RESUMEN
Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proteínas de Arabidopsis/antagonistas & inhibidores , Daño del ADN/fisiología , Proteínas Nucleares/antagonistas & inhibidores , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Ubiquitinación/efectos de los fármacos , Algoritmos , Butiratos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Ensayo de Unidades Formadoras de Colonias , Daño del ADN/genética , ADN de Neoplasias/antagonistas & inhibidores , ADN de Neoplasias/biosíntesis , Resistencia a Antineoplásicos , Electroforesis en Gel de Poliacrilamida , Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Humanos , Indicadores y Reactivos , Compuestos de Fenilurea/farmacología , Pimozida/farmacología , Antígeno Nuclear de Célula en Proliferación/efectos de los fármacos , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Interferente Pequeño/genética , Proteínas Recombinantes/química , Recombinación Genética/efectos de los fármacos , Intercambio de Cromátides Hermanas/efectos de los fármacosRESUMEN
Human cells utilize a variety of complex DNA repair mechanisms in order to combat constant mutagenic and cytotoxic threats from both exogenous and endogenous sources. The RecQ family of DNA helicases, which includes Bloom helicase (BLM), plays an important function in DNA repair by unwinding complementary strands of duplex DNA as well as atypical DNA structures such as Holliday junctions. Mutations of the BLM gene can result in Bloom syndrome, an autosomal recessive disorder associated with cancer predisposition. BLM-deficient cells exhibit increased sensitivity to DNA damaging agents indicating that a selective BLM inhibitor could be useful in potentiating the anticancer activity of these agents. In this work, we describe the medicinal chemistry optimization of the hit molecule following a quantitative high-throughput screen of >355,000 compounds. These efforts lead to the identification of ML216 and related analogs, which possess potent BLM inhibition and exhibit selectivity over related helicases. Moreover, these compounds demonstrated cellular activity by inducing sister chromatid exchanges, a hallmark of Bloom syndrome.
Asunto(s)
Aminas/química , Inhibidores Enzimáticos/síntesis química , Compuestos de Fenilurea/síntesis química , RecQ Helicasas/antagonistas & inhibidores , Tiadiazoles/química , Aminas/síntesis química , Aminas/farmacología , Células CACO-2 , Permeabilidad de la Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , RecQ Helicasas/metabolismo , Relación Estructura-Actividad , Tiadiazoles/síntesis química , Tiadiazoles/farmacologíaRESUMEN
Continued examination of substituted 6-arylquinazolin-4-amines as Clk4 inhibitors resulted in selective inhibitors of Clk1, Clk4, Dyrk1A and Dyrk1B. Several of the most potent inhibitors were validated as being highly selective within a comprehensive kinome scan.
Asunto(s)
Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Concentración 50 Inhibidora , Isoenzimas/antagonistas & inhibidores , Modelos Moleculares , Estructura Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Quinazolinas/síntesis química , Quinazolinas/química , Quinazolinas/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Especificidad por Sustrato , Tiazoles/síntesis química , Tiazoles/química , Tiazoles/farmacología , Quinasas DyrKRESUMEN
In four or five chemical steps from the 1,2,4-trioxane artemisinin, a new series of 23 trioxane dimers has been prepared. Eleven of these new trioxane dimers cure malaria-infected mice via oral dosing at 3 x 30 mg/kg. The clinically used trioxane drug sodium artesunate prolonged mouse average survival to 7.2 days with this oral dose regimen. In comparison, animals receiving no drug die typically on day 6-7 postinfection. At only 3 x 10 mg/kg oral dosing, seven dimers prolong the lifetime of malaria-infected mice to days 14-17, more than double the chemotherapeutic effect of sodium artesunate. Ten new trioxane dimers at only a single oral dose of 30 mg/kg prolong mouse average survival to days 8.7-13.7, and this effect is comparable to that of the fully synthetic trioxolane drug development candidate OZ277, which is in phase II clinical trials.
Asunto(s)
Antimaláricos/síntesis química , Artemisininas/síntesis química , Malaria/tratamiento farmacológico , Animales , Antimaláricos/química , Antimaláricos/farmacología , Artemisininas/química , Artemisininas/uso terapéutico , Ratones , Plasmodium berghei , Relación Estructura-ActividadRESUMEN
We disclose here for the first time the curative activity of a new generation of trioxane dimers, designed logically and prepared easily from the natural trioxane artemisinin in only four or five chemical steps that would be easily accomplished also on a manufacturing scale. Four of these trioxane dimers cure malaria-infected mice after only a single subcutaneous dose, and two other dimers cure after three oral doses.
