RESUMEN
New drugs and new targets are urgently needed to treat tuberculosis. We discovered that d-phenylalanine-benzoxazole Q112 displays potent antibacterial activity against Mycobacterium tuberculosis (Mtb) in multiple media and in macrophage infections. A metabolomic profiling indicates that Q112 has a unique mechanism of action. Q112 perturbs the essential pantothenate/coenzyme A biosynthetic pathway, depleting pantoate while increasing ketopantoate, as would be expected if ketopantoate reductase (KPR) were inhibited. We searched for alternative KPRs, since the enzyme annotated as PanE KPR is not essential in Mtb. The ketol-acid reductoisomerase IlvC catalyzes the KPR reaction in the close Mtb relative Corynebacterium glutamicum, but Mtb IlvC does not display KPR activity. We identified the essential protein Rv3603c as an orthologue of PanG KPR and demonstrated that a purified recombinant Rv3603c has KPR activity. Q112 inhibits Rv3603c, explaining the metabolomic changes. Surprisingly, pantothenate does not rescue Q112-treated bacteria, indicating that Q112 has an additional target(s). Q112-resistant strains contain loss-of-function mutations in the twin arginine translocase TatABC, further underscoring Q112's unique mechanism of action. Loss of TatABC causes a severe fitness deficit attributed to changes in nutrient uptake, suggesting that Q112 resistance may derive from a decrease in uptake.
Asunto(s)
Mycobacterium tuberculosis , Benzoxazoles/farmacología , Vías Biosintéticas , Coenzima A , Mycobacterium tuberculosis/genética , FenilalaninaRESUMEN
Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 µM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. Five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Animales , IMP Deshidrogenasa/genética , Inosina , Ratones , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureusRESUMEN
New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. Mycobacterium tuberculosis ( Mtb) inosine 5'-monophosphate dehydrogenase 2 ( MtbIMPDH2) is a promising yet controversial potential target. The inhibition of MtbIMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure-activity relationship (SAR) for the benzoxazole series of MtbIMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 µM can be achieved. The antibacterial activity of the most promising compound, 17b (Q151), is derived from the inhibition of MtbIMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of 17b, alleviating the concern that guanine salvage can protect Mtb in vivo. These findings suggest that MtbIMPDH2 is a vulnerable target for tuberculosis.
Asunto(s)
Antituberculosos/química , Antituberculosos/farmacología , Benzoxazoles/química , Benzoxazoles/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Línea Celular Tumoral , Diseño de Fármacos , Humanos , IMP Deshidrogenasa/química , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Inosine 5'-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment of Cryptosporidium infections. Here, the structure of C. parvum IMPDH (CpIMPDH) in complex with inosine 5'-monophosphate (IMP) and P131, an inhibitor with in vivo anticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO2 moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is a new strategy for the further optimization of C. parvum inhibitors for both antiparasitic and antibacterial applications.
Asunto(s)
Antiparasitarios/química , Cryptosporidium/química , Inhibidores Enzimáticos/química , IMP Deshidrogenasa/química , Secuencia de Aminoácidos , Antiparasitarios/metabolismo , Cryptosporidium/genética , Cryptosporidium/metabolismo , Inhibidores Enzimáticos/metabolismo , IMP Deshidrogenasa/genética , IMP Deshidrogenasa/metabolismo , Datos de Secuencia Molecular , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization.
Asunto(s)
Antiinfecciosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Antiinfecciosos/química , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/enzimología , Bacillus anthracis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Campylobacter jejuni/efectos de los fármacos , Campylobacter jejuni/enzimología , Campylobacter jejuni/genética , Clostridium perfringens/efectos de los fármacos , Clostridium perfringens/enzimología , Clostridium perfringens/genética , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , IMP Deshidrogenasa/química , IMP Deshidrogenasa/genética , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Cryptosporidium inosine 5'-monophosphate dehydrogenase (CpIMPDH) has emerged as a therapeutic target for treating Cryptosporidium parasites because it catalyzes a critical step in guanine nucleotide biosynthesis. A 4-oxo-[1]benzopyrano[4,3-c]pyrazole derivative was identified as a moderately potent (IC50 = 1.5 µM) inhibitor of CpIMPDH. We report a SAR study for this compound series resulting in 8k (IC50 = 20 ± 4 nM). In addition, an X-ray crystal structure of CpIMPDH·IMP·8k is also presented.
