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1.
J Org Chem ; 88(15): 10996-11002, 2023 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-37471139

RESUMO

Enigmazole B (1) and four new analogues, cis-enigmazole B (2), dehydroenigmazole B (3), enigmimide B (4), and enigmimide A (5), were isolated from the marine sponge Cinachyrella enigmatica. Their planar structures were elucidated by detailed NMR and MS data analyses, which established 1-3 to be oxazole-substituted 18-membered phosphomacrolides, while 4 and 5 were oxazole ring-opened congeners. The relative and absolute configurations in 1 were determined by a combination of chemical transformations and spectroscopic analyses. Photooxidation of the oxazole moiety in 1 gave enigmimide B (4), thus establishing that 4 has the same absolute configuration of 1. Enigmazole B (1) along with analogues 2 and 3 showed cytotoxicity against murine IC-2 mast cells with IC50 values of 3.6-7.0 µM. The enigmimides (4 and 5) and dephosphoenigmazoles did not show cytotoxicity (IC50 > 10 µM), implying that both the oxazole moiety and the phosphate group are necessary for the cytotoxicity of the enigmazole class macrolides.


Assuntos
Poríferos , Animais , Camundongos , Poríferos/química , Macrolídeos/química , Oxazóis/farmacologia , Oxazóis/química , Antibacterianos , Estrutura Molecular
2.
J Am Chem Soc ; 144(11): 4925-4941, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35282679

RESUMO

Germline antibodies, the initial set of antibodies produced by the immune system, are critical for host defense, and information about their binding properties can be useful for designing vaccines, understanding the origins of autoantibodies, and developing monoclonal antibodies. Numerous studies have found that germline antibodies are polyreactive with malleable, flexible binding pockets. While insightful, it remains unclear how broadly this model applies, as there are many families of antibodies that have not yet been studied. In addition, the methods used to obtain germline antibodies typically rely on assumptions and do not work well for many antibodies. Herein, we present a distinct approach for isolating germline antibodies that involves immunizing activation-induced cytidine deaminase (AID) knockout mice. This strategy amplifies antigen-specific B cells, but somatic hypermutation does not occur because AID is absent. Using synthetic haptens, glycoproteins, and whole cells, we obtained germline antibodies to an assortment of clinically important tumor-associated carbohydrate antigens, including Lewis Y, the Tn antigen, sialyl Lewis C, and Lewis X (CD15/SSEA-1). Through glycan microarray profiling and cell binding, we demonstrate that all but one of these germline antibodies had high selectivity for their glycan targets. Using molecular dynamics simulations, we provide insights into the structural basis of glycan recognition. The results have important implications for designing carbohydrate-based vaccines, developing anti-glycan monoclonal antibodies, and understanding antibody evolution within the immune system.


Assuntos
Anticorpos Monoclonais , Antígenos Glicosídicos Associados a Tumores , Animais , Anticorpos Monoclonais/química , Biomarcadores Tumorais , Carboidratos , Células Germinativas , Camundongos , Camundongos Knockout , Polissacarídeos/química
3.
Molecules ; 25(23)2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-33276504

RESUMO

Due to its antiangiogenic and anti-immunomodulatory activity, thalidomide continues to be of clinical interest despite its teratogenic actions, and efforts to synthesize safer, clinically active thalidomide analogs are continually underway. In this study, a cohort of 27 chemically diverse thalidomide analogs was evaluated for antiangiogenic activity in an ex vivo rat aorta ring assay. The protein cereblon has been identified as the target for thalidomide, and in silico pharmacophore analysis and molecular docking with a crystal structure of human cereblon were used to investigate the cereblon binding abilities of the thalidomide analogs. The results suggest that not all antiangiogenic thalidomide analogs can bind cereblon, and multiple targets and mechanisms of action may be involved.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Inibidores da Angiogênese/farmacologia , Aorta/efeitos dos fármacos , Simulação de Acoplamento Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Talidomida/análogos & derivados , Talidomida/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Inibidores da Angiogênese/química , Animais , Simulação por Computador , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
4.
J Biol Chem ; 291(21): 11133-47, 2016 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-27022025

