RESUMEN
BACKGROUND: The ongoing global malaria eradication campaign requires development of potent, safe, and cost-effective drugs lacking cross-resistance with existing chemotherapies. One critical step in drug development is selecting a suitable clinical candidate from late leads. The process used to select the clinical candidate SJ733 from two potent dihydroisoquinolone (DHIQ) late leads, SJ733 and SJ311, based on their physicochemical, pharmacokinetic (PK), and toxicity profiles is described. METHODS: The compounds were tested to define their physicochemical properties including kinetic and thermodynamic solubility, partition coefficient, permeability, ionization constant, and binding to plasma proteins. Metabolic stability was assessed in both microsomes and hepatocytes derived from mice, rats, dogs, and humans. Cytochrome P450 inhibition was assessed using recombinant human cytochrome enzymes. The pharmacokinetic profiles of single intravenous or oral doses were investigated in mice, rats, and dogs. RESULTS: Although both compounds displayed similar physicochemical properties, SJ733 was more permeable but metabolically less stable than SJ311 in vitro. Single dose PK studies of SJ733 in mice, rats, and dogs demonstrated appreciable oral bioavailability (60-100%), whereas SJ311 had lower oral bioavailability (mice 23%, rats 40%) and higher renal clearance (10-30 fold higher than SJ733 in rats and dogs), suggesting less favorable exposure in humans. SJ311 also displayed a narrower range of dose-proportional exposure, with plasma exposure flattening at doses above 200 mg/kg. CONCLUSION: SJ733 was chosen as the candidate based on a more favorable dose proportionality of exposure and stronger expectation of the ability to justify a strong therapeutic index to regulators.
Asunto(s)
Antimaláricos/farmacología , Isoquinolinas/farmacología , Animales , Antimaláricos/farmacocinética , Antimaláricos/toxicidad , Disponibilidad Biológica , Perros , Hepatocitos/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/toxicidad , Humanos , Isoquinolinas/farmacocinética , Isoquinolinas/toxicidad , Ratones , Microsomas Hepáticos/efectos de los fármacos , RatasRESUMEN
A virtual screen was performed to identify anti-malarial compounds targeting Plasmodium falciparum heat shock 90 protein by applying a series of drug-like and commercial availability filters to compounds in the ZINC database, resulting in a virtual library of more than 13 million candidates. The goal of the virtual screen was to identify novel compounds which could serve as a starting point for the development of antimalarials with a mode of action different from anything currently used in the clinic. The screen targeted the ATP binding pocket of the highly conserved Plasmodium heat shock 90 protein, as this protein is critical to the survival of the parasite and has several significant structural differences from the human homolog. The top twelve compounds from the virtual screen were tested in vitro, with all twelve showing no antiproliferative activity against the human fibroblast cell line and three compounds exhibiting single digit or better micromolar antiproliferative activity against the chloroquine-sensitive P. falciparum 3D7 strain.
Asunto(s)
Antimaláricos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/química , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/metabolismo , Relación Estructura-ActividadRESUMEN
Malaria remains one of the deadliest infectious diseases worldwide and continues to infect hundreds of millions of individuals each year. Here we report the discovery and derivatization of a series of 2,6-dibenzylidenecyclohexanones targeting the chloroquine-sensitive 3D7 strain of Plasmodium falciparum . While the initial lead compound displayed significant toxicity in a human cell proliferation assay, we were able to identify a derivative with no detectable toxicity and sub-micromolar potency.
Asunto(s)
Antimaláricos/farmacología , Cloroquina/farmacología , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/síntesis química , Antimaláricos/química , Proliferación Celular/efectos de los fármacos , Cloroquina/síntesis química , Cloroquina/química , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Humanos , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Relación Estructura-ActividadRESUMEN
A series of tetrahydro-ß-carboline derivatives of a lead compound known to target the heat shock 90 protein of Plasmodium falciparum were synthesized and assayed for both potency against the parasite and toxicity against a human cell line. Using a rationalized structure based design strategy, a new lead compound with a potency two orders of magnitude greater than the original lead compound was found. Additional modeling of this new lead compound suggests multiple avenues to further increase potency against this target, potentially paving the path for a therapeutic with a mode of action different than any current clinical treatment.
