RESUMO
Successful cancer immunotherapy entails activation of innate immune receptors to promote dendritic cell (DC) maturation, antigen presentation, up-regulation of costimulatory molecules, and cytokine secretion, leading to activation of tumor antigen-specific cytotoxic T lymphocytes (CTLs). Here we screened a synthetic library of 100,000 compounds for innate immune activators using TNF production by THP-1 cells as a readout. We identified and optimized a potent human and mouse Toll-like receptor (TLR)1/TLR2 agonist, Diprovocim, which exhibited an EC50 of 110 pM in human THP-1 cells and 1.3 nM in primary mouse peritoneal macrophages. In mice, Diprovocim-adjuvanted ovalbumin immunization promoted antigen-specific humoral and CTL responses and synergized with anti-PD-L1 treatment to inhibit tumor growth, generating long-term antitumor memory, curing or prolonging survival of mice engrafted with the murine melanoma B16-OVA. Diprovocim induced greater frequencies of tumor-infiltrating leukocytes than alum, of which CD8 T cells were necessary for the antitumor effect of immunization plus anti-PD-L1 treatment.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Melanoma Experimental/terapia , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas , Animais , Anticorpos Monoclonais/imunologia , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Sinergismo Farmacológico , Humanos , Imunoterapia/métodos , Estimativa de Kaplan-Meier , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Células THP-1 , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismoRESUMO
Structurally disparate molecules reportedly engage and activate Toll-like receptor (TLR) 4 and other TLRs, yet the interactions that mediate binding and activation by dissimilar ligands remain unknown. We describe Neoseptins, chemically synthesized peptidomimetics that bear no structural similarity to the established TLR4 ligand, lipopolysaccharide (LPS), but productively engage the mouse TLR4 (mTLR4)/myeloid differentiation factor 2 (MD-2) complex. Neoseptin-3 activates mTLR4/MD-2 independently of CD14 and triggers canonical myeloid differentiation primary response gene 88 (MyD88)- and Toll-interleukin 1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-dependent signaling. The crystal structure mTLR4/MD-2/Neoseptin-3 at 2.57-Å resolution reveals that Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, inducing an active receptor complex similar to that induced by lipid A. However, Neoseptin-3 and lipid A form dissimilar molecular contacts to achieve receptor activation; hence strong TLR4/MD-2 agonists need not mimic LPS.
Assuntos
Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/agonistas , Peptidomiméticos/farmacologia , Receptor 4 Toll-Like/agonistas , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
A screen conducted with nearly 100000 compounds and a surrogate functional assay for stimulation of an immune response that measured the release of TNF-α from treated human THP-1 myeloid cells differentiated along the macrophage line led to the discovery of the diprovocims. Unique to these efforts and of special interest, the screening leads for this new class of activators of an immune response came from a compound library designed to promote cell-surface receptor dimerization. Subsequent comprehensive structure-activity relationship studies improved the potency 800-fold over that of the screening leads, providing diprovocim-1 and diprovocim-2. The diprovocims act by inducing cell-surface toll-like receptor (TLR)-2 dimerization and activation with TLR1 (TLR1/TLR2 agonist), bear no structural similarity to any known natural or synthetic TLR agonist, and are easy to prepare and synthetically modify, and selected members are active in both human and murine systems. The most potent diprovocim (3, diprovocim-1) elicits full agonist activity at extraordinarily low concentrations (EC50 = 110 pM) in human THP-1 cells, being more potent than the naturally derived TLR1/TLR2 agonist Pam3CSK4 or any other known small molecule TLR agonist.
Assuntos
Antineoplásicos/farmacologia , Melanoma Experimental/tratamento farmacológico , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Melanoma Experimental/patologia , Camundongos , Conformação Molecular , Células THP-1RESUMO
Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumors, and WRN inhibitors are in development. In this study, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth in vitro and in vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic lethal targeting of WRN in MSI cancer and tools to dissect WRN biology. Significance: We report the discovery and characterization of potent, selective WRN helicase inhibitors for MSI cancer treatment, with biomarker analysis and evaluation of efficacy in vivo and in immunotherapy-refractory preclinical models. These findings pave the way to translate WRN inhibition into MSI cancer therapies and provide tools to investigate WRN biology. See related commentary by Wainberg, p. 1369.