RESUMO

The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Substituição de Aminoácidos , Sítios de Ligação/genética , Cristalografia por Raios X , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Dibutirato de 12,13-Forbol/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
J Mol Recognit ; 30(8)2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28233410

RESUMO

In this review, we address a fundamental question: What is the range of conformational energies seen in ligands in protein-ligand crystal structures? This value is important biophysically, for better understanding the protein-ligand binding process; and practically, for providing a parameter to be used in many computational drug design methods such as docking and pharmacophore searches. We synthesize a selection of previously reported conflicting results from computational studies of this issue and conclude that high ligand conformational energies really are present in some crystal structures. The main source of disagreement between different analyses appears to be due to divergent treatments of electrostatics and solvation. At the same time, however, for many ligands, a high conformational energy is in error, due to either crystal structure inaccuracies or incorrect determination of the reference state. Aside from simple chemistry mistakes, we argue that crystal structure error may mainly be because of the heuristic weighting of ligand stereochemical restraints relative to the fit of the structure to the electron density. This problem cannot be fixed with improvements to electron density fitting or with simple ligand geometry checks, though better metrics are needed for evaluating ligand and binding site chemistry in addition to geometry during structure refinement. The ultimate solution for accurately determining ligand conformational energies lies in ultrahigh-resolution crystal structures that can be refined without restraints.


Assuntos
Conformação Proteica , Proteínas/química , Termodinâmica , Animais , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas/agonistas , Proteínas/antagonistas & inibidores , Solubilidade , Eletricidade Estática
6.
Bioorg Med Chem ; 25(12): 2971-2980, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28392275

RESUMO

C1 domain-containing proteins, such as protein kinase C (PKC), have a central role in cellular signal transduction. Their involvement in many diseases, including cancer, cardiovascular disease, and immunological and neurological disorders has been extensively demonstrated and has prompted a search for small molecules to modulate their activity. By employing a diacylglycerol (DAG)-lactone template, we have been able to develop ultra potent analogs of diacylglycerol with nanomolar binding affinities approaching those of complex natural products such as phorbol esters and bryostatins. One current challenge is the development of selective ligands capable of discriminating between different protein family members. Recently, structure-activity relationship studies have shown that the introduction of an indole ring as a DAG-lactone substituent yielded selective Ras guanine nucleotide-releasing protein (RasGRP1) activators when compared to PKCα and PKCε. In the present work, we examine the effects of ligand selectivity relative to the orientation of the indole ring and the nature of the DAG-lactone template itself. Our results show that the indole ring must be attached to the lactone moiety through the sn-2 position in order to achieve RasGRP1 selectivity.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Indóis/química , Indóis/farmacologia , Lactonas/química , Lactonas/farmacologia , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-épsilon/metabolismo , Proteínas de Ligação a DNA/química , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Proteína Quinase C-alfa/química , Proteína Quinase C-épsilon/química , Relação Estrutura-Atividade
7.
J Org Chem ; 81(17): 7862-83, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27494208

RESUMO

As an initial step in designing a simplified bryostatin hybrid molecule, three bryostatin analogues bearing a diacylglycerol lactone-based C-ring, which possessed the requisite pharmacophores for binding to protein kinase C (PKC) together with a modified bryostatin-like A- and B-ring region, were synthesized and evaluated. Merle 46 and Merle 47 exhibited binding affinity to PKC alpha with Ki values of 7000 ± 990 and 4940 ± 470 nM, respectively. Reinstallation of the trans-olefin and gem-dimethyl group present in bryostatin 1 in Merle 48 resulted in improved binding affinity, 363 ± 42 nM. While Merle 46 and 47 were only marginally active biologically, Merle 48 showed sufficient activity on the U937 cells to confirm that it was PMA-like for growth and attachment, as predicted by the substitution pattern of its A- and B-rings.


