RESUMEN
Each year, approximately 50,000 children under 5 die as a result of diarrhea caused by Cryptosporidium parvum, a protozoan parasite. There are currently no effective drugs or vaccines available to cure or prevent Cryptosporidium infection, and there are limited tools for identifying and validating targets for drug or vaccine development. We previously reported a high throughput screening (HTS) of a large compound library against Plasmodium N-myristoyltransferase (NMT), a validated drug target in multiple protozoan parasite species. To identify molecules that could be effective against Cryptosporidium, we counter-screened hits from the Plasmodium NMT HTS against Cryptosporidium NMT. We identified two potential hit compounds and validated them against CpNMT to determine if NMT might be an attractive drug target also for Cryptosporidium. We tested the compounds against Cryptosporidium using both cell-based and NMT enzymatic assays. We then determined the crystal structure of CpNMT bound to Myristoyl-Coenzyme A (MyrCoA) and structures of ternary complexes with MyrCoA and the hit compounds to identify the ligand binding modes. The binding site architectures display different conformational states in the presence of the two inhibitors and provide a basis for rational design of selective inhibitors.
Asunto(s)
Criptosporidiosis , Cryptosporidium , Plasmodium , Niño , Humanos , Criptosporidiosis/tratamiento farmacológico , Desarrollo de MedicamentosRESUMEN
The attachment of myristate to the N-terminal glycine of certain proteins is largely a co-translational modification catalyzed by N-myristoyltransferase (NMT), and involved in protein membrane-localization. Pathogen NMT is a validated therapeutic target in numerous infectious diseases including malaria. In Plasmodium falciparum, NMT substrates are important in essential processes including parasite gliding motility and host cell invasion. Here, we generated parasites resistant to a particular NMT inhibitor series and show that resistance in an in vitro parasite growth assay is mediated by a single amino acid substitution in the NMT substrate-binding pocket. The basis of resistance was validated and analyzed with a structure-guided approach using crystallography, in combination with enzyme activity, stability, and surface plasmon resonance assays, allowing identification of another inhibitor series unaffected by this substitution. We suggest that resistance studies incorporated early in the drug development process help selection of drug combinations to impede rapid evolution of parasite resistance.
Asunto(s)
Aciltransferasas/genética , Aciltransferasas/metabolismo , Aciltransferasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Antimaláricos/química , Inhibidores Enzimáticos/química , Humanos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Polimorfismo de Nucleótido Simple/genética , Procesamiento Proteico-PostraduccionalRESUMEN
In bacteria, the second committed step in the diaminopimelate/lysine anabolic pathways is catalyzed by the enzyme dihydrodipicolinate reductase (DapB). DapB catalyzes the reduction of dihydrodipicolinate to yield tetrahydrodipicolinate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of DapB from the human-pathogenic bacterium Bartonella henselae, the causative bacterium of cat-scratch disease, are reported. Protein crystals were grown in conditions consisting of 5%(w/v) PEG 4000, 200â mM sodium acetate, 100â mM sodium citrate tribasic pH 5.5 and were shown to diffract to â¼2.3â Å resolution. They belonged to space group P4322, with unit-cell parameters a = 109.38, b = 109.38, c = 176.95â Å. Rr.i.m. was 0.11, Rwork was 0.177 and Rfree was 0.208. The three-dimensional structural features of the enzymes show that DapB from B. henselae is a tetramer consisting of four identical polypeptides. In addition, the substrate NADP+ was found to be bound to one monomer, which resulted in a closed conformational change in the N-terminal domain.
Asunto(s)
Proteínas Bacterianas/química , Bartonella henselae/química , Dihidrodipicolinato-Reductasa/química , NADP/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bartonella henselae/enzimología , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Dihidrodipicolinato-Reductasa/genética , Dihidrodipicolinato-Reductasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , NADP/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The effects of the organic flame retardant 1,2-Dibromo-4-(1,2-dibromoethyl) cyclohexane (TBECH) on androgen receptor target gene expression were examined in the human LNCaP prostate cancer cell line. While γ-/δ-TBECH alone led to a significant increase in prostate-specific antigen (PSA) mRNA accumulation, both the α-/-TBECH and γ-/δ-TBECH mixtures repressed androgen-inducible PSA mRNA and protein accumulation in human LNCaP cells. Thus, we hypothesize that isomeric mixtures of TBECH may act as partial agonists of the androgen receptor.
Asunto(s)
Ciclohexanos/farmacología , Disruptores Endocrinos/farmacología , Contaminantes Ambientales/farmacología , Células Epiteliales/efectos de los fármacos , Retardadores de Llama/farmacología , Receptores Androgénicos/genética , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Próstata/citología , Próstata/efectos de los fármacos , Próstata/metabolismo , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal/genética , EstereoisomerismoRESUMEN
The effects of six organophosphate flame retardants (OPFRs) tris(2-butoxyethyl) phosphate, tris(2-chloroethyl) phosphate, tris(1-chloro-2-propyl) phosphate, tris(methylphenyl) phosphate, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), and triethyl phosphate on the activities of androgen receptor (AR), estrogen receptor (ER), and aryl hydrocarbon receptor (AhR) were assessed in human prostate and endometrial cancer cells. OPFRs had no effect on ER or AhR target gene activation in ECC-1 cells. The effect of TDCIPP on mRNA and protein accumulation of AR target genes was examined further. AR-inducible gene and protein expression were significantly altered by TDCIPP exposure and repressed PSA levels in conditioned media of prostate cancer cells. We demonstrated that TDCIPP has no affinity for the AR ligand binding domain (AR-LBD) and exerts its antiandrogenic effects in a noncompetitive fashion. Thus, the clinical relevance of TDCIPP exposure on prostate cancer detection and progression to a therapeutically refractile state ought to be investigated further.