RESUMO
The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems.
Assuntos
Antígeno B7-H1 , Leucócitos Mononucleares , Comunicação Celular , Flavinas , ImunoterapiaRESUMO
Neisseria meningitidis is a Gram-negative opportunistic pathogen that is responsible for causing human diseases with high mortality, such as septicemia and meningitis. The molecular mechanisms N.â meningitidis employ to manipulate the immune system, translocate the mucosal and blood-brain barriers, and exert virulence are largely unknown. Human-associated bacteria encode a variety of bioactive small molecules with growing evidence for N-acyl amides as being important signaling molecules. However, only a small fraction of these metabolites has been identified from the human microbiota thus far. Here, we heterologously expressed an N-acyltransferase encoded in the obligate human pathogen N.â meningitidis and identified 30â N-acyl amides with representative members serving as agonists of the G-protein coupled receptor (GPCR) S1PR4. During this process, we also characterized two mammalian N-acyl amides derived from the bovine medium. Both groups of metabolites suppress anti-inflammatory interleukin-10 signaling in human macrophage cell types, but they also suppress the pro-inflammatory interleukin-17A+ population in TH 17-differentiated CD4+ T cells.
Assuntos
Neisseria meningitidis , Humanos , Bovinos , Animais , Esfingosina , Amidas/farmacologia , Virulência , Transdução de Sinais , MamíferosRESUMO
Natural products represent a rich reservoir of small molecule drug candidates utilized as antimicrobial drugs, anticancer therapies, and immunomodulatory agents. These molecules are microbial secondary metabolites synthesized by co-localized genes termed Biosynthetic Gene Clusters (BGCs). The increase in full microbial genomes and similar resources has led to development of BGC prediction algorithms, although their precision and ability to identify novel BGC classes could be improved. Here we present a deep learning strategy (DeepBGC) that offers reduced false positive rates in BGC identification and an improved ability to extrapolate and identify novel BGC classes compared to existing machine-learning tools. We supplemented this with random forest classifiers that accurately predicted BGC product classes and potential chemical activity. Application of DeepBGC to bacterial genomes uncovered previously undetectable putative BGCs that may code for natural products with novel biologic activities. The improved accuracy and classification ability of DeepBGC represents a major addition to in-silico BGC identification.
Assuntos
Vias Biossintéticas/genética , Biologia Computacional/métodos , Mineração de Dados/métodos , Família Multigênica/genética , Aprendizado Profundo , Genoma , Genoma Bacteriano/genéticaRESUMO
Tapinarof is a stilbene drug that is used to treat psoriasis and atopic dermatitis, and is thought to function through regulation of the AhR and Nrf2 signaling pathways, which have also been linked to inflammatory bowel diseases. It is produced by the gammaproteobacterial Photorhabdus genus, which thus represents a model to probe tapinarof structural and functional transformations. We show that Photorhabdus transforms tapinarof into novel drug metabolism products that kill inflammatory bacteria, and that a cupin enzyme contributes to the conversion of tapinarof and related dietary stilbenes into novel dimers. One dimer has activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE), and another undergoes spontaneous cyclizations to a cyclopropane-bridge-containing hexacyclic framework that exhibits activity against Mycobacterium. These dimers lack efficacy in a colitis mouse model, whereas the monomer reduces disease symptoms.
Assuntos
Antibacterianos/metabolismo , Autoimunidade/efeitos dos fármacos , Fatores Imunológicos/metabolismo , Photorhabdus/metabolismo , Resorcinóis/metabolismo , Estilbenos/metabolismo , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Biotransformação , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Camundongos , Resorcinóis/química , Resorcinóis/farmacologia , Estilbenos/química , Estilbenos/farmacologiaRESUMO
Protein-tyrosine phosphatase 1B (PTP1B) is a key regulatory enzyme in several signal transduction pathways, and its upregulation has been associated with type-2 diabetes, obesity and cancer. Selective determination of the functional significance of PTP1B remains a major challenge because the activity of this crucial enzyme is currently evaluated through the use of fluorescent probes that lack selectivity and are limited to biochemical assays. Here we describe the rational design, synthesis and biological evaluation of new modular PTP1B fluorogenic substrates. The self-immolative 4-hydroxybenzyl alcohol has been used as a key component for the design of phosphotyrosine mimics linked to a latent chromophore, which is released through an enzyme-initiated domino reaction. Preliminary biological investigations showed that, by optimising the stereoelectronic properties and the binding interactions at the enzyme active site, it is possible to achieve substrates with high affinity and promising selectivity. Due to their modular nature, the synthesised fluorogenic probes represent versatile tools; customisation of the different subunits could widen the scope of these probes to a broader range of in vitro assays. Finally, these studies elucidate the critical role played by Asp181 in the PTP1B-catalysed dephosphorylation mechanism: disruption of the native conformation of this key amino acid residue on the WDP loop yields fluorogenic inhibitors, rather than substrates. For this reason, our studies also represent a step forward for the development of improved PTP1B noncovalent inhibitors.
