inhibition of RhoA activation, and extension of neurites in vitro. Neuropathy but not skeletal dysplasia mutations disrupt TRPV4-RhoA binding and cytoskeletal outgrowth. However, inhibition of RhoA restores neurite length in vitro and in a fly model of TRPV4 neuropathy. Together these results identify RhoA as a critical mediator of TRPV4-induced cell structure changes and suggest that disruption of TRPV4-RhoA binding may contribute to tissue-specific toxicity of TRPV4 neuropathy mutations.
Asunto(s)
Neuritas/metabolismo , Enfermedades del Sistema Nervioso Periférico/genética , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Células COS , Calcio/metabolismo , Línea Celular , Chlorocebus aethiops , Drosophila , Células HEK293 , HumanosRESUMEN
The efficient production of many medicinally or synthetically important starting materials suffers from wasteful or toxic precursors for the synthesis. In particular, the aromatic non-protected primary amine function represents a versatile synthetic precursor, but its synthesis typically requires toxic oxidizing agents and transition metal catalysts. The twofold electrochemical amination of activated benzene derivatives via Zincke intermediates provides an alternative sustainable strategy for the formation of new C-N bonds of high synthetic value. As a proof of concept, we use our approach to generate a benzoxazinone scaffold that gained attention as a starting structure against castrate-resistant prostate cancer. Further improvement of the structure led to significantly increased cancer cell line toxicity. Thus, exploiting environmentally benign electrooxidation, we present a new versatile and powerful method based on direct C-H activation that is applicable for example the production of medicinally relevant compounds.
RESUMEN
Lipoic acid (LA) is a redox-active disulphide compound, which functions as a pivotal co-factor for mitochondrial oxidative decarboxylation. LA and chemical derivatives were shown to target mitochondria in cancer cells with altered energy metabolism, thereby inducing cell death. In this study, the impact of LA on the tumor suppressor protein p53 was analyzed in various colorectal cancer (CRC) cell lines, with a focus on the mechanisms driving p53 degradation. First, LA was demonstrated to trigger the depletion of both wildtype and mutant p53 protein in all CRC cells tested without influencing its gene expression and preceded LA-triggered cytotoxicity. Depletion of p53 coincided with a moderate, LA-dependent ROS production, but was not rescued by antioxidant treatment. LA induced the autophagy receptor p62 and differentially modulated autophagosome formation in CRC cells. However, p53 degradation was not mediated via autophagy as shown by chemical inhibition and genetic abrogation of autophagy. LA treatment also stabilized and activated the transcription factor Nrf2 in CRC cells, which was however dispensable for p53 degradation. Mechanistically, p53 was found to be readily ubiquitinylated and degraded by the proteasomal machinery following LA treatment, which did not involve the E3 ubiquitin ligase MDM2. Intriguingly, the combination of LA and anticancer drugs (doxorubicin, 5-fluorouracil) attenuated p53-mediated stabilization of p21 and resulted in synergistic killing in CRC cells in a p53-dependant manner.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ácido Tióctico/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Doxorrubicina/farmacología , Sinergismo Farmacológico , Metabolismo Energético/efectos de los fármacos , Fluorouracilo/farmacología , Células HCT116 , Humanos , Mutación , Proteolisis , Proteína p53 Supresora de Tumor/genética , UbiquitinaciónRESUMEN
Trypanosomal and leishmanial infections claim tens of thousands of lives each year. The metabolism of these unicellular eukaryotic parasites differs from the human host and their enzymes thus constitute promising drug targets. Tryparedoxin (Tpx) from Trypanosoma brucei is the essential oxidoreductase in the parasite's hydroperoxide-clearance cascade. Inâ vitro and inâ vivo functional assays show that a small, selective inhibitor efficiently inhibits Tpx. With X-ray crystallography, SAXS, analytical SEC, SEC-MALS, MD simulations, ITC, and NMR spectroscopy, we show how covalent binding of this monofunctional inhibitor leads to Tpx dimerization. Intra- and intermolecular inhibitor-inhibitor, protein-protein, and inhibitor-protein interactions stabilize the dimer. The behavior of this efficient antitrypanosomal molecule thus constitutes an exquisite example of chemically induced dimerization with a small, monovalent ligand that can be exploited for future drug design.