Asunto(s)
Adenosina Trifosfato/química , Antimaláricos/farmacología , Carbolinas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/síntesis química , Antimaláricos/química , Sitios de Unión/efectos de los fármacos , Carbolinas/síntesis química , Carbolinas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Plasmodium falciparum/química , Plasmodium falciparum/citología , Relación Estructura-ActividadRESUMEN
The 4-(heteroarylthio)thieno[2,3-d]pyrimidine (TTP) series of antimalarials, represented by 1 and 17, potently inhibit proliferation of the 3D7 strain of P. falciparum (EC50 70-100 nM), but suffer from oxidative metabolism. The 1,1-cyclopropylidene isosteres 6 and 16 were designed to obviate this drawback. They were prepared by a short route that features a combined Peterson methylenation / cyclopropanation transformation of, e. g., ketone 7. Isosteres 6 and 16 possess significantly attenuated antimalarial potency relative to parents 1 and 17. This outcome can be rationalized based on the increased out-of-plane steric demands of the latter two. In support of this hypothesis, the relatively flat ketone 7 retains some of the potency of 1, even though it appears to be a comparatively inferior mimic with respect to electronics and bond lengths and angles. We also demonstrate crystallographically and computationally an apparent increase in the strength of the intramolecular sulfur hole interaction of 1 upon protonation.
Asunto(s)
Antimaláricos/farmacología , Ciclopropanos/farmacología , Plasmodium falciparum/efectos de los fármacos , Pirimidinas/farmacología , Antimaláricos/síntesis química , Antimaláricos/química , Células Cultivadas , Cristalografía por Rayos X , Ciclopropanos/síntesis química , Ciclopropanos/química , Teoría Funcional de la Densidad , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-ActividadRESUMEN
N-terminal acetylation is an abundant modification influencing protein functions. Because â¼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Acetilación/efectos de los fármacos , Sitios de Unión , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Proteína NEDD8 , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
The bacterial cell division protein FtsZ polymerizes in a GTP-dependent manner to form a Z-ring that marks the plane of division. As a validated antimicrobial target, considerable efforts have been devoted to identify small molecule FtsZ inhibitors. We recently discovered the chrysophaentins, a novel suite of marine natural products that inhibit FtsZ activity in vitro. These natural products along with a synthetic hemi-chrysophaentin exhibit strong antimicrobial activity toward a broad spectrum of Gram-positive pathogens. To define their mechanisms of FtsZ inhibition and determine their in vivo effects in live bacteria, we used GTPase assays and fluorescence anisotropy to show that hemi-chrysophaentin competitively inhibits FtsZ activity. Furthermore, we developed a model system using a permeable Escherichia coli strain, envA1, together with an inducible FtsZ-yellow fluorescent protein construct to show by fluorescence microscopy that both chrysophaentin A and hemi-chrysophaentin disrupt Z-rings in live bacteria. We tested the E. coli system further by reproducing phenotypes observed for zantrins Z1 and Z3, and demonstrate that the alkaloid berberine, a reported FtsZ inhibitor, exhibits auto-fluorescence, making it incompatible with systems that employ GFP or YFP tagged FtsZ. These studies describe unique examples of nonnucleotide, competitive FtsZ inhibitors that disrupt FtsZ in vivo, together with a model system that should be useful for in vivo testing of FtsZ inhibitor leads that have been identified through in vitro screens but are unable to penetrate the Gram-negative outer membrane.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/antagonistas & inhibidores , Compuestos de Bencilo/química , Proteínas del Citoesqueleto/antagonistas & inhibidores , Éteres Cíclicos/química , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Compuestos de Bencilo/síntesis química , Compuestos de Bencilo/aislamiento & purificación , Compuestos de Bencilo/farmacología , Berberina/química , Berberina/farmacología , Proteínas del Citoesqueleto/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Éteres Cíclicos/aislamiento & purificación , Éteres Cíclicos/farmacología , GTP Fosfohidrolasas/metabolismo , Bacterias Grampositivas/efectos de los fármacos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Leishmaniasis, a neglected tropical disease caused by Leishmania species parasites, annually affects over 1 million individuals worldwide. Treatment options for leishmaniasis are limited due to high cost, severe adverse effects, poor efficacy, difficulty of use, and emerging drug resistance to all approved therapies. We discovered 2,4,5-trisubstituted benzamides (4) that possess potent antileishmanial activity but poor aqueous solubility. Herein, we disclose our optimization of the physicochemical and metabolic properties of 2,4,5-trisubstituted benzamide that retains potency. Extensive structure-activity and structure-property relationship studies allowed selection of early leads with suitable potency, microsomal stability, and improved solubility for progression. Early lead 79 exhibited an 80% oral bioavailability and potently blocked proliferation of Leishmania in murine models. These benzamide early leads are suitable for development as orally available antileishmanial drugs.
