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1.
Front Immunol ; 11: 832, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32457754

RESUMO

Inhibitors that block the programmed cell death-1 (PD-1) pathway can potentiate endogenous antitumor immunity and have markedly improved cancer survival rates across a broad range of indications. However, these treatments work for only a minority of patients. The efficacy of anti-PD-1 inhibitors may be extended by cytokines, however, the incorporation of cytokines into therapeutic regimens has significant challenges. In their natural form when administered as recombinant proteins, cytokine treatments are often associated with low response rates. Most cytokines have a short half-life which limits their exposure and efficacy. In addition, cytokines can activate counterregulatory pathways, in the case of immune-potentiating cytokines this can lead to immune suppression and thereby diminish their potential efficacy. Improving the drug-like properties of natural cytokines using protein engineering can yield synthetic cytokines with improved bioavailability and tissue targeting, allowing for enhanced efficacy and reduced off-target effects. Using structure guided engineering we have designed a novel class of antibody-cytokine fusion proteins consisting of a PD-1 targeting antibody fused together with an interleukin-21 (IL-21) cytokine mutein. Our bifunctional fusion proteins can block PD-1/programmed death-ligand 1 (PD-L1) interaction whilst simultaneously delivering IL-21 cytokine to PD-1 expressing T cells. Targeted delivery of IL-21 can improve T cell function in a manner that is superior to anti-PD-1 monotherapy. Fusion of engineered IL-21 variants to anti-PD1 antibodies can improve the drug-like properties of IL-21 cytokine leading to improved cytokine serum half-life allowing for less frequent dosing. In addition, we show that targeted delivery of IL-21 can minimize any potential detrimental effect on local antigen-presenting cells. A highly attenuated IL-21 mutein variant (R9E:R76A) fused to a PD-1 antibody provides protection in a humanized mouse model of cancer that is refractory to anti-PD-1 monotherapy. Collectively, our preclinical data demonstrate that this approach may improve upon and extend the utility of anti-PD-1 therapeutics currently in the clinic.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Imunoterapia , Interleucinas/uso terapêutico , Neoplasias/terapia , Animais , Anticorpos Monoclonais/imunologia , Antígeno B7-H1/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Interleucinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias/imunologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico
2.
Bioorg Med Chem Lett ; 23(16): 4608-16, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23845219

RESUMO

Sphingosine-1-phosphate (S1P) signaling plays a vital role in mitogenesis, cell migration and angiogenesis. Sphingosine kinases (SphKs) catalyze a key step in sphingomyelin metabolism that leads to the production of S1P. There are two isoforms of SphK and observations made with SphK deficient mice show the two isoforms can compensate for each other's loss. Thus, inhibition of both isoforms is likely required to block SphK dependent angiogenesis. A structure based approach was used to design and synthesize a series of SphK inhibitors resulting in the identification of the first potent inhibitors of both isoforms of human SphK. Additionally, to our knowledge, this series of inhibitors contains the only sufficiently potent inhibitors of murine SphK1 with suitable physico-chemical properties to pharmacologically interrogate the role of SphK1 in rodent models and to reproduce the phenotype of SphK1 (-/-) mice.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/química , Bibliotecas de Moléculas Pequenas/síntese química , Animais , Células Cultivadas , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Estrutura Molecular , Isoformas de Proteínas/química , Ratos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
3.
J Med Chem ; 55(8): 3837-51, 2012 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-22458568

RESUMO

The eukaryotic initiation factor 4E (eIF4E) plays a central role in the initiation of gene translation and subsequent protein synthesis by binding the 5' terminal mRNA cap structure. We designed and synthesized a series of novel compounds that display potent binding affinity against eIF4E despite their lack of a ribose moiety, phosphate, and positive charge as present in m7-GMP. The biochemical activity of compound 33 is 95 nM for eIF4E in an SPA binding assay. More importantly, the compound has an IC(50) of 2.5 µM for inhibiting cap-dependent mRNA translation in a rabbit reticular cell extract assay (RRL-IVT). This series of potent, truncated analogues could serve as a promising new starting point toward the design of neutral eIF4E inhibitors with physicochemical properties suitable for cellular activity assessment.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Guanina/análogos & derivados , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/farmacologia , Organofosfonatos/síntese química , Capuzes de RNA/metabolismo , Animais , Cristalografia por Raios X , Desenho de Fármacos , Fator de Iniciação 4E em Eucariotos/química , Guanina/síntese química , Guanina/farmacologia , Guanosina Monofosfato/síntese química , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Organofosfonatos/farmacologia , Ácidos Fosforosos , Biossíntese de Proteínas/efeitos dos fármacos , Capuzes de RNA/química , Coelhos , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Relação Estrutura-Atividade
4.
J Am Chem Soc ; 131(50): 18139-46, 2009 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-19924990

RESUMO

Eukaryotic mRNAs are appended at the 5' end, with the 7-methylguanosine cap linked by a 5'-5'-triphosphate bridge to the first transcribed nucleoside (m7GpppX). Initiation of cap-dependent translation of mRNA requires direct interaction between the cap structure and the eukaryotic translation initiation factor eIF4E. Biophysical studies of the association between eIF4E and various cap analogs have demonstrated that m(7)GTP binds to the protein ca. -5.0 kcal/mol more favorably than unmethylated GTP. In this work, a thermodynamic analysis of the binding process between eIF4E and several cap analogs has been conducted using Monte Carlo (MC) simulations in conjunction with free energy perturbation (FEP) calculations. To address the role of the 7-methyl group in the eIF4E/m7GpppX cap interaction, binding free energies have been computed for m(7)GTP, GTP, protonated GTP at N(7), the 7-methyldeazaguanosine 5'-triphosphate (m(7)DTP), and 7-deazaguanosine 5'-triphosphate (DTP) cap analogs. The MC/FEP simulations for the GTP-->m(7)DTP transformation demonstrate that half of the binding free energy gain of m(7)GTP with respect to GTP can be attributed to favorable van der Waals interactions with Trp166 and reduced desolvation penalty due to the N(7) methyl group. The methyl group both eliminates the desolvation penalty of the N(7) atom upon binding and creates a larger cavity within the solvent that further facilitates the desolvation step. Analysis of the pair m(7)GTP-m(7)DTP suggests that the remaining gain in affinity is related to the positive charge created on the guanine moiety due to the N(7) methylation. The charge provides favorable cation-pi interactions with Trp56 and Trp102 and decreases the negative molecular charge, which helps the transfer from the solvent, a more polar environment, to the protein.


Assuntos
Fator de Iniciação 4E em Eucariotos/química , Capuzes de RNA/química , Termodinâmica , Biologia Computacional , Simulação por Computador , Cristalografia por Raios X , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Ligação de Hidrogênio , Metilação , Modelos Moleculares , Método de Monte Carlo , Ligação Proteica , Análogos de Capuz de RNA/química , Capuzes de RNA/metabolismo
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