Assuntos
Helicase da Síndrome de Werner , Humanos , Helicase da Síndrome de Werner/genética , Camundongos , Animais , Instabilidade de Microssatélites , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêuticoRESUMO
The cyclic prodigiosins are an important family of bioactive natural products that continue to be the subject of numerous structural, synthetic, and biosynthetic studies. In particular, the structural assignments of the isomeric cyclic prodigiosins butylcycloheptylprodigiosin (BCHP) and streptorubin B have been the cause of significant confusion. Herein, we report detailed studies regarding the electron impact (EI) mass spectra of synthetic BCHP and streptorubin B that have allowed us to distinguish the two compounds in the absence of quality historical isolation NMR data. On the basis of these fragmentation differences, the status of BCHP as a natural product is challenged. The proposed mechanism of fragmentation is supported by the EI mass spectra of synthetic pentyl-chain analogues of BCHP and streptorubin B, X-ray crystallography, and DFT calculations. Elimination of BCHP from the prodigiosin family supports a proposed evolutionary hypothesis for the surprising biosynthesis of cyclic prodigiosins.
Assuntos
Produtos Biológicos , Prodigiosina , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Cristalografia por Raios X , Conformação Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Prodigiosina/análogos & derivados , Prodigiosina/biossíntese , Prodigiosina/químicaRESUMO
In this multi-year study, we taught English/Language Arts teachers of students with learning disabilities in middle school to incorporate 15 min of daily vocabulary activities with students in their intact special education English/Language Arts classes. During Year 1, teachers taught 48 words to their sixth grade students, who learned and retained the words significantly better than the students in business-as-usual (BAU) control classes. In the current study, we report the second year results, as the sixth grade students entered seventh grade. Students (n = 42) in treatment classes again learned 48 new vocabulary words significantly better than similar students in BAU (n = 21) special education classes. In seventh grade, students also outperformed BAU students on maintenance of these age-appropriate words (p < .001) and on a standardized measure of vocabulary (p = .04).
Assuntos
Idioma , Vocabulário , Educação Inclusiva , Humanos , Estudos de Linguagem , Instituições Acadêmicas , EnsinoRESUMO
Diprovocim is a recently discovered exceptionally potent, synthetic small molecule agonist of TLR2/TLR1 and has shown significant adjuvant activity in anticancer vaccination against murine melanoma. Since Diprovocim bears no structural similarity to the canonical lipopeptide ligands of TLR2/TLR1, we investigated how Diprovocim interacts with TLR2/TLR1 through in vitro biophysical, structural, and computational approaches. We found that Diprovocim induced the formation of TLR2/TLR1 heterodimers as well as TLR2 homodimers in vitro. We determined the crystal structure of Diprovocim in a complex with a TLR2 ectodomain, which revealed, unexpectedly, two Diprovocim molecules bound to the ligand binding pocket formed between two TLR2 ectodomains. Extensive hydrophobic interactions and a hydrogen-bonding network between the protein and Diprovocim molecules are observed within the defined ligand binding pocket and likely underlie the high potency of Diprovocim. Our work shed first light into the activation mechanism of TLR2/TLR1 by a noncanonical agonist. The structural information obtained here may be exploited to manipulate TLR2/TLR1-dependent signaling.
Assuntos
Ciclopropanos/farmacologia , Pirazóis/farmacologia , Pirrolidinas/farmacologia , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas , Animais , Linhagem Celular , Cristalografia por Raios X , Ciclopropanos/química , Dimerização , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Camundongos , Estrutura Molecular , Pirazóis/química , Pirrolidinas/química , Transdução de Sinais/efeitos dos fármacos , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/metabolismoRESUMO
Herein, we report studies leading to the discovery of the neoseptins and a comprehensive examination of the structure-activity relationships (SARs) of this new class of small-molecule mouse Toll-like receptor 4 (mTLR4) agonists. The compounds in this class, which emerged from screening an α-helix mimetic library, stimulate the immune response, act by a well-defined mechanism (mouse TLR4 agonist), are easy to produce and structurally manipulate, exhibit exquisite SARs, are nontoxic, and elicit improved and qualitatively different responses compared to lipopolysaccharide, even though they share the same receptor.