Asunto(s)
Antiprotozoarios , Leishmania , Leishmaniasis , Humanos , Animales , Ratones , Leishmaniasis/tratamiento farmacológico , Leishmaniasis/inducido químicamente , Leishmaniasis/parasitología , Antiprotozoarios/química , Benzamidas/farmacología , Benzamidas/uso terapéuticoRESUMEN
Drug-resistant Staphylococcus aureus is a continuing public health concern, both in the hospital and community settings. Antibacterial compounds that possess novel structural scaffolds and are effective against multiple S. aureus strains, including current drug-resistant ones, are needed. Previously, we have described the chrysophaentins, a family of bisdiarylbutene macrocycles from the chrysophyte alga Chrysophaeum taylori that inhibit the growth of S. aureus and methicillin-resistant S. aureus (MRSA). In this study we have analyzed the geographic variability of chrysophaentin production in C. taylori located at different sites on the island of St. John, U.S. Virgin Islands, and identified two new linear chrysophaentin analogs, E2 and E3. In addition, we have expanded the structure activity relationship through synthesis of fragments comprising conserved portions of the chrysophaentins, and determined the antimicrobial activity of natural chrysophaentins and their synthetic analogs against five diverse S. aureus strains. We find that the chrysophaentins show similar activity against all S. aureus strains, regardless of their drug sensitivity profiles. The synthetic chrysophaentin fragments indeed mimic the natural compounds in their spectrum of antibacterial activity, and therefore represent logical starting points for future medicinal chemistry studies of the natural products and their analogs.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Staphylococcus aureus Resistente a Meticilina/química , Antibacterianos/síntesis química , Chrysophyta/química , Geografía , Compuestos Macrocíclicos/síntesis química , Pruebas de Sensibilidad Microbiana/métodos , Islas Virgenes de los Estados UnidosRESUMEN
BACKGROUND: SJ733, a newly developed inhibitor of P. falciparum ATP4, has a favorable safety profile and rapid antiparasitic effect but insufficient duration to deliver a single-dose cure of malaria. We investigated the safety, tolerability, and pharmacokinetics of a multidose SJ733 regimen and a single-dose pharmacoboost approach using cobicistat to inhibit CYP3A4, thereby increasing exposure. METHODS: Two multidose unboosted cohorts (nâ¯=â¯9) (SJ733, 300â¯mg and 600â¯mg daily for 3 days) followed by three single-dose boosted cohorts combining SJ733 (nâ¯=â¯18) (75-, 300-, or 600-mg single dose) with cobicistat (150-mg single dose) as a pharmacokinetic booster were evaluated in healthy volunteers (ClinicalTrials.gov: NCT02661373). FINDINGS: All participants tolerated SJ733 well, with no serious adverse events (AEs), dose-limiting toxicity, or clinically significant electrocardiogram or laboratory test findings. All reported AEs were Grade 1, clinically insignificant, and considered unlikely or unrelated to SJ733. Compared to unboosted cohorts, the SJ733/cobicistat-boosted cohorts showed a median increase in area under the curve and maximum concentration of 3·9â¯×â¯and 2·6 ×, respectively, and a median decrease in the ratio of the major CYP3A-produced metabolite SJ506 to parent drug of 4·6â¯×â¯. Incorporating these data in a model of parasite dynamics indicated that a 3-day regimen of SJ733/cobicistat (600â¯mg/150â¯mg daily) relative to a single 600-mg dose ± cobicistat would increase parasite clearance from 106 to 1012 parasites/µL. INTERPRETATION: The multidose and pharmacoboosted approaches to delivering SJ733 were well-tolerated and significantly increased drug exposure and prediction of cure. This study supports the further development of SJ733 and demonstrates an innovative pharmacoboost approach for an antimalarial. FUNDING: Global Health Innovative Technology Fund, Medicines for Malaria Venture, National Institutes of Health, and American Lebanese Syrian Associated Charities.
Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria Falciparum , Malaria , Antimaláricos/efectos adversos , Cobicistat/uso terapéutico , Compuestos Heterocíclicos de 4 o más Anillos , Humanos , Isoquinolinas , Malaria/tratamiento farmacológico , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Plasmodium falciparumRESUMEN
The cullin-RING ubiquitin ligases (CRLs) are ubiquitin E3 enzymes that play a key role in controlling proteasomal degradation and are activated by neddylation. We previously reported inhibitors that target CRL activation by disrupting the interaction of defective in cullin neddylation 1 (DCN1), a CRL neddylation co-E3, and UBE2M, a neddylation E2. Our first-generation inhibitors possessed poor oral bioavailability and fairly rapid clearance that hindered the study of acute inhibition of DCN-controlled CRL activity in vivo. Herein, we report studies to improve the pharmacokinetic performance of the pyrazolo-pyridone inhibitors. The current best inhibitor, 40, inhibits the interaction of DCN1 and UBE2M, blocks NEDD8 transfer in biochemical assays, thermally stabilizes cellular DCN1, and inhibits anchorage-independent growth in a DCN1 amplified squamous cell carcinoma cell line. Additionally, we demonstrate that a single oral 50 mg/kg dose sustains plasma exposures above the biochemical IC90 for 24 h in mice.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pirazoles/química , Piridinas/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Administración Oral , Animales , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Estabilidad de Medicamentos , Semivida , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Ratones , Simulación de Dinámica Molecular , Pirazoles/metabolismo , Pirazoles/farmacología , Piridinas/metabolismo , Piridinas/farmacología , Relación Estructura-Actividad , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidoresRESUMEN
Leishmaniasis, a disease caused by protozoa of the Leishmania species, afflicts roughly 12 million individuals worldwide. Most existing drugs for leishmaniasis are toxic, expensive, difficult to administer, and subject to drug resistance. We report a new class of antileishmanial leads, the 3-arylquinolines, that potently block proliferation of the intramacrophage amastigote form of Leishmania parasites with good selectivity relative to the host macrophages. Early lead 34 was rapidly acting and possessed good potency against L. mexicana (EC50 = 120 nM), 30-fold selectivity for the parasite relative to the macrophage (EC50 = 3.7 µM), and also blocked proliferation of Leishmania donovani parasites resistant to antimonial drugs. Finally, another early lead, 27, which exhibited reasonable in vivo tolerability, impaired disease progression during the dosing period in a murine model of cutaneous leishmaniasis. These results suggest that the arylquinolines provide a fruitful departure point for the development of new antileishmanial drugs.
Asunto(s)
Leishmaniasis Cutánea/tratamiento farmacológico , Quinolinas/uso terapéutico , Tripanocidas/uso terapéutico , Animales , Femenino , Leishmania/efectos de los fármacos , Ratones Endogámicos BALB C , Microsomas Hepáticos/metabolismo , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/metabolismo , Quinolinas/farmacocinética , Relación Estructura-Actividad , Tripanocidas/síntesis química , Tripanocidas/metabolismo , Tripanocidas/farmacocinéticaRESUMEN
Genetically incorporated unnatural amino acid (UAA) technologies are powerful tools that are greatly enhancing our ability to study and engineer biological systems. Using these techniques, researchers can precisely control the position and number of novel chemical moieties in a protein, via introducing the novel R group of UAAs, that are genetically encoded in the protein's primary structure. The substrate recognition properties of a natural aminoacyl-tRNA synthetase (aaRS) must be modified in order to incorporate UAAs into proteins. Protocols to do so are technically simple but require time and optimization, which has significantly limited the accessibility of this important technology. At present, engineered unnatural aminoacyl-tRNA synthetases (UaaRS) are evaluated on their translational efficiency (the extent to which they allow for incorporation of UAAs into protein) and fidelity (the extent to which they prevent incorporation of natural amino acids). We propose that a third parameter of substrate recognition, permissivity, is equally important. Permissive UaaRSs, whose relaxed substrate recognition properties allow them to incorporate multiple unnatural amino acids (but not natural amino acids), would eliminate the need to generate new UaaRSs for many new UAAs. Here, we outline methods for quickly and easily assessing the permissivity of existing UaaRSs and for generating permissive UaaRSs. In proof of principle experiments, we determined the degree of permissivity of two UaaRSs for a family of structurally related fluorinated UAAs ((19)F-UAAs). We then increased the permissivity of the initial UaaRSs to allow for incorporation of the family of (19)F-UAAs. Finally, we validated the utility of these new (19)F-UAAs as probes for fluorine NMR studies of protein structure and dynamics. We expect that results of this work will increase the accessibility of UAA technology and the use of new UAAs in proteins.
