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Invest New Drugs ; 31(3): 576-86, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23179338


Deletions or mutations in the tumor suppressor gene DPC4 (deleted in pancreatic carcinoma locus 4) are common in colon and pancreatic cancers. Using the Target-related Affinity Profiling (TRAP) chemical library screening method, a novel agent, UA8967, was selected for further studies because it showed greater potency in DPC4-deleted HCT-116 colon cancer cells. Cytotoxicity studies in six pancreatic cancer cell lines (MiaPaca-2, Panc-1, BxPC3, CF-PAC1, AsPC1, and T3M4), one normal human pancreatic ductal epithelial line (HPDE-6) and the HCT-116 DPC4(+/+) and HCT-116 DPC4(-/-) colon cancer cells showed IC50s ranging from 12-61 µM for exposure times of 72 h. Analysis of schedule dependence showed no advantage for long drug exposure times. There was also no selective inhibition of DNA, RNA or protein synthesis after exposure to UA8967. At 24-48 h, there was an accumulation of cells in G0/G1-phase and a proportionate reduction in S-phase cells. Within 1-6 h of exposure, cells were found to undergo an autophagic response, followed at 24 h by a low level of caspase-independent apoptosis with some necrosis. Because of the relatively non-specific mechanistic effects of UA8967, plasma membrane viability was evaluated using uptake of trypan blue and Sytox® Green dyes, and leakage of LDH. There was a dose dependent increase in Sytox® Green staining, trypan blue uptake and LDH leakage with increasing concentrations of UA8967, suggesting that UA8967 is affecting the plasma membrane. The DPC4(-/-) cells were more sensitive to UA8967 but not to DMSO, suggesting a drug-specific effect on cell membrane integrity.

Antineoplásicos/farmacologia , Indóis/farmacologia , Piperazinas/farmacologia , Proteína Smad4/genética , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos
J Med Chem ; 54(15): 5403-13, 2011 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-21692479


The metabolism of poly(ADP-ribose) (PAR) in response to DNA strand breaks, which involves the concerted activities of poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG), modulates cell recovery or cell death depending upon the level of DNA damage. While PARP inhibitors show high promise in clinical trials because of their low toxicity and selectivity for BRCA related cancers, evaluation of the therapeutic potential of PARG is limited by the lack of well-validated cell permeable inhibitors. In this study, target-related affinity profiling (TRAP), an alternative to high-throughput screening, was used to identify a number of druglike compounds from several chemical classes that demonstrated PARG inhibition in the low-micromolar range. A number of analogues of one of the most active chemotypes were synthesized to explore the structure-activity relationship (SAR) for that series. This led to the discovery of a putative pharmacophore for PARG inhibition that contains a modified salicylanilide structure. Interestingly, these compounds also inhibit PARP-1, indicating strong homology in the active sites of PARG and PARP-1 and raising a new challenge for development of PARG specific inhibitors. The cellular activity of a lead inhibitor was demonstrated by the inhibition of both PARP and PARG activity in squamous cell carcinoma cells, although preferential inhibition of PARG relative to PARP was observed. The ability of inhibitors to modulate PAR metabolism via simultaneous effects on PARPs and PARG may represent a new approach for therapeutic development.

Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Salicilanilidas/farmacologia , Carcinoma de Células Escamosas/metabolismo , Inibidores Enzimáticos/síntese química , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases , Salicilanilidas/síntese química , Relação Estrutura-Atividade
Curr Top Med Chem ; 5(4): 371-81, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15892680


Target-Related Affinity Profiling (TRAP) is a computational drug discovery technology that is based on 'affinity fingerprints', which are molecular descriptors derived from the protein binding preferences of small molecules. The underlying concepts of TRAP are reviewed. Affinity fingerprints are compared to molecular descriptors derived from chemical structures and shown to be a useful alternative for lead discovery. The TRAP screening process is described and two example applications are presented: I. the discovery of novel inhibitors of human intestinal carboxylesterase, and II. the discovery of novel inhibitors of cyclooxygenase-1 through the use of the affinity fingerprints of known cyclooxygenase-1 inhibitors. A summary of the complementary advantages of TRAP screening technology compared to traditional approaches to drug lead discovery concludes the review.

