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1.
Proc Natl Acad Sci U S A ; 121(19): e2317307121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38683990

RESUMO

Directing antibodies to a particular epitope among many possible on a target protein is a significant challenge. Here, we present a simple and general method for epitope-directed selection (EDS) using a differential phage selection strategy. This involves engineering the protein of interest (POI) with the epitope of interest (EOI) mutated using a systematic bioinformatics algorithm to guide the local design of an EOI decoy variant. Using several alternating rounds of negative selection with the EOI decoy variant followed by positive selection on the wild-type POI, we were able to identify highly specific and potent antibodies to five different EOI antigens that bind and functionally block known sites of proteolysis. Among these, we developed highly specific antibodies that target the proteolytic site on the CUB domain containing protein 1 (CDCP1) to prevent its proteolysis allowing us to study the cellular maturation of this event that triggers malignancy. We generated antibodies that recognize the junction between the pro- and catalytic domains for three different matrix metalloproteases (MMPs), MMP1, MMP3, and MMP9, that selectively block activation of each of these enzymes and impair cell migration. We targeted a proteolytic epitope on the cell surface receptor, EPH Receptor A2 (EphA2), that is known to transform it from a tumor suppressor to an oncoprotein. We believe that the EDS method greatly facilitates the generation of antibodies to specific EOIs on a wide range of proteins and enzymes for broad therapeutic and diagnostic applications.


Assuntos
Epitopos , Epitopos/imunologia , Humanos , Proteólise , Ligação Proteica , Engenharia de Proteínas/métodos , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz/imunologia , Anticorpos/imunologia , Biblioteca de Peptídeos
2.
Development ; 151(9)2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38587174

RESUMO

The gastrointestinal (GI) tract is complex and consists of multiple organs with unique functions. Rare gene variants can cause congenital malformations of the human GI tract, although the molecular basis of these has been poorly studied. We identified a patient with compound-heterozygous variants in RFX6 presenting with duodenal malrotation and atresia, implicating RFX6 in development of the proximal intestine. To identify how mutations in RFX6 impact intestinal patterning and function, we derived induced pluripotent stem cells from this patient to generate human intestinal organoids (HIOs). We identified that the duodenal HIOs and human tissues had mixed regional identity, with gastric and ileal features. CRISPR-mediated correction of RFX6 restored duodenal identity. We then used gain- and loss-of-function and transcriptomic approaches in HIOs and Xenopus embryos to identify that PDX1 is a downstream transcriptional target of RFX6 required for duodenal development. However, RFX6 had additional PDX1-independent transcriptional targets involving multiple components of signaling pathways that are required for establishing early regional identity in the GI tract. In summary, we have identified RFX6 as a key regulator in intestinal patterning that acts by regulating transcriptional and signaling pathways.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio , Organoides , Fatores de Transcrição de Fator Regulador X , Transativadores , Humanos , Fatores de Transcrição de Fator Regulador X/genética , Fatores de Transcrição de Fator Regulador X/metabolismo , Animais , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Transativadores/metabolismo , Transativadores/genética , Organoides/metabolismo , Organoides/embriologia , Duodeno/metabolismo , Duodeno/embriologia , Intestinos/embriologia , Atresia Intestinal/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Padronização Corporal/genética , Transdução de Sinais/genética , Mutação/genética
3.
Cancer Immunol Res ; 12(5): 575-591, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38588410

RESUMO

Poliovirus receptor-related 2 (PVRL2, also known as nectin-2 or CD112) is believed to act as an immune checkpoint protein in cancer; however, most insight into its role is inferred from studies on its known receptor, poliovirus receptor (PVR)-related immunoglobulin domain protein (PVRIG, also known as CD112R). Here, we study PVRL2 itself. PVRL2 levels were found to be high in tumor cells and tumor-derived exosomes. Deletion of PVRL2 in multiple syngeneic mouse models of cancer showed a dramatic reduction in tumor growth that was immune dependent. This effect was even greater than that seen with deletion of PD-L1. PVRL2 was shown to function by suppressing CD8+ T and natural killer cells in the tumor microenvironment. The loss of PVRL2 suppressed tumor growth even in the absence of PVRIG. In contrast, PVRIG loss showed no additive effect in the absence of PVRL2. T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade combined with PVRL2 deletion resulted in a near complete block in tumor growth. This effect was not recapitulated by the combined deletion of PVRL2 with its paralog, PVR, which is the ligand for TIGIT. These data uncover PVRL2 as a distinct inhibitor of the antitumor immune response with functions beyond that of its known receptor PVRIG. Moreover, the data provide a strong rationale for combinatorial targeting of PVRL2 and TIGIT for cancer immunotherapy.