Asunto(s)
Antimaláricos/síntesis química , Artemisininas/síntesis química , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Malaria/tratamiento farmacológico , Animales , Antimaláricos/química , Antimaláricos/uso terapéutico , Artemisininas/química , Artemisininas/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Malaria/mortalidad , Ratones , Plasmodium berghei , Relación Estructura-ActividadRESUMEN
Deregulation of ubiquitin conjugation or deconjugation has been implicated in the pathogenesis of many human diseases including cancer. The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1), in association with UAF1 (USP1-associated factor 1), is a known regulator of DNA damage response and has been shown as a promising anticancer target. To further evaluate USP1/UAF1 as a therapeutic target, we conducted a quantitative high throughput screen of >400000 compounds and subsequent medicinal chemistry optimization of small molecules that inhibit the deubiquitinating activity of USP1/UAF1. Ultimately, these efforts led to the identification of ML323 (70) and related N-benzyl-2-phenylpyrimidin-4-amine derivatives, which possess nanomolar USP1/UAF1 inhibitory potency. Moreover, we demonstrate a strong correlation between compound IC50 values for USP1/UAF1 inhibition and activity in nonsmall cell lung cancer cells, specifically increased monoubiquitinated PCNA (Ub-PCNA) levels and decreased cell survival. Our results establish the druggability of the USP1/UAF1 deubiquitinase complex and its potential as a molecular target for anticancer therapies.
Asunto(s)
Antineoplásicos/síntesis química , Proteínas de Arabidopsis/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Pirimidinas/síntesis química , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Pirimidinas/farmacología , Relación Estructura-Actividad , UbiquitinaciónRESUMEN
Drug screening against novel targets is warranted to generate biochemical probes and new therapeutic drug leads. TDP1 and TDP2 are two DNA repair enzymes that have yet to be successfully targeted. TDP1 repairs topoisomerase I-, alkylation-, and chain terminator-induced DNA damage, whereas TDP2 repairs topoisomerase II-induced DNA damage. Here, we report the quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository using recombinant human TDP1. We also developed a secondary screening method using a multiple loading gel-based assay where recombinant TDP1 is replaced by whole cell extract (WCE) from genetically engineered DT40 cells. While developing this assay, we determined the importance of buffer conditions for testing TDP1, and most notably the possible interference of phosphate-based buffers. The high specificity of endogenous TDP1 in WCE allowed the evaluation of a large number of hits with up to 600 samples analyzed per gel via multiple loadings. The increased stringency of the WCE assay eliminated a large fraction of the initial hits collected from the qHTS. Finally, inclusion of a TDP2 counter-screening assay allowed the identification of two novel series of selective TDP1 inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/química , Animales , Antineoplásicos/química , Camptotecina/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Pollos , Sinergismo Farmacológico , Humanos , Fragmentos de Péptidos/química , Inhibidores de Fosfodiesterasa/químicaRESUMEN
The Bloom's syndrome protein, BLM, is a member of the conserved RecQ helicase family. Although cell lines lacking BLM exist, these exhibit progressive genomic instability that makes distinguishing primary from secondary effects of BLM loss problematic. In order to be able to acutely disable BLM function in cells, we undertook a high throughput screen of a chemical compound library for small molecule inhibitors of BLM. We present ML216, a potent inhibitor of the DNA unwinding activity of BLM. ML216 shows cell-based activity and can induce sister chromatid exchanges, enhance the toxicity of aphidicolin, and exert antiproliferative activity in cells expressing BLM, but not those lacking BLM. These data indicate that ML216 shows strong selectivity for BLM in cultured cells. We discuss the potential utility of such a BLM-targeting compound as an anticancer agent.