Asunto(s)
Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Flúor/química , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Ingeniería de Proteínas , Especificidad por SustratoRESUMEN
Functionalized bicyclo[3.3.1]non-3-en-2-ones are obtained from commercially available phenols by a hypervalent iodine oxidation, enone epoxidation, epoxide thiolysis, and intramolecular aldol reaction sequence. Reaction optimization studies identified room temperature as well as microwave-mediated procedures, providing moderate to good yields (57%-88%) in the thiophenol-mediated epoxide opening and intramolecular aldol reaction. In addition, the isolation of a key intermediate and in situ NMR studies supported the mechanistic hypothesis. The bicyclic ring products occupy novel chemical space according to ChemGPS and Chemaxon chemical diversity and cheminformatics analyses.
RESUMEN
Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of deaths each year, primarily in young children and pregnant mothers. Here, we report the discovery and derivatization of a series of pyrazolo[3,4-b]pyridines targeting Plasmodium falciparum, the deadliest species of the malaria parasite. Hit compounds in this series display sub-micromolar in vitro activity against the intraerythrocytic stage of the parasite as well as little to no toxicity against the human fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the liver stage of the parasite but little activity against the gametocyte stage. Parasitological profiles, including rate of killing, docking, and molecular dynamics studies, suggest that our compounds may target the Qo binding site of cytochrome bc1.
Asunto(s)
Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Pirazoles/farmacología , Piridinas/farmacología , Antimaláricos/síntesis química , Antimaláricos/química , Línea Celular , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Modelos Moleculares , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Pirazoles/síntesis química , Pirazoles/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
The experimental study of protein folding is enhanced by the use of nonintrusive probes that are sensitive to local conformational changes in the protein structure. Here, we report the selection of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of two unnatural amino acids that can function as fluorescence resonance energy transfer (FRET) donors with Trp to probe the disruption of the hydrophobic core upon protein unfolding. l-4-Cyanophenylalanine (pCNPhe) and 4-ethynylphenylalanine (pENPhe) were incorporated into the hydrophobic core of the 171-residue protein, T4 lysozyme. The FRET donor ability of pCNPhe and pENPhe is evident by the overlap of the emission spectra of pCNPhe and pENPhe with the absorbance spectrum of Trp. The incorporation of both unnatural amino acids in place of a phenylalanine in the hydrophobic core of T4 lysozyme was well tolerated by the protein, due in part to the small size of the cyano and ethynyl groups. The hydrophobic nature of the ethynyl group of pENPhe suggests that this unnatural amino acid is a more conservative substitution into the hydrophobic core of the protein compared to pCNPhe. The urea-induced disruption of the hydrophobic core of the protein was probed by the change in FRET efficiency between either pCNPhe or pENPhe and the Trp residues in T4 lysozyme. The methodology for the study of protein conformational changes using FRET presented here is of general applicability to the study of protein structural changes, since the incorporation of the unnatural amino acids is not inherently limited by the size of the protein.
