RESUMEN
The discovery of genetic drivers of lung cancer in patient sub-groups has led to their use as predictive biomarkers and as targets for selective drug therapy. Some of the most important lung cancer drivers are mutations in the EGFR gene, for example, the exon 19 deletions and the L858R variant that confer sensitivity to the front line drugs erlotinib and gefitinib; the acquired T790M variants confer drug resistance and a poor prognosis. A challenge then in targeting EGFR is to produce drugs that inhibit both sensitising variants and resistance variants, leaving wild type protein in healthy cells unaffected. One such agent is AstraZeneca's "breakthrough" AZD9291 molecule that shows a 200-fold selectivity for T790M/L858R over wild type EGFR. Our X-ray crystal structure reveals the binding mode of AZD9291 to the kinase domain of wild type EGFR.
Asunto(s)
Acrilamidas/farmacología , Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/farmacología , Acrilamidas/metabolismo , Compuestos de Anilina/metabolismo , Cristalografía por Rayos X , Resistencia a Antineoplásicos/genética , Receptores ErbB/ultraestructura , Clorhidrato de Erlotinib/farmacología , Gefitinib , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Unión Proteica/fisiología , Quinazolinas/farmacologíaRESUMEN
Inhibitors of the aldo-keto reductase enzyme AKR1C3 are of interest as potential drugs for leukemia and hormone-related cancers. A series of non-carboxylate morpholino(phenylpiperazin-1-yl)methanones were prepared by palladium-catalysed coupling of substituted phenyl or pyridyl bromides with the known morpholino(piperazin-1-yl)methanone, and shown to be potent (IC50â¼100nM) and very isoform-selective inhibitors of AKR1C3. Lipophilic electron-withdrawing substituents on the phenyl ring were positive for activity, as was an H-bond acceptor on the other terminal ring, and the ketone moiety (as a urea) was essential. These structure-activity relationships are consistent with an X-ray structure of a representative compound bound in the AKR1C3 active site, which showed H-bonding between the carbonyl oxygen of the drug and Tyr55 and His117 in the 'oxyanion hole' of the enzyme, with the piperazine bridging unit providing the correct twist to allow the terminal benzene ring to occupy the lipophilic pocket and align with Phe311.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Dominio Catalítico , Técnicas de Química Sintética , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Enlace de Hidrógeno , Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Morfolinas/química , Relación Estructura-ActividadRESUMEN
Off-target aerobic activation of PR-104A by human aldo-keto reductase 1C3 (AKR1C3) has confounded the development of this dual hypoxia/gene therapy prodrug. Previous attempts to design prodrugs resistant to AKR1C3 activation have resulted in candidates that require further optimization. Herein we report the evaluation of a lipophilic series of PR-104A analogues in which a piperazine moiety has been introduced to improve drug-like properties. Octanol-water partition coefficients (LogD7.4) spanned >2 orders of magnitude. 2D antiproliferative and 3D multicellular clonogenic assays using isogenic HCT116 and H1299 cells confirmed that all examples were resistant to AKR1C3 metabolism while producing an E. coli NfsA nitroreductase-mediated bystander effect. Prodrugs 16, 17, and 20 demonstrated efficacy in H1299 xenografts where only a minority of tumor cells express NfsA. These prodrugs and their bromo/mesylate counterparts (25-27) were also evaluated for hypoxia-selective cell killing in vitro. These results in conjunction with stability assays recommended prodrug 26 (CP-506) for Phase I/II clinical trial.
RESUMEN
PR-104A is a dual hypoxia/nitroreductase gene therapy prodrug by virtue of its ability to undergo either one- or two-electron reduction to its cytotoxic species. It has been evaluated extensively in pre-clinical GDEPT studies, yet off-target human aldo-keto reductase AKR1C3-mediated activation has limited its use. Re-evaluation of this chemical scaffold has previously identified SN29176 as an improved hypoxia-activated prodrug analogue of PR-104A that is free from AKR1C3 activation. However, optimization of the bystander effect of SN29176 is required for use in a GDEPT setting to compensate for the non-uniform distribution of therapeutic gene transfer that is often observed with current gene therapy vectors. A lipophilic series of eight analogues were synthesized from commercially available 3,4-difluorobenzaldehyde. Calculated octanol-water partition coefficients (LogD7.4) spanned > 2 orders of magnitude. 2D anti-proliferative and 3D multicellular layer assays were performed using isogenic HCT116 cells expressing E. coli NfsA nitroreductase (NfsA_Ec) or AKR1C3 to determine enzyme activity and measure bystander effect. A variation in potency for NfsA_Ec was observed, while all prodrugs appeared AKR1C3-resistant by 2D assay. However, 3D assays indicated that increasing prodrug lipophilicity correlated with increased AKR1C3 activation and NfsA_Ec activity, suggesting that metabolite loss from the cell of origin into media during 2D monolayer assays could mask cytotoxicity. Three prodrugs were identified as bono fide AKR1C3-negative candidates whilst maintaining activity with NfsA_Ec. These were converted to their phosphate ester pre-prodrugs before being taken forward into in vivo therapeutic efficacy studies. Ultimately, 2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyl dihydrogen phosphate possessed a significant 156% improvement in median survival in mixed NfsA_Ec/WT tumors compared to untreated controls (p = 0.005), whilst still maintaining hypoxia selectivity comparable to PR-104A.