Assuntos
Corantes Fluorescentes/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Sítios de Ligação , Biocatálise , Domínio Catalítico , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The allosteric inhibitor of the mechanistic target of rapamycin (mTOR) everolimus reduces seizures in tuberous sclerosis complex (TSC) patients through partial inhibition of mTOR functions. Due to its limited brain permeability, we sought to develop a catalytic mTOR inhibitor optimized for central nervous system (CNS) indications. We recently reported an mTOR inhibitor (1) that is able to block mTOR functions in the mouse brain and extend the survival of mice with neuronal-specific ablation of the Tsc1 gene. However, 1 showed the risk of genotoxicity in vitro. Through structure-activity relationship (SAR) optimization, we identified compounds 9 and 11 without genotoxicity risk. In neuronal cell-based models of mTOR hyperactivity, both corrected aberrant mTOR activity and significantly improved the survival rate of mice in the Tsc1 gene knockout model. Unfortunately, 9 and 11 showed limited oral exposures in higher species and dose-limiting toxicities in cynomolgus macaque, respectively. However, they remain optimal tools to explore mTOR hyperactivity in CNS disease models.
Assuntos
Inibidores de MTOR , Sirolimo , Camundongos , Animais , Síndrome , Sistema Nervoso Central/metabolismo , Encéfalo/metabolismo , Serina-Treonina Quinases TOR , Trifosfato de AdenosinaRESUMO
The continuing emergence of antibacterial resistance reduces the effectiveness of antibiotics and drives an ongoing search for effective replacements. Screening compound libraries for antibacterial activity in standard growth media has been extensively explored and may be showing diminishing returns. Inhibition of bacterial targets that are selectively important under in vivo (infection) conditions and, therefore, would be missed by conventional in vitro screens might be an alternative. Surrogate host models of infection, however, are often not suitable for high-throughput screens. Here, we adapted a medium-throughput Tetrahymena pyriformis surrogate host model that was successfully used to identify inhibitors of a hyperviscous Klebsiella pneumoniae strain to a high-throughput format and screened circa 1.2 million compounds. The screen was robust and identified confirmed hits from different chemical classes with potent inhibition of K. pneumoniae growth in the presence of T. pyriformis that lacked any appreciable direct antibacterial activity. Several of these appeared to inhibit capsule/mucoidy, which are key virulence factors in hypervirulent K. pneumoniae. A weakly antibacterial inhibitor of LpxC (essential for the synthesis of the lipid A moiety of lipopolysaccharides) also appeared to be more active in the presence of T. pyriformis, which is consistent with the role of LPS in virulence as well as viability in K. pneumoniae.
RESUMO
Escherichia coli broadly colonize the intestinal tract of humans and produce a variety of small molecule signals. However, many of these small molecules remain unknown. Here, we describe a family of widely distributed bacterial metabolites termed the "indolokines." In E. coli, the indolokines are upregulated in response to a redox stressor via aspC and tyrB transaminases. Although indolokine 1 represents a previously unreported metabolite, four of the indolokines (2-5) were previously shown to be derived from indole-3-carbonyl nitrile (ICN) in the plant pathogen defense response. We show that the indolokines are produced in a convergent evolutionary manner relative to plants, enhance E. coli persister cell formation, outperform ICN protection in an Arabidopsis thaliana-Pseudomonas syringae infection model, trigger a hallmark plant innate immune response, and activate distinct immunological responses in primary human tissues. Our molecular studies link a family of cellular stress-induced metabolites to defensive responses across bacteria, plants, and humans.
Assuntos
Escherichia coli/metabolismo , Indóis/metabolismo , Regulação para Cima , Animais , Arabidopsis/metabolismo , Escherichia coli/citologia , Fezes/microbiologia , Humanos , Indóis/química , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Estresse Oxidativo , Transdução de SinaisRESUMO
Escherichia coli is an important model organism in microbiology and a prominent member of the human microbiota1. Environmental isolates readily colonize the gastrointestinal tract of humans and other animals, and they can serve diverse probiotic, commensal and pathogenic roles in the host2-4. Although certain strains have been associated with the severity of inflammatory bowel disease (IBD)2,5, the diverse immunomodulatory phenotypes remain largely unknown at the molecular level. Here, we decode a previously unknown E. coli metabolic pathway that produces a family of hybrid pterin-phenylpyruvate conjugates, which we named the colipterins. The metabolites are upregulated by subinhibitory levels of the antifolate sulfamethoxazole, which is used to treat infections including in patients with IBD6,7. The genes folX/M and aspC/tyrB involved in monapterin biosynthesis8-10 and aromatic amino acid transamination,11 respectively, were required to initiate the colipterin pathway. We show that the colipterins are antioxidants, harbour diverse immunological activities in primary human tissues, activate anti-inflammatory interleukin-10 and improve colitis symptoms in a colitis mouse model. Our study defines an antifolate stress response in E. coli and links its associated metabolites to a major immunological marker of IBD.