Assuntos
Briostatinas/síntese química , Briostatinas/farmacologia , Diglicerídeos/química , Lactonas/química , Briostatinas/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Proteína Quinase C/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato
8.
J Chem Inf Model ; 56(11): 2149-2161, 2016 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-27669079

RESUMO

We investigated how many cases of the same chemical sold as different products (at possibly different prices) occurred in a prototypical large aggregated database and simultaneously tested the tautomerism definitions in the chemoinformatics toolkit CACTVS. We applied the standard CACTVS tautomeric transforms plus a set of recently developed ring-chain transforms to the Aldrich Market Select (AMS) database of 6 million screening samples and building blocks. In 30 000 cases, two or more AMS products were found to be just different tautomeric forms of the same compound. We purchased and analyzed 166 such tautomer pairs and triplets by 1H and 13C NMR to determine whether the CACTVS transforms accurately predicted what is the same "stuff in the bottle". Essentially all prototropic transforms with examples in the AMS were confirmed. Some of the ring-chain transforms were found to be too "aggressive", i.e. to equate structures with one another that were different compounds.


Assuntos
Bases de Dados Factuais , Informática/métodos , Compostos Orgânicos/química , Bases de Dados Factuais/economia , Isomerismo
9.
J Org Chem ; 80(20): 9900-9, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26372257

RESUMO

Warfarin, an important anticoagulant drug, can exist in solution in 40 distinct tautomeric forms through both prototropic tautomerism and ring-chain tautomerism. We have investigated all warfarin tautomers with computational and NMR approaches. Relative energies calculated at the B3LYP/6-311G++(d,p) level of theory indicate that the 4-hydroxycoumarin cyclic hemiketal tautomer is the most stable tautomer in aqueous solution, followed by the 4-hydroxycoumarin open-chain tautomer. This is in agreement with our NMR experiments where the spectral assignments indicate that warfarin exists mainly as a mixture of cyclic hemiketal diastereomers, with an open-chain tautomer as a minor component. We present a diagram of the interconversion of warfarin created taking into account the calculated equilibrium constants (pK(T)) for all tautomeric reactions. These findings help with gaining further understanding of proton transfer and ring closure tautomerization processes. We also discuss the results in the context of chemoinformatics rules for handling tautomerism.


Assuntos
Anticoagulantes/química , Simulação de Dinâmica Molecular , Teoria Quântica , Varfarina/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Estereoisomerismo
10.
Bioorg Med Chem Lett ; 25(10): 2146-51, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25881829

RESUMO

Protein sumoylation is a dynamic posttranslational modification that regulates a diverse subset of the proteome. The mechanism by which sumoylation enzymes recognize their cognate substrates is unclear, and the consequences of sumoylation remain difficult to predict. While small molecule probes of the sumoylation process could be valuable for understanding SUMO biology, few small molecules that modulate this process exist. Here, we report the synthesis and evaluation of over 600 oxime-containing peptide sumoylation substrates. Our work demonstrates that higher modification efficiency can be achieved with non-natural side chains that deviate substantially from the consensus site requirement of a hydrophobic substituent. Furthermore, docking studies suggest that these improved substrates mimic binding interactions that are used by other endogenous protein sequences through tertiary interactions. The development of these high efficiency substrates provides key mechanistic insights toward specific recognition of low molecular weight species in the sumoylation pathway.


Assuntos
Sequência Consenso , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Sumoilação , Especificidade por Substrato
11.
J Am Chem Soc ; 136(38): 13202-8, 2014 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-25207434

RESUMO

A convergent synthesis of a des-B-ring bryostatin analogue is described. This analogue was found to undergo an unexpected ring expansion of the bryolactone core to generate the corresponding 21-membered macrocycle. The parent analogue and the ring-expanded product both displayed nanomolar binding affinity for PKC. Despite containing A-ring substitution identical to that of bryostatin 1 and displaying bryostatin-like biological function, the des-B-ring analogues displayed a phorbol-like biological function in cells. These studies shed new light on the role of the bryostatin B-ring in conferring bryo-like biological function to bryostatin analogues.