RESUMEN
PURPOSE: Approved therapies for EGFR exon 20, ERBB2 mutations, and NRG1 fusions are currently lacking for non-small cell lung cancer and other cancers. Tarloxotinib is a prodrug that harnesses tumor hypoxia to generate high levels of a potent, covalent pan-HER tyrosine kinase inhibitor, tarloxotinib-effector (tarloxotinib-E), within the tumor microenvironment. This tumor-selective delivery mechanism was designed to minimize the dose-limiting toxicities that are characteristic of systemic inhibition of wild-type EGFR. EXPERIMENTAL DESIGN: Novel and existing patient-derived cell lines and xenografts harboring EGFR exon 20 insertion mutations, ERBB2 mutations and amplification, and NRG1 fusions were tested in vitro and in vivo with tarloxotinib to determine its impact on cancer cell proliferation, apoptosis, and cell signaling. RESULTS: Tarloxotinib-E inhibited cell signaling and proliferation in patient-derived cancer models in vitro by directly inhibiting phosphorylation and activation of EGFR, HER2, and HER2/HER3 heterodimers. In vivo, tarloxotinib induced tumor regression or growth inhibition in multiple murine xenograft models. Pharmacokinetic analysis confirmed markedly higher levels of tarloxotinib-E in tumor tissue than plasma or skin. Finally, a patient with lung adenocarcinoma harboring an ERBB2 exon 20 p.A775_G776insYVMA mutation demonstrated a dramatic clinical response to tarloxotinib. CONCLUSIONS: Experimental data with tarloxotinib validate the novel mechanism of action of a hypoxia-activated prodrug in cancer models by concentrating active drug in the tumor versus normal tissue, and this activity can translate into clinical activity in patients.
Asunto(s)
Adenocarcinoma del Pulmón/tratamiento farmacológico , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Hipoxia/fisiopatología , Inhibidores de Proteínas Quinasas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Adulto , Animales , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular , Resistencia a Antineoplásicos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Mutación , Fosforilación , Pronóstico , Células Tumorales Cultivadas , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PR-104 is a phosphate ester pre-prodrug that is converted in vivo to its cognate alcohol, PR-104A, a latent alkylator which forms potent cytotoxins upon bioreduction. Hypoxia selectivity results from one-electron nitro reduction of PR-104A, in which cytochrome P450 oxidoreductase (POR) plays an important role. However, PR-104A also undergoes 'off-target' two-electron reduction by human aldo-keto reductase 1C3 (AKR1C3), resulting in activation in oxygenated tissues. AKR1C3 expression in human myeloid progenitor cells probably accounts for the dose-limiting myelotoxicity of PR-104 documented in clinical trials, resulting in human PR-104A plasma exposure levels 3.4- to 9.6-fold lower than can be achieved in murine models. Structure-based design to eliminate AKR1C3 activation thus represents a strategy for restoring the therapeutic window of this class of agent in humans. Here, we identified SN29176, a PR-104A analogue resistant to human AKR1C3 activation. SN29176 retains hypoxia selectivity in vitro with aerobic/hypoxic IC50 ratios of 9 to 145, remains a substrate for POR and triggers γH2AX induction and cell cycle arrest in a comparable manner to PR-104A. SN35141, the soluble phosphate pre-prodrug of SN29176, exhibited superior hypoxic tumour log cell kill (>4.0) to PR-104 (2.5-3.7) in vivo at doses predicted to be achievable in humans. Orthologues of human AKR1C3 from mouse, rat and dog were incapable of reducing PR-104A, thus identifying an underlying cause for the discrepancy in PR-104 tolerance in pre-clinical models versus humans. In contrast, the macaque AKR1C3 gene orthologue was able to metabolise PR-104A, indicating that this species may be suitable for evaluating the toxicokinetics of PR-104 analogues for clinical development. We confirmed that SN29176 was not a substrate for AKR1C3 orthologues across all four pre-clinical species, demonstrating that this prodrug analogue class is suitable for further development. Based on these findings, a prodrug candidate was subsequently identified for clinical trials.