Asunto(s)
Acetanilidas/síntesis química , Acetanilidas/farmacología , Cumarinas/síntesis química , Cumarinas/farmacología , Cryptosporidium parvum/enzimología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Pirazoles/química , Cryptosporidium parvum/efectos de los fármacos , Cristalografía por Rayos X , Técnicas In Vitro , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the pivotal step in guanine nucleotide biosynthesis. IMPDH is a target for immunosuppressive, antiviral, and anticancer drugs, but, as of yet, has not been exploited for antimicrobial therapy. We have previously reported potent inhibitors of IMPDH from the protozoan parasite Cryptosporidium parvum (CpIMPDH). Many pathogenic bacteria, including Bacillus anthracis, Staphylococcus aureus, and Listeria monocytogenes, contain IMPDHs that should also be inhibited by these compounds. Herein, we present the structure-activity relationships for the inhibition of B. anthracis IMPDH (BaIMPDH) and antibacterial activity of 140 compounds from five structurally distinct compound series. Many potent inhibitors of BaIMPDH were identified (78% with IC50 ≤ 1 µM). Four compounds had minimum inhibitory concentrations (MIC) of less than 2 µM against B. anthracis Sterne 770. These compounds also displayed antibacterial activity against S. aureus and L. monocytogenes.
RESUMEN
Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5'-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (CpIMPDH) identified by high-throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC(50) < 2 nM) against CpIMPDH, excellent selectivity >1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model.
Asunto(s)
Cryptosporidium parvum/enzimología , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/metabolismo , Urea/farmacología , Humanos , IMP Deshidrogenasa/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of Bacillus anthracis IMPDH, in a phosphate ion-bound (termed "apo") form and in complex with its substrate, inosine 5'-monophosphate (IMP), and product, xanthosine 5'-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known Cryptosporidium parvum IMPDH inhibitors was examined for the B. anthracis enzyme and compared with those of three bacterial IMPDHs from Campylobacter jejuni, Clostridium perfringens, and Vibrio cholerae. The structures contribute to the characterization of the active site and design of inhibitors that specifically target B. anthracis and other microbial IMPDH enzymes.
Asunto(s)
Bacillus anthracis/enzimología , IMP Deshidrogenasa/química , IMP Deshidrogenasa/metabolismo , Inosina Monofosfato/metabolismo , Ribonucleótidos/metabolismo , Secuencia de Aminoácidos , Apoenzimas/antagonistas & inhibidores , Apoenzimas/química , Apoenzimas/metabolismo , Bencimidazoles/química , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Dominio Catalítico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Modelos Moleculares , Datos de Secuencia Molecular , Ácido Micofenólico/metabolismo , NAD/metabolismo , Unión Proteica , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacología , XantinaRESUMEN
The discovery of drugs that cause the degradation of their target proteins has been largely serendipitous. Here we report that the tert-butyl carbamate-protected arginine (Boc(3)Arg) moiety provides a general strategy for the design of degradation-inducing inhibitors. The covalent inactivators ethacrynic acid and thiobenzofurazan cause the specific degradation of glutathione-S-transferase when linked to Boc(3)Arg. Similarly, the degradation of dihydrofolate reductase is induced when cells are treated with the noncovalent inhibitor trimethoprim linked to Boc(3)Arg. Degradation is rapid and robust, with 30%-80% of these abundant target proteins consumed within 1.3-5 hr. The proteasome is required for Boc(3)Arg-mediated degradation, but ATP is not necessary and the ubiquitin pathways do not appear to be involved. These results suggest that the Boc(3)Arg moiety may provide a general strategy to construct inhibitors that induce targeted protein degradation.