Assuntos
Antineoplásicos/química , Produtos Biológicos/química , Briostatinas/química , Briozoários/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Produtos Biológicos/síntese química , Produtos Biológicos/farmacologia , Briostatinas/síntese química , Briostatinas/farmacologia , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Forbóis/farmacologia , Proteína Quinase C/metabolismo
12.
J Am Chem Soc ; 136(38): 13209-16, 2014 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-25207655

RESUMO

The seco-B-ring bryostatin analogue, macrodiolide WN-1, was prepared in 17 steps (longest linear sequence) and 30 total steps with three bonds formed via hydrogen-mediated C-C coupling. This synthetic route features a palladium-catalyzed alkoxycarbonylation of a C2-symmetric diol to form the C9-deoxygenated bryostatin A-ring. WN-1 binds to PKCα (Ki = 16.1 nM) and inhibits the growth of multiple leukemia cell lines. Although structural features of the WN-1 A-ring and C-ring are shared by analogues that display bryostatin-like behavior, WN-1 displays PMA-like behavior in U937 cell attachment and proliferation assays, as well as in K562 and MV-4-11 proliferation assays. Molecular modeling studies suggest the pattern of internal hydrogen bonds evident in bryostatin 1 is preserved in WN-1, and that upon docking WN-1 into the crystal structure of the C1b domain of PKCδ, the binding mode of bryostatin 1 is reproduced. The collective data emphasize the critical contribution of the B-ring to the function of the upper portion of the molecule in conferring a bryostatin-like pattern of biological activity.


Assuntos
Antineoplásicos/química , Briostatinas/química , Briozoários/química , Acetato de Tetradecanoilforbol/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Briostatinas/síntese química , Briostatinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Hidrogenação , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Modelos Moleculares , Proteína Quinase C-alfa/metabolismo , Acetato de Tetradecanoilforbol/síntese química , Acetato de Tetradecanoilforbol/farmacologia , Células U937
13.
J Chem Inf Model ; 54(3): 705-12, 2014 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-24524735

RESUMO

Many of the structures in PubChem are annotated with activities determined in high-throughput screening (HTS) assays. Because of the nature of these assays, the activity data are typically strongly imbalanced, with a small number of active compounds contrasting with a very large number of inactive compounds. We have used several such imbalanced PubChem HTS assays to test and develop strategies to efficiently build robust QSAR models from imbalanced data sets. Different descriptor types [Quantitative Neighborhoods of Atoms (QNA) and "biological" descriptors] were used to generate a variety of QSAR models in the program GUSAR. The models obtained were compared using external test and validation sets. We also report on our efforts to incorporate the most predictive of our models in the publicly available NCI/CADD Group Web services ( http://cactus.nci.nih.gov/chemical/apps/cap).


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Relação Quantitativa Estrutura-Atividade , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Algoritmos , Bases de Dados de Compostos Químicos , Células HEK293 , Humanos , Modelos Biológicos , Software
14.
J Chem Inf Model ; 54(3): 713-9, 2014 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-24451033

RESUMO

We describe a novel approach to RBF approximation, which combines two new elements: (1) linear radial basis functions and (2) weighting the model by each descriptor's contribution. Linear radial basis functions allow one to achieve more accurate predictions for diverse data sets. Taking into account the contribution of each descriptor produces more accurate similarity values used for model development. The method was validated on 14 public data sets comprising nine physicochemical properties and five toxicity endpoints. We also compared the new method with five different QSAR methods implemented in the EPA T.E.S.T. program. Our approach, implemented in the program GUSAR, showed a reasonable accuracy of prediction and high coverage for all external test sets, providing more accurate prediction results than the comparison methods and even the consensus of these methods. Using our new method, we have created models for physicochemical and toxicity endpoints, which we have made freely available in the form of an online service at http://cactus.nci.nih.gov/chemical/apps/cap.