Assuntos
Nectinas , Receptores de Superfície Celular , Receptores Imunológicos , Microambiente Tumoral , Animais , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Nectinas/metabolismo , Camundongos , Humanos , Microambiente Tumoral/imunologia , Linhagem Celular Tumoral , Transdução de Sinais , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo
4.
bioRxiv ; 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38562834

RESUMO

New epitopes for immune recognition provide the basis of anticancer immunity. Due to the high concentration of extracellular adenosine triphosphate in the tumor microenvironment, we hypothesized that extracellular kinases (ectokinases) could have dysregulated activity and introduce aberrant phosphorylation sites on cell surface proteins. We engineered a cell-tethered version of the extracellular kinase CK2α, demonstrated it was active on cells under tumor-relevant conditions, and profiled its substrate scope using a chemoproteomic workflow. We then demonstrated that mice developed polyreactive antisera in response to syngeneic tumor cells that had been subjected to surface hyperphosphorylation with CK2α. Interestingly, these mice developed B cell and CD4+ T cell responses in response to these antigens but failed to develop a CD8+ T cell response. This work provides a workflow for probing the extracellular phosphoproteome and demonstrates that extracellular phosphoproteins are immunogenic even in a syngeneic system.

5.
Sci Total Environ ; 927: 172190, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38575025

RESUMO

Identification of methods for the standardized assessment of bacterial pathogens and antimicrobial resistance (AMR) in environmental water can improve the quality of monitoring and data collected, support global surveillance efforts, and enhance the understanding of environmental water sources. We conducted a systematic review to assemble and synthesize available literature that identified methods for assessment of prevalence and abundance of bacterial fecal indicators and pathogens in water for the purposes of monitoring bacterial pathogens and AMR. After screening for quality, 175 unique publications were identified from 15 databases, and data were extracted for analysis. This review identifies the most common and robust methods, and media used to isolate target organisms from surface water sources, summarizes methodological trends, and recognizes knowledge gaps. The information presented in this review will be useful when establishing standardized methods for monitoring bacterial pathogens and AMR in water in the United States and globally.


Assuntos
Enterococcus , Monitoramento Ambiental , Escherichia coli , Salmonella , Microbiologia da Água , Enterococcus/isolamento & purificação , Salmonella/isolamento & purificação , Monitoramento Ambiental/métodos , Escherichia coli/isolamento & purificação
6.
Front Immunol ; 15: 1298721, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38469294

RESUMO

Subunit vaccines hold substantial promise in controlling infectious diseases, due to their superior safety profile, specific immunogenicity, simplified manufacturing processes, and well-defined chemical compositions. One of the most important end-targets of vaccines is a subset of lymphocytes originating from the thymus, known as T cells, which possess the ability to mount an antigen-specific immune response. Furthermore, vaccines confer long-term immunity through the generation of memory T cell pools. Dendritic cells are essential for the activation of T cells and the induction of adaptive immunity, making them key for the in vitro evaluation of vaccine efficacy. Upon internalization by dendritic cells, vaccine-bearing antigens are processed, and suitable fragments are presented to T cells by major histocompatibility complex (MHC) molecules. In addition, DCs can secrete various cytokines to crosstalk with T cells to coordinate subsequent immune responses. Here, we generated an in vitro model using the immortalized murine dendritic cell line, DC2.4, to recapitulate the process of antigen uptake and DC maturation, measured as the elevation of CD40, MHC-II, CD80 and CD86 on the cell surface. The levels of key DC cytokines, tumor necrosis alpha (TNF-α) and interleukin-10 (IL-10) were measured to better define DC activation. This information served as a cost-effective and rapid proxy for assessing the antigen presentation efficacy of various vaccine formulations, demonstrating a strong correlation with previously published in vivo study outcomes. Hence, our assay enables the selection of the lead vaccine candidates based on DC activation capacity prior to in vivo animal studies.


Assuntos
Apresentação de Antígeno , Células Dendríticas , Animais , Camundongos , Antígenos CD40/metabolismo , Citocinas/metabolismo , Vacinas de Subunidades Antigênicas/metabolismo
7.
Sci Rep ; 14(1): 6362, 2024 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-38493204

RESUMO

Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter- and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of antigens. In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface antigens that could be targeted by antibodies and chimeric antigen receptor-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas) and 9166 normal tissue samples (from the Genotype-Tissue Expression project), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN, which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative antigens. Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.