Desenho de Fármacos , Receptores de Droga/química , Animais , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Inibidores de Ciclo-Oxigenase/síntese química , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores Enzimáticos/farmacocinética , Humanos , Biologia Molecular , Prostaglandina-Endoperóxido Sintases/metabolismo , Ligação Proteica
J Med Chem ; 48(8): 2906-15, 2005 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-15828829


Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2) > 0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.

Carboxilesterase/antagonistas & inibidores , Fenilglioxal/análogos & derivados , Fenilglioxal/química , Acetilcolinesterase/química , Animais , Butirilcolinesterase/química , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/química , Inibidores da Colinesterase/química , Bases de Dados Factuais , Humanos , Intestinos/enzimologia , Modelos Moleculares , Fenilglioxal/síntese química , Relação Quantitativa Estrutura-Atividade , Ratos , Relação Estrutura-Atividade , Umbeliferonas/química
J Med Chem ; 47(20): 4875-80, 2004 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-15369391


We used protein affinity fingerprints to discover structurally novel inhibitors of cyclooxygenase-1 (COX-1) by screening a selected number of compounds, thus providing an alternative to extensive screening. From the affinity fingerprints of 19 known COX-1 inhibitors, a computational model for COX-1 inhibition was constructed and used to select candidate inhibitors from our compound library to be tested in the COX-1 assay. Subsequent refinement of the model by including affinity fingerprints of inactive compounds identified three molecules that were more potent than ibuprofen, a commonly used COX-1 inhibitor. These compounds are structurally distinct from those used to build the model and were discovered by testing only 62 library compounds. The discovery of these leads demonstrates the efficiency with which affinity fingerprints can identify novel bioactive chemotypes from known drugs.

Técnicas de Química Combinatória/métodos , Inibidores de Ciclo-Oxigenase/química , Inibidores de Ciclo-Oxigenase/farmacologia , Isoenzimas/antagonistas & inibidores , Modelos Teóricos , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Ciclo-Oxigenase 1 , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Ibuprofeno/química , Ibuprofeno/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Estudos Prospectivos , Prostaglandina-Endoperóxido Sintases , Relação Quantitativa Estrutura-Atividade
Mol Pharmacol ; 65(6): 1336-43, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15155827


The dose-limiting toxicity of the highly effective anticancer agent 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin (irinotecan; CPT-11) is delayed diarrhea. This is thought to be caused by either bacteria-mediated hydrolysis of the glucuronide conjugate of the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) or direct conversion of CPT-11 to SN-38 by carboxylesterases (CE) in the small intestine. After drug administration, a very high level of CPT-11 is present in the bile; this is deposited into the duodenum, the region of the gut with the highest levels of CE activity. Hence, it is likely that direct conversion of the drug to SN-38 is partially responsible for the diarrhea associated with this agent. In an attempt to ameliorate this toxicity, we have applied Target-Related Affinity Profiling to identify novel CE inhibitors that are selective inhibitors of the human intestinal enzyme (hiCE). Seven inhibitors, all sulfonamide derivatives, demonstrated greater than 200-fold selectivity for hiCE compared with the human liver CE hCE1, and none was an inhibitor of human acetylcholinesterase or butyrylcholinesterase. Quantitative structure-activity relationship (QSAR) analysis demonstrated excellent correlations with the predicted versus experimental Ki values (r2 = 0.944) for hiCE. Additionally, design and synthesis of a tetrafluorine-substituted sulfonamide analog, which QSAR indicated would demonstrate improved inhibition of hiCE, validated the computer predictive analyses. These and other phenyl-substituted sulfonamides compounds are regarded as lead compounds for the development of effective, selective CE inhibitors for clinical applications.

Camptotecina/análogos & derivados , Camptotecina/efeitos adversos , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Diarreia/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Antineoplásicos Fitogênicos/efeitos adversos , Antineoplásicos Fitogênicos/metabolismo , Camptotecina/metabolismo , Diarreia/induzido quimicamente , Inibidores Enzimáticos/química , Humanos , Intestinos/enzimologia , Irinotecano , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Coelhos , Sulfonamidas/síntese química , Sulfonamidas/química