Assuntos
Algoritmos , Modelos Biológicos , Relação Quantitativa Estrutura-Atividade , Software , Animais , Simulação por Computador , Cyprinidae/fisiologia , Daphnia/efeitos dos fármacos , Daphnia/fisiologia , Bases de Dados Factuais , Internet , Redes Neurais de Computação , Ratos , Tetrahymena/efeitos dos fármacos , Tetrahymena/fisiologia , Testes de Toxicidade
15.
Bioorg Med Chem ; 22(12): 3123-40, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24794745

RESUMO

The development of selective agents capable of discriminating between protein kinase C (PKC) isoforms and other diacylglycerol (DAG)-responsive C1 domain-containing proteins represents an important challenge. Recent studies have highlighted the role that Ras guanine nucleotide-releasing protein (RasGRP) isoforms play both in immune responses as well as in the development of prostate cancer and melanoma, suggesting that the discovery of selective ligands could have potential therapeutic value. Thus far, the N-methyl-substituted indololactone 1 is the agonist with the highest reported potency and selectivity for RasGRP relative to PKC. Here we present the synthesis, binding studies, cellular assays and biophysical analysis of interactions with model membranes of a family of regioisomers of 1 (compounds 2-5) that differ in the position of the linkage between the indole ring and the lactone moiety. These structural variations were studied to explore the interaction of the active complex (C1 domain-ligand) with cellular membranes, which is believed to be an important factor for selectivity in the activation of DAG-responsive C1 domain containing signaling proteins. All compounds were potent and selective activators of RasGRP when compared to PKCα with selectivities ranging from 6 to 65 fold. However, the parent compound 1 was appreciably more selective than any of the other isomers. In intact cells, modest differences in the patterns of translocation of the C1 domain targets were observed. Biophysical studies using giant vesicles as model membranes did show substantial differences in terms of molecular interactions impacting lipid organization, dynamics and membrane insertion. However, these differences did not yield correspondingly large changes in patterns of biological response, at least for the parameters examined.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Diglicerídeos/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Indóis/farmacologia , Lactonas/farmacologia , Neoplasias da Próstata/patologia , Proteína Quinase C/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Células Cultivadas , Cricetulus , Diglicerídeos/química , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Indóis/química , Lactonas/química , Masculino , Modelos Moleculares , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Isoformas de Proteínas
16.
Mar Drugs ; 12(8): 4593-601, 2014 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-25196934

RESUMO

A cell-based high-throughput screen that assessed the cellular stability of a tumor suppressor protein PDCD4 (Programmed cell death 4) was used to identify a new guanidine-containing marine alkaloid mirabilin K (3), as well as the known compounds mirabilin G (1) and netamine M (2). The structures of these tricyclic guanidine alkaloids were established from extensive spectroscopic analyses. Compounds 1 and 2 inhibited cellular degradation of PDCD4 with EC50 values of 1.8 µg/mL and 2.8 µg/mL, respectively. Mirabilin G (1) and netamine M (2) are the first marine natural products reported to stabilize PDCD4 under tumor promoting conditions.


Assuntos
Alcaloides/química , Alcaloides/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Guanidina/química , Guanidina/farmacologia , Poríferos/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Células HEK293 , Humanos , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/química , Saponinas/química
17.
Nat Commun ; 15(1): 1703, 2024 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-38402212

RESUMO

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.


Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Criança , Humanos , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma Alveolar/genética , Linhagem Celular Tumoral , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases/metabolismo
18.
J Biol Chem ; 287(16): 13137-58, 2012 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-22351766

RESUMO

C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologous to that of the typical C1 domains of PKCs. This study clarifies the basis for its failure to bind ligands. Substituting Vav1-specific residues into the C1b domain of PKCδ, we identified five crucial residues (Glu(9), Glu(10), Thr(11), Thr(24), and Tyr(26)) along the rim of the binding cleft that weaken binding potency in a cumulative fashion. Reciprocally, replacing these incompatible residues in the Vav1 C1 domain with the corresponding residues from PKCδ C1b (δC1b) conferred high potency for phorbol ester binding. Computer modeling predicts that these unique residues in Vav1 increase the hydrophilicity of the rim of the binding pocket, impairing membrane association and thereby preventing formation of the ternary C1-ligand-membrane binding complex. The initial design of diacylglycerol-lactones to exploit these Vav1 unique residues showed enhanced selectivity for C1 domains incorporating these residues, suggesting a strategy for the development of ligands targeting Vav1.