Assuntos
Glioblastoma , Glioma , Humanos , Processamento Alternativo , Antígenos de Superfície , Glioma/genética , Antígenos de Histocompatibilidade , RNA , Antígenos de Neoplasias/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores
8.
Nature ; 627(8004): 680-687, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38448587

RESUMO

Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics1-3. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity3,4. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues that participate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.


Assuntos
Cátions , Ciclização , Indicadores e Reagentes , Proteínas , Triptofano , Cátions/química , Indicadores e Reagentes/química , Oxirredução , Proteoma/química , Triptofano/química , Peptídeos/química , Química Click , Proteínas/química
9.
Sci Rep ; 14(1): 4112, 2024 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-38374190

RESUMO

Arginine, a semi-essential amino acid, is critical for cell growth. Typically, de novo synthesis of arginine is sufficient to support cellular processes, however, it becomes vital for cancer cells that are unable to synthesise arginine due to enzyme deficiencies. Targeting this need, arginine depletion with enzymes such as arginase (ARG) has emerged as a potential cancer therapeutic strategy. Studies have proposed using high dose insulin to induce a state of hypoaminoacidaemia in the body, thereby further reducing circulating arginine levels. However, the mitogenic and metabolic properties of insulin could potentially counteract the therapeutic effects of ARG. Our study examined the combined impact of insulin and ARG on breast, lung, and ovarian cell lines, focusing on cell proliferation, metabolism, apoptosis, and autophagy. Our results showed that the influence of insulin on ARG uptake varied between cell lines but failed to promote the proliferation of ARG-treated cells or aid recovery post-ARG treatment. Moreover, insulin was largely ineffective in altering ARG-induced metabolic changes and did not prevent apoptosis. In vitro, at least, these findings imply that insulin does not offer a growth or survival benefit to cancer cells being treated with ARG.


Assuntos
Arginase , Insulina , Neoplasias , Humanos , Apoptose , Arginase/metabolismo , Arginina/metabolismo , Insulina/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo
10.
Transpl Int ; 37: 12019, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38323070

RESUMO

Solid organ transplant recipients are at an increased risk of developing skin cancers due to chronic immunosuppression, particularly with calcineurin inhibitors. Tacrolimus is the most prescribed calcineurin inhibitor in this patient cohort, and understanding tacrolimus concentrations in the skin will facilitate the development of anti-cancer preventive and therapeutic strategies. Here, we show that in mice, tacrolimus blood levels peaked rapidly ∼1 h post last oral dose while skin levels rose more slowly and remained high for at least 6 h. Subsequently, tacrolimus skin and blood concentrations were assessed in 15 kidney transplant recipients. The mean age was 61 years, the average time post-transplant was 7 years (range 0-21 years) and 87% were male. The average skin sampling time post tacrolimus dosing was 6 h 32 min. Skin tacrolimus concentrations ranged from 7.1 ng/g to 71.2 ng/g and correlated with blood concentrations (r = 0.6). Mouse and human mean skin concentrations were in a similar range. Our data suggests that tacrolimus measurements in the blood may be used to approximate tacrolimus concentrations in the skin of kidney transplant recipients, and further exploited for the delivery of anti-cancer therapies designed to antagonize the immunosuppressive effects of tacrolimus in the skin.


Assuntos
Transplante de Rim , Tacrolimo , Adulto , Masculino , Humanos , Animais , Camundongos , Recém-Nascido , Lactente , Pré-Escolar , Criança , Adolescente , Adulto Jovem , Feminino , Imunossupressores/uso terapêutico , Estudos de Viabilidade , Inibidores de Calcineurina , Transplantados
11.
bioRxiv ; 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38352573

RESUMO

Background and Aims: We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn's Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). Methods: Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. Results: iPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation of fibroblast activation genes including ACTA2 and COL1A1 , and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. Conclusions: ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.

