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1.
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
2.
J Am Chem Soc ; 144(9): 4017-4025, 2022 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-35195411

RESUMO

Protein kinases are therapeutic targets for many human diseases, but the lack of user-friendly quantitative assays limits the ability to follow the activities of numerous kinases at once (multiplexing). To develop such an assay, we report an array of sulfonamido-oxine (SOX)-labeled peptides showing cross-reactivity to different mitogen-activated protein kinases (MAPKs) for use in a differential sensing scheme. We first verified using linear discriminant analysis that the array could differentiate MAPK isoforms. Then, using principal component analysis, the array was optimized based on the discrimination imparted by each SOX-peptide. Next, the activity of individual MAPK families in ternary mixtures was quantified by support vector machine regression. Finally, we multiplexed the quantification of three MAPK families using partial least squares regression in A549 cell lysates, which has possible interference from other kinase classes. Thus, our method simultaneously quantifies the activity of multiple kinases. The technique could be applied to other protein kinase families and the monitoring of diseases.


Assuntos
Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peptídeos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
3.
Bioorg Chem ; 116: 105344, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34598088

RESUMO

Src kinase activity controls diverse cellular functions, including cell growth, migration, adhesion, and survival. It is de-regulated in several cancers, including breast cancer, where it is highly expressed and phosphorylated. Thus, targeting Src by a small molecule is a feasible strategy for managing different breast cancer types. Several Src kinase inhibitors are available, including the FDA-approved drug (dasatinib). However, they are primarily ATP-competitive inhibitors that have been reported to lack specificity towards Src. We have a long-time interest in discovering protein kinase inhibitors that are non-competitive for ATP. In this project, three groups of 2'-aminospiro[pyrano[3,2-c]quinoline]-3'-carbonitrile derivatives were designed and synthesized, hypothesizing that small molecules with a spiro scaffold appended to a pyrano[3,2-c]quinoline analog could act as non-ATP competitive Src kinase inhibitors. 3b, 3c, and 3d inhibited Src kinase activity with IC50s of 4.9, 5.9, and 0.9 µM, respectively. At the same time, they did not impact the MDM2/p53 interaction in HEK293 cells, which has been reported to be affected by some spirocyclic compounds. 25 µM of 3b, 3c, or 3d did not inhibit the kinase activity of ERK2, JNK1, or p38-alpha in an in-vitro kinase assay. Steady-state kinetic studies for the effect of 3d on the ability of recombinant Src to phosphorylate its substrate (Srctide) revealed a non-ATP competitive inhibition mechanism. 1.6 µM of 3d was enough to diminish Src, Fak, and paxillin phosphorylation in the breast cancer cell lines MDA-MB-231 and MCF7. In the NCI screening, 3d induced broad tumor cytotoxicity for the NCI-60 cell lines, including all the breast cancer cell lines. The potency of 3b, 3c, and 3d to inhibit migration, proliferation, and colony formation of MDA-MB-231 and proliferation of MCF7 cells correlates with their potency to suppress Src kinase activity in the same cell line. Noticeably, the cell growth suppression and apoptosis induction in the tested cell lines can be attributed to the ability of the new derivatives to suppress the ERK and Akt survival pathways downstream of Src.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Desenvolvimento de Medicamentos , Inibidores de Proteínas Quinases/farmacologia , Piranos/farmacologia , Quinolinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Piranos/síntese química , Piranos/química , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-Atividade , Quinases da Família src/metabolismo
4.
ACS Chem Biol ; 15(1): 83-92, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31775004

RESUMO

The understanding of complex biological systems requires an ability to evaluate interacting networks of genes, proteins, and cellular reactions. Enabling technologies that support the rapid quantification of these networks will facilitate the development of biological models and help to identify treatment targets and to assess treatment plans. The biochemical process of protein phosphorylation, which underlies almost all aspects of cell signaling, is typically evaluated by immunoblotting procedures (Western blot) or more recently proteomics procedures, which provide qualitative estimates of the concentration of proteins and their modifications in cells. However, protein modifications are difficult to correlate with activity, and while immunoblotting and proteomics approaches have the potential to be quantitative, they require a complex series of steps that diminish reproducibility. Here, a complementary approach is presented that allows for the rapid quantification of a protein kinase activity in cell lysates and tumor samples. Using the activity of cellular ERK (extracellular signal-regulated kinase) as a test case, an array sensing approach that utilizes a library of differential peptide-based biosensors and chemometric tools was used to rapidly quantify nanograms of active ERK in micrograms of unfractionated cell lysates and tumor extracts. This approach has the potential both for high-throughput and for quantifying the activities of multiple protein kinases in a single biological sample. The critical advantages of this differential sensing approach over others are that it removes the need for the addition of exogenous inhibitors to suppress the activities of major off-target kinases and allows us to quantitate the amount of active kinase in tested samples rather than measuring the changes in its activity upon induction or inhibition.


Assuntos
Técnicas Biossensoriais/métodos , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Extratos Celulares/química , Linhagem Celular Tumoral , Humanos , Immunoblotting/métodos , Cinética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Reprodutibilidade dos Testes , Transdução de Sinais
5.
Nat Commun ; 10(1): 5232, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31745079

RESUMO

Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies. In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK. The DRS is a conserved region that lies distal to the active site and mediates ERK-protein interactions. We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo. BI-78D3 does not covalently modify p38MAPK, JNK or ERK5. BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation. These studies provide the basis for designing modulators of protein-protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers.


Assuntos
Dioxanos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/tratamento farmacológico , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Tiazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Cisteína/genética , Cisteína/metabolismo , Dioxanos/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Melanoma/genética , Melanoma/metabolismo , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/metabolismo
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