Asunto(s)
Diseño de Fármacos , Glutatión Transferasa/metabolismo , Fenilcarbamatos/química , Fenilcarbamatos/farmacología , Proteolisis/efectos de los fármacos , Tetrahidrofolato Deshidrogenasa/metabolismo , Células HeLa , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Ubiquitina/metabolismoRESUMEN
Photoinduced charge separation processes of three-layer supramolecular hybrids, fullerene-porphyrin-SWCNT, which are constructed from semiconducting (7,6)- and (6,5)-enriched SWCNTs and self-assembled via π-π interacting long alkyl chain substituted porphyrins (tetrakis(4-dodecyloxyphenyl)porphyrins; abbreviated as MP(alkyl)(4)) (M = Zn and H(2)), to which imidazole functionalized fullerene[60] (C(60)Im) is coordinated, have been investigated in organic solvents. The intermolecular alkyl-π and π-π interactions between the MP(alkyl)(4) and SWCNTs, in addition, coordination between C(60)Im and Zn ion in the porphyrin cavity are visualized using DFT calculations at the B3LYP/3-21G(*) level, predicting donor-acceptor interactions between them in the ground and excited states. The donor-acceptor nanohybrids thus formed are characterized by TEM imaging, steady-state absorption and fluorescence spectra. The time-resolved fluorescence studies of MP(alkyl)(4) in two-layered nanohybrids (MP(alkyl)(4)/SWCNT) revealed efficient quenching of the singlet excited states of MP(alkyl)(4) ((1)MP*(alkyl)(4)) with the rate constants of charge separation (k(CS)) in the range of (1-9) × 10(9) s(-1). A nanosecond transient absorption technique confirmed the electron transfer products, MPË(+)(alkyl)(4)/SWCNTË(-) and/or MPË(-)(alkyl)(4)/SWCNTË(+) for the two-layer nanohybrids. Upon further coordination of C(60)Im to ZnP, acceleration of charge separation via(1)ZnP* in C(60)ImâZnP(alkyl)(4)/SWCNT is observed to form C(60)Ë(-)ImâZnPË(+)(alkyl)(4)/SWCNT and C(60)Ë(-)ImâZnP(alkyl)(4)/SWCNTË(+) charge separated states as supported by the transient absorption spectra. These characteristic absorptions decay with rate constants due to charge recombination (k(CR)) in the range of (6-10) × 10(6) s(-1), corresponding to the lifetimes of the radical ion-pairs of 100-170 ns. The electron transfer in the nanohybrids has further been utilized for light-to-electricity conversion by the construction of proof-of-concept photoelectrochemical solar cells.
RESUMEN
Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.
Asunto(s)
GMP-Reductasa/metabolismo , IMP Deshidrogenasa/metabolismo , Biocatálisis , Cristalografía por Rayos X , GMP-Reductasa/química , Guanosina Monofosfato/biosíntesis , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Humanos , IMP Deshidrogenasa/química , Inosina Monofosfato/química , Inosina Monofosfato/metabolismo , Cinética , Modelos Moleculares , Estructura Molecular , NADP/química , NADP/metabolismo , Teoría Cuántica , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismoRESUMEN
IMP dehydrogenase (IMPDH) catalyzes a critical step in guanine nucleotide biosynthesis. IMPDH also has biological roles that are distinct from its enzymatic function. We report a biotin-linked reagent that selectively labels IMPDH and is released by dithiothreitol. This reagent will be invaluable in elucidating the moonlighting functions of IMPDH.
Asunto(s)
IMP Deshidrogenasa/química , Biotinilación , Ditiotreitol/química , Humanos , Modelos Moleculares , Estructura MolecularRESUMEN
The protozoan parasite Cryptosporidium parvum is a major cause of gastrointestinal disease; no effective drug therapy exists to treat this infection. Curiously, C. parvum IMPDH (CpIMPDH) is most closely related to prokaryotic IMPDHs, suggesting that the parasite obtained its IMPDH gene via horizontal transfer. We previously identified inhibitors of CpIMPDH that do not inhibit human IMPDHs. Here, we show that these compounds also inhibit IMPDHs from Helicobacter pylori, Borrelia burgdorferi, and Streptococcus pyogenes, but not from Escherichia coli. Residues Ala165 and Tyr358 comprise a structural motif that defines susceptible enzymes. Importantly, a second-generation CpIMPDH inhibitor has bacteriocidal activity on H. pylori but not E. coli. We propose that CpIMPDH-targeted inhibitors can be developed into a new class of antibiotics that will spare some commensal bacteria.