12.
bioRxiv ; 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38370768

RESUMO

To investigate the co-development of vasculature, mesenchyme, and epithelium crucial for organogenesis and the acquisition of organ-specific characteristics, we constructed a human pluripotent stem cell-derived organoid system comprising lung or intestinal epithelium surrounded by organotypic mesenchyme and vasculature. We demonstrated the pivotal role of co-differentiating mesoderm and endoderm via precise BMP regulation in generating multilineage organoids and gut tube patterning. Single-cell RNA-seq analysis revealed organ specificity in endothelium and mesenchyme, and uncovered key ligands driving endothelial specification in the lung (e.g., WNT2B and Semaphorins) or intestine (e.g., GDF15). Upon transplantation under the kidney capsule in mice, these organoids further matured and developed perfusable human-specific sub-epithelial capillaries. Additionally, our model recapitulated the abnormal endothelial-epithelial crosstalk in patients with FOXF1 deletion or mutations. Multilineage organoids provide a unique platform to study developmental cues guiding endothelial and mesenchymal cell fate determination, and investigate intricate cell-cell communications in human organogenesis and disease. Highlights: BMP signaling fine-tunes the co-differentiation of mesoderm and endoderm.The cellular composition in multilineage organoids resembles that of human fetal organs.Mesenchyme and endothelium co-developed within the organoids adopt organ-specific characteristics.Multilineage organoids recapitulate abnormal endothelial-epithelial crosstalk in FOXF1-associated disorders.

13.
Nat Chem Biol ; 20(6): 742-750, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38308046

RESUMO

Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryogenic-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights into their mechanisms of action. Rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PAD4 activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.


Assuntos
Proteína-Arginina Desiminase do Tipo 4 , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Proteína-Arginina Desiminase do Tipo 4/química , Humanos , Domínio Catalítico , Microscopia Crioeletrônica , Modelos Moleculares , Anticorpos/química , Anticorpos/imunologia , Anticorpos/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/tratamento farmacológico , Hidrolases/metabolismo , Hidrolases/química , Desiminases de Arginina em Proteínas/metabolismo , Desiminases de Arginina em Proteínas/química
14.
J Immunother Cancer ; 12(1)2024 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-38296598

RESUMO

BACKGROUND: Skin cancers, particularly keratinocyte cancers, are the most commonly diagnosed tumors. Although surgery is often effective in early-stage disease, skin tumors are not always easily accessible, can reoccur and have the ability to metastasize. More recently, immunotherapies, including intravenously administered checkpoint inhibitors, have been shown to control some skin cancers, but with off-target toxicities when used in combination. Our study investigated whether peritumoral administration of an antibody combination targeting PD-1, 4-1BB (CD137) and VISTA might control skin tumors and lead to circulating antitumor immunity without off-target toxicity. METHODS: The efficacy of combination immunotherapy administered peritumorally or intravenously was tested using transplantable tumor models injected into mouse ears (primary tumors) or subcutaneously in flank skin (secondary tumors). Changes to the tumor microenvironment were tracked using flow cytometry while tumor-specific, CD8 T cells were identified through enzyme-linked immunospot (ELISPOT) assays. Off-target toxicity of the combination immunotherapy was assessed via serum alanine aminotransferase ELISA and histological analysis of liver sections. RESULTS: The data showed that local administration of antibody therapy eliminated syngeneic murine tumors transplanted in the ear skin at a lower dose than required intravenously, and without measured hepatic toxicity. Tumor elimination was dependent on CD8 T cells and was associated with an increased percentage of CD8 T cells expressing granzyme B, KLRG1 and Eomes, and a decreased population of CD4 T cells including CD4+FoxP3+ cells in the treated tumor microenvironment. Importantly, untreated, distal tumors regressed following antibody treatment of a primary tumor, and immune memory prevented growth of subcutaneous flank tumors administered 50 days after regression of a primary tumor. CONCLUSIONS: Together, these data suggest that peritumoral immunotherapy for skin tumors offers advantages over conventional intravenous delivery, allowing antibody dose sparing, improved safety and inducing long-term systemic memory. Future clinical trials of immunotherapy for primary skin cancer should focus on peritumoral delivery of combinations of immune checkpoint antibodies.