Asunto(s)
Sustitución de Aminoácidos/genética , Transferencia Resonante de Energía de Fluorescencia , Fenilalanina-ARNt Ligasa/química , Pliegue de Proteína , ARN de Transferencia de Triptófano/química , Triptófano-ARNt Ligasa/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Fenilalanina-ARNt Ligasa/genética , Fenilalanina-ARNt Ligasa/metabolismo , Conformación Proteica , Desnaturalización Proteica , ARN de Transferencia de Triptófano/genética , ARN de Transferencia de Triptófano/metabolismo , Triptófano-ARNt Ligasa/genética , Triptófano-ARNt Ligasa/metabolismoRESUMEN
Chemical control of cullin neddylation is attracting increased attention based largely on the successes of the NEDD8-activating enzyme (E1) inhibitor pevonedistat. Recently reported chemical probes enable selective and time-dependent inhibition of downstream members of the neddylation trienzymatic cascade including the co-E3, DCN1. In this work, we report the optimization of a novel class of small molecule inhibitors of the DCN1-UBE2M interaction. Rational X-ray co-structure enabled optimization afforded a 25-fold improvement in potency relative to the initial screening hit. The potency gains are largely attributed to additional hydrophobic interactions mimicking the N-terminal acetyl group that drives binding of UBE2M to DCN1. The compounds inhibit the protein-protein interaction, block NEDD8 transfer in biochemical assays, engage DCN1 in cells, and selectively reduce the steady-state neddylation of Cul1 and Cul3 in two squamous carcinoma cell lines harboring DCN1 amplification.
Asunto(s)
Proteínas Cullin/química , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteína NEDD8/química , Pirazoles/química , Piridonas/química , Amidas/química , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Ciclopentanos/farmacología , Diseño de Fármacos , Fibroblastos/metabolismo , Glicina/química , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Dominios Proteicos , Mapeo de Interacción de Proteínas , Pirimidinas/farmacología , Especies Reactivas de Oxígeno/química , Relación Estructura-Actividad , Enzimas Ubiquitina-Conjugadoras/químicaRESUMEN
We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin neddylation 1 (DCN1)-dependent neddylation of cullins. Here, we report preliminary structure-activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1-UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit. The optimized compounds inhibit the DCN1-UBE2M protein-protein interaction in our TR-FRET binding assay and inhibit cullin neddylation in our pulse-chase NEDD8 transfer assay. The optimized compounds bind to DCN1 and selectively reduce steady-state levels of neddylated CUL1 and CUL3 in a squamous cell carcinoma cell line. Ultimately, we anticipate that these studies will identify early lead compounds for clinical development for the treatment of lung squamous cell carcinomas and other cancers.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proteínas Cullin/antagonistas & inhibidores , Proteína NEDD8/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Cristalografía por Rayos X , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares/tratamiento farmacológico , Modelos Moleculares , Conformación Molecular , Proteína NEDD8/metabolismo , Unión Proteica , Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidoresRESUMEN
We previously reported the discovery, validation, and structure-activity relationships of a series of piperidinyl ureas that potently inhibit the DCN1-UBE2M interaction. We demonstrated that compound 7 inhibits both the DCN1-UBE2M protein-protein interaction and DCN1-mediated cullin neddylation in biochemical assays and reduces levels of steady-state cullin neddylation in a squamous carcinoma cell line harboring DCN1 amplification. Although compound 7 exhibits good solubility and permeability, it is rapidly metabolized in microsomal models (CLint = 170 mL/min/kg). This work lays out the discovery of an orally bioavailable analogue, NAcM-OPT (67). Compound 67 retains the favorable biochemical and cellular activity of compound 7 but is significantly more stable both in vitro and in vivo. Compound 67 is orally bioavailable, well tolerated in mice, and currently used to study the effects of acute pharmacologic inhibition of the DCN1-UBE2M interaction on the NEDD8/CUL pathway.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proteínas Cullin/efectos de los fármacos , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Proteína NEDD8/antagonistas & inhibidores , Proteína NEDD8/efectos de los fármacos , Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Relación Estructura-Actividad , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Urea/análogos & derivados , Urea/químicaRESUMEN
The majority of frontline therapies for the treatment of malaria are combination drugs containing artemisinin (or its semisynthetic analogs), known as artemisinin combination therapies (ACTs). While generally efficacious, ACTs and the first generation fully synthetic ozonide, arterolane (OZ277, 1), suffer from rapid clearance requiring 3-day dosing regimens. Extensive structure-activity studies led to the discovery of a second-generation ozonide, artefenomel (OZ439, 2), which has overcome this limitation, maintaining the rapid onset of action and potent activity of the artemisinin derivatives while exhibiting greatly improved pharmacokinetics, low projected cost of goods, prophylactic activity, and the potential for a single dose cure.