Assuntos
Antioxidantes/metabolismo , Escherichia coli/metabolismo , Imunomodulação , Pteridinas/metabolismo , Sulfametoxazol/metabolismo , Animais , Antioxidantes/administração & dosagem , Antioxidantes/química , Antioxidantes/farmacologia , Células Cultivadas , Colite/tratamento farmacológico , Colite/microbiologia , Modelos Animais de Doenças , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Microbioma Gastrointestinal , Humanos , Interleucina-10/metabolismo , Redes e Vias Metabólicas , Camundongos , Oxirredução , Pteridinas/administração & dosagem , Pteridinas/química , Pteridinas/farmacologia , Estresse Fisiológico , Sulfametoxazol/administração & dosagemRESUMO
Escherichia coli is a common inhabitant of the human microbiota and a beacon model organism in biology. However, an understanding of its signaling systems that regulate population-level phenotypes known as quorum sensing remain incomplete. Here, we define the structure and biosynthesis of autoinducer-3 (AI-3), a metabolite of previously unknown structure involved in the pathogenesis of enterohemorrhagic E. coli (EHEC). We demonstrate that novel AI-3 analogs are derived from threonine dehydrogenase (Tdh) products and "abortive" tRNA synthetase reactions, and they are distributed across a variety of Gram-negative and Gram-positive bacterial pathogens. In addition to regulating virulence genes in EHEC, we show that the metabolites exert diverse immunological effects on primary human tissues. The discovery of AI-3 metabolites and their biochemical origins now provides a molecular foundation for investigating the diverse biological roles of these elusive yet widely distributed bacterial signaling molecules.
RESUMO
Recent clinical evaluation of everolimus for seizure reduction in patients with tuberous sclerosis complex (TSC), a disease with overactivated mechanistic target of rapamycin (mTOR) signaling, has demonstrated the therapeutic value of mTOR inhibitors for central nervous system (CNS) indications. Given that everolimus is an incomplete inhibitor of the mTOR function, we sought to develop a new mTOR inhibitor that has improved properties and is suitable for CNS disorders. Starting from an in-house purine-based compound, optimization of the physicochemical properties of a thiazolopyrimidine series led to the discovery of the small molecule 7, a potent and selective brain-penetrant ATP-competitive mTOR inhibitor. In neuronal cell-based models of mTOR hyperactivity, 7 corrected the mTOR pathway activity and the resulting neuronal overgrowth phenotype. The new mTOR inhibitor 7 showed good brain exposure and significantly improved the survival rate of mice with neuronal-specific ablation of the Tsc1 gene. These results demonstrate the potential utility of this tool compound to test therapeutic hypotheses that depend on mTOR hyperactivity in the CNS.
Assuntos
Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Convulsões/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Tiazóis/uso terapêutico , Animais , Anticonvulsivantes/metabolismo , Anticonvulsivantes/farmacocinética , Anticonvulsivantes/uso terapêutico , Sítios de Ligação , Encéfalo/efeitos dos fármacos , Descoberta de Drogas , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratos , Serina-Treonina Quinases TOR/química , Serina-Treonina Quinases TOR/metabolismo , Tiazóis/metabolismo , Tiazóis/farmacocinética , Proteína 1 do Complexo Esclerose Tuberosa/genéticaRESUMO
We report our full account of the enantioselective total synthesis of (-)-acylfulvene (1) and (-)-irofulven (2), which features metathesis reactions for the rapid assembly of the molecular framework of these antitumor agents. We discuss (1) the application of an Evans Cu-catalyzed aldol addition reaction using a strained cyclopropyl ketenethioacetal, (2) an efficient enyne ring-closing metathesis cascade reaction in a challenging setting, (3) the reagent IPNBSH for a late-stage reductive allylic transposition reaction, and (4) the final RCM/dehydrogenation sequence for the formation of (-)-acylfulvene (1) and (-)-irofulven (2).