Assuntos
Aminobutiratos/farmacologia , Benzamidas/farmacologia , Descoberta de Drogas , Receptor 4 Toll-Like/agonistas , Aminobutiratos/síntese química , Aminobutiratos/química , Animais , Benzamidas/síntese química , Benzamidas/química , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Ovalbumina/imunologia , Relação Estrutura-AtividadeRESUMO
PURPOSE: Extrahepatic biliary duct injuries such as transections, stenoses, and biliary leaks are well-known complications of upper abdominal surgeries. The popularization of laparoscopic cholecystectomies in the early 1990's resulted in an increase in the numbers of these reported injuries. The surgical repair of these injuries may be challenging. In this feasibility study, we were presented with the opportunity to evaluate a novel polytetrafluoroethylene (PTFE) covered stent graft that could be useful in common bile duct reconstructions. The long-term goal of this research is to offer the surgeon a new technique for reconstructing the biliary duct or repairing biliary strictures.John G. Zografakis MD, was the first place winner in the Basic Sciences Resident Competition at the Ohio American College of Surgeons meeting. METHODS: Seven dogs were originally enrolled in the study. After general endotracheal anesthesia and open cholecystectomy, the common bile duct was identified in each dog. A guide wire was then passed through the neck of the cystic duct, anterograde into the common bile duct, through the Ampulla of Vater and into the duodenum. A stent graft delivery system was placed over the wire, and the covered stent graft was deployed within the lumen of the common bile duct. Study outcomes included graft patency and assessment of the bio-incorporation of the graft and the effectiveness of the graft to drain the biliary system as determined by liver enzyme tests. RESULTS: Three implants were harvested at 1 month, and 2 grafts were harvested each at 3 months and 6 months postoperatively. All of the stent grafts were patent. Liver enzyme tests revealed that all dogs had increased serum levels of alkaline phosphatase, alanine aminotransferase (ALT) serum glutamate pyruvate transaminase (SGPT) and aspartate aminotransferase (AST) serum glutamic oxaloacetic transaminase (SGOT). Four dogs had increased total bilirubin. These increases were all measured in the immediate postoperative period. Peak levels for each measure were reached between 4 and 10 days and then gradually trended toward baselines by 1 month postoperatively. We did not observe meaningful changes in serum albumin or total protein. One dog suffered a tear in the common bile duct due to balloon overinflation. This tear was suture repaired when the graft was implanted. However, bile leakage was found when the graft was harvested at 1 month postoperatively. There appeared to be minimal bio-incorporation of the stent-grafts into the biliary duct wall, and there was no pronounced inflammatory response found in the duct wall or surrounding tissues. CONCLUSIONS: We are encouraged by these early results. Additional studies are planned to evaluate a self-expanding PTFE covered stent graft and a percutaneous delivery system.
Assuntos
Colangiopancreatografia Retrógrada Endoscópica/métodos , Ducto Colédoco/lesões , Ducto Colédoco/cirurgia , Stents , Animais , Colecistectomia Laparoscópica/métodos , Modelos Animais de Doenças , Cães , Estudos de Viabilidade , Feminino , Sobrevivência de Enxerto , Masculino , Procedimentos Cirúrgicos Minimamente Invasivos , Politetrafluoretileno , Procedimentos de Cirurgia Plástica/métodos , Sensibilidade e EspecificidadeRESUMO
The first total synthesis of propolisbenzofuran B, a bioactive natural product isolated from honeybee propolis resin, is reported. The convergent synthesis makes use of a silicon-tether controlled oxidative ketone-ketone cross-coupling and a novel benzofuran-generating cascade reaction to deliver the core structure of the natural product from readily prepared precursors.
RESUMO
A method for the oxidative alkylation of ketones through intramolecular allyl-group transfer within preformed allyldimethylsilyl enol ethers is reported. A number of examples are detailed, including a study into the effects of resident sterocenters within cyclic enol ethers.