Asunto(s)
Inestabilidad Cromosómica/efectos de los fármacos , RecQ Helicasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , ADN/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Unión Proteica/efectos de los fármacos , RecQ Helicasas/metabolismoRESUMEN
We recently reported that two artemisinin-derived dimers (dimer primary alcohol 606 and dimer sulfone 4-carbamate 832-4) are significantly more potent in inhibiting human cytomegalovirus (CMV) replication than artemisinin-derived monomers. In our continued evaluation of the activities of artemisinins in CMV inhibition, twelve artemisinin-derived dimers and five artemisinin-derived monomers were used. Dimers as a group were found to be potent inhibitors of CMV replication. Comparison of CMV inhibition and the slope parameter of dimers and monomers suggest that dimers are distinct in their anti-CMV activities. A deoxy dimer (574), lacking the endoperoxide bridge, did not have any effect on CMV replication, suggesting a role for the endoperoxide bridge in CMV inhibition. Differences in anti-CMV activity were observed among three structural analogs of dimer sulfone 4-carbamate 832-4 indicating that the exact placement and oxidation state of the sulfur atom may contribute to its anti-CMV activity. Of all tested dimers, artemisinin-derived diphenyl phosphate dimer 838 proved to be the most potent inhibitor of CMV replication, with a selectivity index of approximately 1500, compared to our previously reported dimer sulfone 4-carbamate 832-4 with a selectivity index of about 900. Diphenyl phosphate dimer 838 was highly active against a Ganciclovir-resistant CMV strain and was also the most active dimer in inhibition of cancer cell growth. Thus, diphenyl phosphate dimer 838 may represent a lead for development of a highly potent and safe anti-CMV compound.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Antivirales/química , Antivirales/farmacología , Artemisininas/química , Citomegalovirus/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células HeLa , HumanosRESUMEN
The antimalarial trioxanes, exemplified by the naturally occurring sesquiterpene lactone artemisinin and its semi-synthetic derivatives, contain an endoperoxide pharmacophore that lends tremendous potency against Plasmodium parasites. Despite decades of research, their mechanism of action remains unresolved. A leading model of anti-plasmodial activity hypothesizes that iron-mediated cleavage of the endoperoxide bridge generates cytotoxic drug metabolites capable of damaging cellular macromolecules. To probe the malarial targets of the endoperoxide drugs, we studied the distribution of fluorescent dansyl trioxane derivatives in living, intraerythrocytic-stage Plasmodium falciparum parasites using microscopic imaging. The fluorescent trioxanes rapidly accumulated in parasitized erythrocytes, localizing within digestive vacuole-associated neutral lipid bodies of trophozoites and schizonts, and surrounding the developing merozoite membranes. Artemisinin pre-treatment significantly reduced fluorescent labeling of neutral lipid bodies, while iron chelation increased non-specific cytoplasmic localization. To further explore the effects of endoperoxides on cellular lipids, we used an oxidation-sensitive BODIPY lipid probe to show the presence of artemisinin-induced peroxyl radicals in parasite membranes. Lipid extracts from artemisinin-exposed parasites contained increased amounts of free fatty acids and a novel cholesteryl ester. The cellular accumulation patterns and effects on lipids were entirely endoperoxide-dependent, as inactive dioxolane analogs lacking the endoperoxide moiety failed to label neutral lipid bodies or induce oxidative membrane damage. In the parasite digestive vacuole, neutral lipids closely associate with heme and promote hemozoin formation. We propose that the trioxane artemisinin and its derivatives are activated by heme-iron within the neutral lipid environment where they initiate oxidation reactions that damage parasite membranes.
Asunto(s)
Antimaláricos/metabolismo , Artemisininas/metabolismo , Metabolismo de los Lípidos , Peróxidos/metabolismo , Plasmodium falciparum/metabolismo , Animales , Antimaláricos/química , Artemisininas/química , Cromatografía en Capa Delgada , Peroxidación de Lípido , Espectroscopía de Resonancia Magnética , Microscopía FluorescenteRESUMEN
A new series of 6 dimeric trioxane sulfones has been prepared from the natural trioxane artemisinin in five or six chemical steps. One of these thermally and hydrolytically stable new chemical entities (4c) completely cured malaria-infected mice via a single oral dose of 144 mg/kg. At a much lower single oral dose of only 54 mg/kg combined with 13 mg/kg of mefloquine hydrochloride, this trioxane dimer 4c as well as its parent trioxane dimer 4b also completely cured malaria-infected mice. Both dimers 4c and 4b were potently and selectively cytotoxic toward five cancer cell lines.