Assuntos
Antineoplásicos/síntese química , Sesquiterpenos/síntese química , Compostos Alílicos/química , Antineoplásicos/química , Catálise , Cobre/química , Hidrogênio/química , Espectroscopia de Ressonância Magnética , Modelos Químicos , Sesquiterpenos/química , Compostos de Espiro/síntese química , Compostos de Espiro/química , EstereoisomerismoRESUMO
Observations concerning the synthesis of the core spirocyclic AB-ring system of illudins using an enyne ring closing metathesis (EYRCM) cascade are discussed. Substituent effects, in addition to optimization of the reaction conditions and the olefin tether for the key EYRCM reaction, are examined.
RESUMO
Targeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody-drug conjugate (ADC). Preliminary studies were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbamate, or MC-VC-PABC cleavable linker. However, the resulting ADCs lacked antigen-specificity in vivo, probably from premature release of the payload. Second-generation ADCs were then developed, using noncleavable linkers, and the resulting conjugates (ADC-4 and ADC-10) led to in vivo efficacy in an HER-2 expressing (SK-OV-3ip) mouse xenograft model while ADC-11 led to in vivo efficacy in an anti-c-KIT (NCI-H526) mouse xenograft model in a target-dependent manner.
RESUMO
Antibody-drug conjugates (ADCs) are a novel modality that allows targeted delivery of potent therapeutic agents to the desired site. Herein we report our discovery of NAMPT inhibitors as a novel nonantimitotic payload for ADCs. The resulting anti-c-Kit conjugates (ADC-3 and ADC-4) demonstrated in vivo efficacy in the c-Kit positive gastrointestinal stromal tumor GIST-T1 xenograft model in a target-dependent manner.
RESUMO
Over the past several decades, the frequency of antibacterial resistance in hospitals, including multidrug resistance (MDR) and its association with serious infectious diseases, has increased at alarming rates. Pseudomonas aeruginosa is a leading cause of nosocomial infections, and resistance to virtually all approved antibacterial agents is emerging in this pathogen. To address the need for new agents to treat MDR P. aeruginosa, we focused on inhibiting the first committed step in the biosynthesis of lipid A, the deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by the enzyme LpxC. We approached this through the design, synthesis, and biological evaluation of novel hydroxamic acid LpxC inhibitors, exemplified by 1, where cytotoxicity against mammalian cell lines was reduced, solubility and plasma-protein binding were improved while retaining potent anti-pseudomonal activity in vitro and in vivo.
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Amidoidrolases/química , Animais , Antibacterianos/síntese química , Técnicas de Química Sintética , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Feminino , Células Hep G2/efeitos dos fármacos , Humanos , Células K562/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Relação Estrutura-AtividadeRESUMO
[reaction: see text] The synthesis of the fully fuctionalized A/B ring of ouabain has been accomplished efficiently from commercially available starting materials. A key Robinson annulation allows for the building of the desired carbon framework in one high-yielding step. Directed epoxidation followed by selective epoxide opening furnished the final tetraol with the desired all-cis stereochemistry.
Assuntos
Cardiotônicos/síntese química , Ouabaína/síntese química , Compostos de Epóxi/química , Cinética , EstereoisomerismoRESUMO
Two 4-silyl-3-buten-2-ones, 2a and 2c, underwent an interesting rearrangement involving migration of the allyl or phenyl group on the silicon atom to the adjacent enone carbon when treated with various bases.
RESUMO
Several novel hydroxylated cis-decalin derivatives, potential intermediates for the synthesis of the AB ring system of the important cardiotonic steroid ouabain, have been synthesized from commercially available starting materials. The first step in the preparation of these highly functionalized intermediates is a Robinson annulation of the beta-keto ester 6 and the 4-silyl-3-buten-2-one 5 to furnish the octalone 4 with good diastereoselectivity in fair yield (due to competition with a novel silicon-to-carbon phenyl migration). Reduction of the epoxy alcohol 3 (derived from 4 in two high-yielding steps) with LiAlH(4) gave a mixture of the desired triol 11 along with the product of an unusual reductive opening at the tertiary carbon, namely the triol 12. A plausible mechanism for this unusual reduction is presented as are possible methods for avoiding it. In particular, reduction of the corrresponding epoxy ketone 15 with aluminum amalgam proceeded in good yield to give the hydroxy ketone 16. Also reduction of the epoxide ester having the inverted stereochemistry at C3 afforded the desired tertiary alcohol 33 in good yield. Another approach using the beta,gamma-unsaturated ketal 38 permitted the formation of the tertiary alcohol 40. Fleming oxidation of the related, very functionalized silane 39 afforded the desired 1beta-alcohol 41 in fair yield. Finally a novel rearrangement was observed when the epoxy alcohol 24 was treated with DIBAL to effect loss of the angular hydroxymethyl group to produce the tetrasubstitued alkene 29 in high yield.