Assuntos
Linfócitos T CD8-Positivos , Neoplasias Cutâneas , Animais , Camundongos , Imunomodulação , Anticorpos/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Imunoterapia , Microambiente Tumoral
15.
ACS Cent Sci ; 10(1): 199-208, 2024 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-38292613

RESUMO

The cell surface proteome (surfaceome) plays a pivotal role in virtually all extracellular biology, and yet we are only beginning to understand the protein complexes formed in this crowded environment. Recently, a high-resolution approach (µMap) was described that utilizes multiple iridium-photocatalysts attached to a secondary antibody, directed to a primary antibody of a protein of interest, to identify proximal neighbors by light-activated conversion of a biotin-diazirine to a highly reactive carbene followed by LC/MS (Geri, J. B.; Oakley, J. V.; Reyes-Robles, T.; Wang, T.; McCarver, S. J.; White, C. H.; Rodriguez-Rivera, F. P.; Parker, D. L.; Hett, E. C.; Fadeyi, O. O.; Oslund, R. C.; MacMillan, D. W. C. Science2020, 367, 1091-1097). Here we calibrated the spatial resolution for carbene labeling using site-specific conjugation of a single photocatalyst to a primary antibody drug, trastuzumab (Traz), in complex with its structurally well-characterized oncogene target, HER2. We observed relatively uniform carbene labeling across all amino acids, and a maximum distance of ∼110 Å from the fixed photocatalyst. When targeting HER2 overexpression cells, we identified 20 highly enriched HER2 neighbors, compared to a nonspecific membrane tethered catalyst. These studies identify new HER2 interactors and calibrate the radius of carbene photoprobe labeling for the surfaceome.

16.
Nat Chem Biol ; 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38233584

RESUMO

BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). In this study, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by triggering ligands or point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. These data inform a pharmacologic strategy for suppressing apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.

18.
Pigment Cell Melanoma Res ; 37(1): 45-50, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37614154

RESUMO

Treatment of melanomas with targeted and immunotherapies has proven effective, but resistance to both treatments is a common outcome leaving a high proportion of patients without effective alternative treatment options. Replication stress is a common feature of melanomas, and this is effectively targeted using a combination of checkpoint kinase 1 (CHK1) inhibitor and low-dose hydroxyurea (LDHU). This combination also promotes inflammatory and anti-tumour immune responses in vivo. Melanoma cell lines resistant to BRAF inhibitor (BRAFi) or immune checkpoint inhibitors (ICI) retain their sensitivity to CHK1i + LDHU, with sensitivity similar to that of parental tumours. In vivo, BRAFi-resistant and BRAFi-sensitive parental tumours produce an identical immune response with treatment.


Assuntos
Melanoma , Humanos , Melanoma/patologia , Hidroxiureia/farmacologia , Hidroxiureia/uso terapêutico , Proteínas Proto-Oncogênicas B-raf , Quinase 1 do Ponto de Checagem/uso terapêutico , Inibidores de Checkpoint Imunológico/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Linhagem Celular Tumoral
19.
Nat Chem Biol ; 20(1): 30-41, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37400538

RESUMO

Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP). Biologic inhibitors have not yet been reported and could have substantial therapeutic advantages over current small molecules because they can be recombinantly engineered into multifunctional formats and immunotherapies. Here we used phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1 and discovered a VH domain that allosterically inhibited the hydrolysis of cGAMP and adenosine triphosphate (ATP). We solved a 3.2 Å-resolution cryo-electron microscopy structure for the VH inhibitor complexed with ENPP1 that confirmed its new allosteric binding pose. Finally, we engineered the VH domain into multispecific formats and immunotherapies, including a bispecific fusion with an anti-PD-L1 checkpoint inhibitor that showed potent cellular activity.


Assuntos
Diester Fosfórico Hidrolases , Anticorpos de Domínio Único , Diester Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases , Microscopia Crioeletrônica
20.
Nat Rev Drug Discov ; 23(2): 126-140, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38062152

RESUMO

Targeted protein degradation (TPD) has emerged in the past decade as a major new drug modality to remove intracellular proteins with bispecific small molecules that recruit the protein of interest (POI) to an E3 ligase for degradation in the proteasome. Unlike classic occupancy-based drugs, intracellular TPD (iTPD) eliminates the target and works catalytically, and so can be more effective and sustained, with lower dose requirements. Recently, this approach has been expanded to the extracellular proteome, including both secreted and membrane proteins. Extracellular targeted protein degradation (eTPD) uses bispecific antibodies, conjugates or small molecules to degrade extracellular POIs by trafficking them to the lysosome for degradation. Here, we focus on recent advances in eTPD, covering degrader systems, targets, molecular designs and parameters to advance them. Now almost any protein, intracellular or extracellular, is addressable in principle with TPD.


Assuntos
Proteínas de Membrana , Complexo de Endopeptidases do Proteassoma , Humanos , Proteólise , Descoberta de Drogas , Ubiquitina-Proteína Ligases
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