RESUMEN
The clinical stage anti-cancer agent PR-104 has potential utility as a cytotoxic prodrug for exogenous bacterial nitroreductases expressed from replicating vector platforms. However substrate selectivity is compromised due to metabolism by the human one- and two-electron oxidoreductases cytochrome P450 oxidoreductase (POR) and aldo-keto reductase 1C3 (AKR1C3). Using rational drug design we developed a novel mono-nitro analog of PR-104A that is essentially free of this off-target activity in vitro and in vivo. Unlike PR-104A, there was no biologically relevant cytotoxicity in cells engineered to express AKR1C3 or POR, under aerobic or anoxic conditions, respectively. We screened this inert prodrug analog, SN34507, against a type I bacterial nitroreductase library and identified E. coli NfsA as an efficient bioactivator using a DNA damage response assay and recombinant enzyme kinetics. Expression of E. coli NfsA in human colorectal cancer cells led to selective cytotoxicity to SN34507 that was associated with cell cycle arrest and generated a robust 'bystander effect' at tissue-like cell densities when only 3% of cells were NfsA positive. Anti-tumor activity of SN35539, the phosphate pre-prodrug of SN34507, was established in 'mixed' tumors harboring a minority of NfsA-positive cells and demonstrated marked tumor control following heterogeneous suicide gene expression. These experiments demonstrate that off-target metabolism of PR-104 can be avoided and identify the suicide gene/prodrug partnership of E. coli NfsA/SN35539 as a promising combination for development in armed vectors.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Antineoplásicos Alquilantes/uso terapéutico , Benzamidas/uso terapéutico , Carcinoma/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Diseño de Fármacos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Mesilatos/uso terapéutico , Modelos Moleculares , Organofosfonatos/uso terapéutico , Profármacos/uso terapéutico , 3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , Activación Metabólica/efectos de los fármacos , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Animales , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacología , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacología , Carcinoma/metabolismo , Carcinoma/patología , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HCT116 , Humanos , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/genética , Mesilatos/química , Mesilatos/metabolismo , Mesilatos/farmacología , Ratones Desnudos , Simulación del Acoplamiento Molecular , Nitrorreductasas/genética , Nitrorreductasas/metabolismo , Organofosfonatos/química , Organofosfonatos/metabolismo , Organofosfonatos/farmacología , Profármacos/química , Profármacos/metabolismo , Profármacos/farmacología , Distribución Aleatoria , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Organismos Libres de Patógenos Específicos , Especificidad por Sustrato , Análisis de Supervivencia , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
High expression of the aldo-keto reductase enzyme AKR1C3 in the human prostate and breast has implicated it in the development and progression of leukemias and of prostate and breast cancers. Inhibitors are thus of interest as potential drugs. Most inhibitors of AKR1C3 are carboxylic acids, whose transport into cells is likely dominated by carrier-mediated processes. We describe here a series of (piperidinosulfonamidophenyl)pyrrolidin-2-ones as potent (<100 nM) and isoform-selective non-carboxylate inhibitors of AKR1C3. Structure-activity relationships identified the sulfonamide was critical, and a crystal structure showed the 2-pyrrolidinone does not interact directly with residues in the oxyanion hole. Variations in the position, co-planarity or electronic nature of the pyrrolidinone ring severely diminished activity, as did altering the size or polarity of the piperidino ring. There was a broad correlation between the enzyme potencies of the compounds and their effectiveness at inhibiting AKR1C3 activity in cells.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Pirrolidinonas/farmacología , Sulfonamidas/farmacología , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HCT116 , Humanos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Modelos Moleculares , Estructura Molecular , Pirrolidinonas/síntesis química , Pirrolidinonas/química , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
A high-throughput screen identified 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acid as a novel, highly potent (low nM), and isoform-selective (1500-fold) inhibitor of aldo-keto reductase AKR1C3: a target of interest in both breast and prostate cancer. Crystal structure studies showed that the carboxylate group occupies the oxyanion hole in the enzyme, while the sulfonamide provides the correct twist to allow the dihydroisoquinoline to bind in an adjacent hydrophobic pocket. SAR studies around this lead showed that the positioning of the carboxylate was critical, although it could be substituted by acid isosteres and amides. Small substituents on the dihydroisoquinoline gave improvements in potency. A set of "reverse sulfonamides" showed a 12-fold preference for the R stereoisomer. The compounds showed good cellular potency, as measured by inhibition of AKR1C3 metabolism of a known dinitrobenzamide substrate, with a broad rank order between enzymic and cellular activity, but amide analogues were more effective than predicted by the cellular assay.