Assuntos
Diglicerídeos/metabolismo , Ésteres de Forbol/metabolismo , Proteínas Proto-Oncogênicas c-vav/química , Proteínas Proto-Oncogênicas c-vav/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Lactonas/metabolismo , Ligantes , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfolipídeos/metabolismo , Neoplasias da Próstata , Ligação Proteica/fisiologia , Proteína Quinase C-delta/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-vav/genética , Transdução de Sinais/fisiologia
19.
J Chem Inf Model ; 52(3): 739-56, 2012 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-22303903

RESUMO

We present here a greatly updated version of an earlier study on the conformational energies of protein-ligand complexes in the Protein Data Bank (PDB) [Nicklaus et al. Bioorg. Med. Chem. 1995, 3, 411-428], with the goal of improving on all possible aspects such as number and selection of ligand instances, energy calculations performed, and additional analyses conducted. Starting from about 357,000 ligand instances deposited in the 2008 version of the Ligand Expo database of the experimental 3D coordinates of all small-molecule instances in the PDB, we created a "high-quality" subset of ligand instances by various filtering steps including application of crystallographic quality criteria and structural unambiguousness. Submission of 640 Gaussian 03 jobs yielded a set of about 415 successfully concluded runs. We used a stepwise optimization of internal degrees of freedom at the DFT level of theory with the B3LYP/6-31G(d) basis set and a single-point energy calculation at B3LYP/6-311++G(3df,2p) after each round of (partial) optimization to separate energy changes due to bond length stretches vs bond angle changes vs torsion changes. Even for the most "conservative" choice of all the possible conformational energies-the energy difference between the conformation in which all internal degrees of freedom except torsions have been optimized and the fully optimized conformer-significant energy values were found. The range of 0 to ~25 kcal/mol was populated quite evenly and independently of the crystallographic resolution. A smaller number of "outliers" of yet higher energies were seen only at resolutions above 1.3 Å. The energies showed some correlation with molecular size and flexibility but not with crystallographic quality metrics such as the Cruickshank diffraction-component precision index (DPI) and R(free)-R, or with the ligand instance-specific metrics such as occupancy-weighted B-factor (OWAB), real-space R factor (RSR), and real-space correlation coefficient (RSCC). We repeated these calculations with the solvent model IEFPCM, which yielded energy differences that were generally somewhat lower than the corresponding vacuum results but did not produce a qualitatively different picture. Torsional sampling around the crystal conformation at the molecular mechanics level using the MMFF94s force field typically led to an increase in energy.


Assuntos
Bases de Dados de Proteínas , Conformação Molecular , Teoria Quântica , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Solventes/química , Termodinâmica
20.
J Med Chem ; 64(15): 11418-11431, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34279947

RESUMO

DAG-lactones represent useful templates for the design of potent and selective C1 domain ligands for PKC isozymes. The ester moiety at the sn-1 position, a common feature in this template, is relevant for C1 domain interactions, but it represents a labile group susceptible to endogenous esterases. An interesting challenge involves replacing the ester group of these ligands while still maintaining biological activity. Here, we present the synthesis and functional characterization of novel diacylglycerol-lactones containing heterocyclic ring substituents at the sn-1 position. Our results showed that the new compound 10B12, a DAG-lactone with an isoxazole ring, binds PKCα and PKCε with nanomolar affinity. Remarkably, 10B12 displays preferential selectivity for PKCε translocation in cells and induces a PKCε-dependent reorganization of the actin cytoskeleton into peripheral ruffles in lung cancer cells. We conclude that introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a novel structural approach to achieve PKC isozyme selectivity.


Assuntos
Diglicerídeos/farmacologia , Desenho de Fármacos , Compostos Heterocíclicos/farmacologia , Lactonas/farmacologia , Proteína Quinase C/metabolismo , Diglicerídeos/síntese química , Diglicerídeos/química , Relação Dose-Resposta a Droga , Células HeLa , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Humanos , Isoenzimas/metabolismo , Lactonas/síntese química , Lactonas/química , Estrutura Molecular , Relação Estrutura-Atividade
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