Asunto(s)
Antiprotozoarios/química , Proteínas Bacterianas/química , Inhibidores Enzimáticos/química , Oxidorreductasas/química , Tiorredoxinas/química , Trypanosoma brucei brucei/enzimología , Animales , Antiprotozoarios/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Glutatión/análogos & derivados , Glutatión/química , Humanos , Peróxido de Hidrógeno/metabolismo , Simulación de Dinámica Molecular , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Espermidina/análogos & derivados , Espermidina/química , Trypanosoma/metabolismo , Trypanosoma/parasitologíaRESUMEN
Transient receptor potential (TRP) channels are polymodally regulated ion channels. TRPV4 (vanilloid 4) is sensitized by PIP2 and desensitized by Syndapin3/PACSIN3, which bind to the structurally uncharacterized TRPV4 N terminus. We determined the nuclear magnetic resonance structure of the Syndapin3/PACSIN3 SH3 domain in complex with the TRPV4 N-terminal proline-rich region (PRR), which binds as a class I polyproline II (PPII) helix. This PPII conformation is broken by a conserved proline in a cis conformation. Beyond the PPII, we find that the proximal TRPV4 N terminus is unstructured, a feature conserved across species thus explaining the difficulties in resolving it in previous structural studies. Syndapin/PACSIN SH3 domain binding leads to rigidification of both the PRR and the adjacent PIP2 binding site. We determined the affinities of the TRPV4 N terminus for PACSIN1, 2, and 3 SH3 domains and PIP2 and deduce a hierarchical interaction network where Syndapin/PACSIN binding influences the PIP2 binding site but not vice versa.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Sitios de Unión , Pollos , Dicroismo Circular , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Dominios ProteicosRESUMEN
Colorectal cancer (CRC) is one of the most common tumor entities, which is causally linked to DNA repair defects and inflammatory bowel disease (IBD). Here, we studied the role of the DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) in CRC. Tissue microarray analysis revealed PARP-1 overexpression in human CRC, correlating with disease progression. To elucidate its function in CRC, PARP-1 deficient (PARP-1-/-) and wild-type animals (WT) were subjected to azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. Miniendoscopy showed significantly more tumors in WT than in PARP-1-/- mice. Although the lack of PARP-1 moderately increased DNA damage, both genotypes exhibited comparable levels of AOM-induced autophagy and cell death. Interestingly, miniendoscopy revealed a higher AOM/DSS-triggered intestinal inflammation in WT animals, which was associated with increased levels of innate immune cells and proinflammatory cytokines. Tumors in WT animals were more aggressive, showing higher levels of STAT3 activation and cyclin D1 up-regulation. PARP-1-/- animals were then crossed with O6-methylguanine-DNA methyltransferase (MGMT)-deficient animals hypersensitive to AOM. Intriguingly, PARP-1-/-/MGMT-/- double knockout (DKO) mice developed more, but much smaller tumors than MGMT-/- animals. In contrast to MGMT-deficient mice, DKO animals showed strongly reduced AOM-dependent colonic cell death despite similar O6-methylguanine levels. Studies with PARP-1-/- cells provided evidence for increased alkylation-induced DNA strand break formation when MGMT was inhibited, suggesting a role of PARP-1 in the response to O6-methylguanine adducts. Our findings reveal PARP-1 as a double-edged sword in colorectal carcinogenesis, which suppresses tumor initiation following DNA alkylation in a MGMT-dependent manner, but promotes inflammation-driven tumor progression.
Asunto(s)
Neoplasias Colorrectales/enzimología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/prevención & control , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Ratones , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasa-1/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Tryparedoxin (Tpx) is a pivotal protein in the redox-metabolism of trypanosomatid parasites. Tpx has previously been identified as a potential target for drug development in the fight against human African sleeping sickness caused by Trypanosoma brucei. Tpx belongs to the thioredoxin superfamily and acts as an oxidoreductase in the parasite's cytoplasm. It contains a WCPPC active site motif, which enables the protein to undergo thiol-disulfide exchange. To promote future protein-drug interaction analyses, we report the 1H, 13C and 15N backbone chemical shift assignments for both the oxidized and reduced states of Tpx. The redox state of the protein has a significant impact on the chemical shifts of the residues at the active site of the protein, especially on the two redox active site cysteines. The NMR assignments presented here will be a prerequisite for investigating drug binding to Tpx in molecular detail and to drive further drug optimization.