Asunto(s)
Inhibidores Enzimáticos/química , IMP Deshidrogenasa/antagonistas & inhibidores , Sitios de Unión , Borrelia burgdorferi/efectos de los fármacos , Borrelia burgdorferi/enzimología , Simulación por Computador , Cryptosporidium parvum/enzimología , Inhibidores Enzimáticos/farmacología , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/enzimología , Humanos , IMP Deshidrogenasa/clasificación , IMP Deshidrogenasa/metabolismo , Cinética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/enzimologíaRESUMEN
BACKGROUND: The protozoan parasite Cryptosporidium parvum is responsible for significant disease burden among children in developing countries. In addition Cryptosporidiosis can result in chronic and life-threatening enteritis in AIDS patients, and the currently available drugs lack efficacy in treating these severe conditions. The discovery and development of novel anti-cryptosporidial therapeutics has been hampered by the poor experimental tractability of this pathogen. While the genome sequencing effort has identified several intriguing new targets including a unique inosine monophosphate dehydrogenase (IMPDH), pursuing these targets and testing inhibitors has been frustratingly difficult. METHODOLOGY AND PRINCIPAL FINDINGS: Here we have developed a pipeline of tools to accelerate the in vivo screening of inhibitors of C. parvum IMPDH. We have genetically engineered the related parasite Toxoplasma gondii to serve as a model of C. parvum infection as the first screen. This assay provides crucial target validation and a large signal window that is currently not possible in assays involving C. parvum. To further develop compounds that pass this first filter, we established a fluorescence-based assay of host cell proliferation, and a C. parvum growth assay that utilizes automated high-content imaging analysis for enhanced throughput. CONCLUSIONS AND SIGNIFICANCE: We have used these assays to evaluate C. parvum IMPDH inhibitors emerging from our ongoing medicinal chemistry effort and have identified a subset of 1,2,3-triazole ethers that exhibit excellent in vivo selectivity in the T. gondii model and improved anti-cryptosporidial activity.
Asunto(s)
Antiprotozoarios/farmacología , Cryptosporidium parvum/efectos de los fármacos , Cryptosporidium parvum/enzimología , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Triazoles/farmacología , Antiprotozoarios/aislamiento & purificación , Automatización , Inhibidores Enzimáticos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Procesamiento de Imagen Asistido por Computador , Coloración y Etiquetado , Toxoplasma/enzimología , Toxoplasma/genética , Triazoles/aislamiento & purificaciónRESUMEN
Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. This protozoan parasite cannot salvage guanine or guanosine and therefore relies on inosine 5'-monophosphate dehydrogenase (IMPDH) for biosynthesis of guanine nucleotides and hence for survival. Because C. parvum IMPDH is highly divergent from the host counterpart, selective inhibitors could potentially be used to treat cryptosporidiosis with minimal effects on its mammalian host. A series of 1,2,3-triazole containing ether CpIMPDH inhibitors are described. A structure-activity relationship study revealed that a small alkyl group on the alpha-position of the ether was required, with the (R)-enantiomer significantly more active than the (S)-enantiomer. Electron-withdrawing groups in the 3- and/or 4-positions of the pendent phenyl ring were best, and conversion of the quinoline containing inhibitors to quinoline-N-oxides retained inhibitory activity both in the presence and absence of bovine serum albumin. The 1,2,3-triazole CpIMPDH inhibitors provide new tools for elucidating the role of IMPDH in C. parvum and may serve as potential therapeutics for treating cryptosporidiosis.
Asunto(s)
Antiprotozoarios/síntesis química , Cryptosporidium parvum/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , IMP Deshidrogenasa/antagonistas & inhibidores , Triazoles/síntesis química , Animales , Antiprotozoarios/farmacología , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium parvum/enzimología , Inhibidores Enzimáticos/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Triazoles/farmacologíaRESUMEN
RPE65 is essential for the biosynthesis of 11-cis-retinal, the chromophore of rhodopsin. Here, we show that the membrane-associated form (mRPE65) is triply palmitoylated and is a chaperone for all-trans-retinyl esters, allowing their entry into the visual cycle for processing into 11-cis-retinal. The soluble form of RPE65 (sRPE65) is not palmitoylated and is a chaperone for vitamin A, rather than all-trans-retinyl esters. Thus, the palmitoylation of RPE65 controls its ligand binding selectivity. The two chaperones are interconverted by lecithin retinol acyl transferase (LRAT) acting as a molecular switch. Here mRPE65 is a palmitoyl donor, revealing a new acyl carrier protein role for palmitoylated proteins. When chromophore synthesis is not required, mRPE65 is converted into sRPE65 by LRAT, and further chromophore synthesis is blocked. The studies reveal new roles for palmitoylated proteins as molecular switches and LRAT as a palmitoyl transferase whose role is to catalyze the mRPE65 to sRPE65 conversion.
Asunto(s)
Ácidos Grasos Monoinsaturados/metabolismo , Células Fotorreceptoras/metabolismo , Proteínas/metabolismo , Retinaldehído/biosíntesis , Visión Ocular/fisiología , Animales , Bovinos , Línea Celular , Proteínas del Ojo , Ligandos , Chaperonas Moleculares/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Unión Proteica/fisiología , Isoformas de Proteínas/metabolismo , Rodopsina/biosíntesis , Tretinoina/metabolismo , Vitamina A/metabolismoRESUMEN
RPE65 is essential in the operation of the visual cycle and functions as a chaperone for all-trans-retinyl esters, the substrates for isomerization in the visual cycle. RPE65 stereospecifically binds all-trans-retinyl esters with a K(D) of 47 nM. It is shown here by using a quantitative fluorescence technique, that Accutane (13-cis-retinoic acid), a drug used in the treatment of acne but that causes night blindness, binds to RPE65 with a K(D) of 195 nM. All-trans-retinoic acid binds with a K(D) of 109 nM. The binding of the retinoic acids to RPE65 is competitive with all-trans-retinyl ester binding, and this competition inhibits visual cycle function. A retinoic acid analog that binds weakly to RPE65 is not inhibitory. These data suggest that RPE65 function is rate-limiting in visual cycle function. They also reveal the target through which the retinoic acids induce night blindness. Finally, certain forms of retinal and macular degeneration are caused by the accumulation of vitamin A-based retinotoxic products, called the retinyl pigment epithelium-lipofuscin. These retinotoxic products accumulate during the normal course of rhodopsin bleaching and regeneration after the operation of the visual cycle. Drugs such as Accutane may represent an important approach to reducing the accumulation of the retinotoxic lipofuscin by inhibiting visual cycle function. The identification of RPE65 as the visual cycle target for the retinoic acids makes it feasible to develop useful drugs to treat retinal and macular degeneration while avoiding the substantial side effects of the retinoic acids.
Asunto(s)
Degeneración Macular/tratamiento farmacológico , Proteínas/metabolismo , Tretinoina/metabolismo , Vitamina A/análogos & derivados , Animales , Bovinos , Diterpenos , Proteínas del Ojo , Proteínas/antagonistas & inhibidores , Ésteres de Retinilo , Tretinoina/farmacología , Tretinoina/uso terapéutico , Vitamina A/biosíntesis , Vitamina A/metabolismo , Vitamina A/farmacologíaRESUMEN
The biochemical pathway to visual chromophore biosynthesis in rod-dominated animals involves minimally a two component system in which all-trans-retinyl esters, generated by the action of lecithin retinol acyltransferase (LRAT) on vitamin A, are processed into 11-cis-retinol by isomerohydrolase. Possible differences in retinoid metabolism in cone-dominated animals have been noted in the literature, so it was of interest to explore whether these differences are tangential or fundamental. Central to this issue is whether cone-dominated animals use an isomerohydrolase (IMH)-based mechanism in the predominant pathway to 11-cis-retinoids. Here, it is shown that all-trans-retinyl esters (tREs) are the direct precursors of 11-cis-retinol formation in chicken retinyl pigment epithelium/retina preparations. This conclusion is based on at least three avenues of evidence. First, reagents that block tRE synthesis from vitamin A also block 11-cis-retinol synthesis. Second, pulse-chase experiments also establish that tREs are the precursors to 11-cis-retinol. Finally, 11-cis-retinyl-bromoacetate, a known affinity-labeling agent of isomerohydrolase, also blocks chromophore biosynthesis in the cone system.