RESUMO
Due to the CD1d restricted recognition of altered glycolipids, Vα24-invariant natural killer T (iNKT) cells are excellent tools for cancer immunotherapy with a significantly reduced risk for graft-versus-host disease when applied as off-the shelf-therapeutics across Human Leukocyte Antigen (HLA) barriers. To maximally harness their therapeutic potential for multiple myeloma (MM) treatment, we here armed iNKT cells with chimeric antigen receptors (CAR) directed against the MM-associated antigen CD38 and the plasma cell specific B cell maturation antigen (BCMA). We demonstrate that both CD38- and BCMA-CAR iNKT cells effectively eliminated MM cells in a CAR-dependent manner, without losing their T cell receptor (TCR)-mediated cytotoxic activity. Importantly, iNKT cells expressing either BCMA-CARs or affinity-optimized CD38-CARs spared normal hematopoietic cells and displayed a Th1-like cytokine profile, indicating their therapeutic utility. While the costimulatory domain of CD38-CARs had no influence on the cytotoxic functions of iNKT cells, CARs containing the 4-1BB domain showed a better expansion capacity. Interestingly, when stimulated only via CD1d+ dendritic cells (DCs) loaded with α-galactosylceramide (α-GalCer), both CD38- and BCMA-CAR iNKT cells expanded well, without losing their CAR- or TCR-dependent cytotoxic activities. This suggests the possibility of developing an off-the-shelf therapy with CAR iNKT cells, which might even be boostable in vivo by administration α-GalCer pulsed DCs.
Assuntos
ADP-Ribosil Ciclase 1/química , Antígeno de Maturação de Linfócitos B/química , Imunoterapia Adotiva , Células Matadoras Naturais/citologia , Glicoproteínas de Membrana/química , Mieloma Múltiplo/metabolismo , Células T Matadoras Naturais/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Antígeno de Maturação de Linfócitos B/metabolismo , Células da Medula Óssea/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica , Células Dendríticas/metabolismo , Galactosilceramidas/química , Antígenos HLA/química , Células-Tronco Hematopoéticas/citologia , Humanos , Leucócitos Mononucleares/citologia , Glicoproteínas de Membrana/metabolismo , Domínios Proteicos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Risco , Células Th1/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/químicaRESUMO
Multiple myeloma is a blood cancer characterized by the plasma cell malignancy in the bone marrow, resulting in the destruction of bone tissue. Recently, the US FDA approved two antibody drugs for the treatment of multiple myeloma, daratumumab and isatuximab, targeting CD38, a type II transmembrane glycoprotein highly expressed in plasma cells and multiple myeloma cells. Here, we report the crystal structure of CD38 in complex with the Fab fragment of daratumumab, providing its exact epitope on CD38 and the structural insights into the mechanism of action of the antibody drug. Daratumumab binds to a specific discontinuous region on CD38 that includes residues located opposite to the active site of CD38. All the six complementarity determining regions of daratumumab are involved in the CD38 interaction. The epitopes of daratumumab and isatuximab do not overlap at all and their bindings to CD38 induce different structural changes within the CD38 protein. This structural study can facilitate the design of improved biologics or effective combination therapies for the treatment of multiple myeloma.
Assuntos
ADP-Ribosil Ciclase 1/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Sequência de Aminoácidos , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/uso terapêutico , Domínio Catalítico , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/química , Ligação ProteicaRESUMO
CD38 is a molecule that can act as an enzyme, with NAD-depleting and intracellular signaling activity, or as a receptor with adhesive functions. CD38 can be found expressed either on the cell surface, where it may face the extracellular milieu or the cytosol, or in intracellular compartments, such as endoplasmic reticulum, nuclear membrane, and mitochondria. The main expression of CD38 is observed in hematopoietic cells, with some cell-type specific differences between mouse and human. The role of CD38 in immune cells ranges from modulating cell differentiation to effector functions during inflammation, where CD38 may regulate cell recruitment, cytokine release, and NAD availability. In line with a role in inflammation, CD38 appears to also play a critical role in inflammatory processes during autoimmunity, although whether CD38 has pathogenic or regulatory effects varies depending on the disease, immune cell, or animal model analyzed. Given the complexity of the physiology of CD38 it has been difficult to completely understand the biology of this molecule during autoimmune inflammation. In this review, we analyze current knowledge and controversies regarding the role of CD38 during inflammation and autoimmunity and novel molecular tools that may clarify current gaps in the field.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Autoimunidade , Imunomodulação , Inflamação/etiologia , Inflamação/metabolismo , Glicoproteínas de Membrana/metabolismo , ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/genética , Animais , Apresentação de Antígeno/imunologia , Biomarcadores , Movimento Celular , Citocinas/metabolismo , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Fagocitose , Transporte ProteicoRESUMO
BACKGROUND: Drug-loaded nanoparticles have established their benefits in the fight against multiple myeloma; however, ligand-targeted nanomedicine has yet to successfully translate to the clinic due to insufficient efficacies reported in preclinical studies. METHODS: In this study, liposomal nanoparticles targeting multiple myeloma via CD38 or CD138 receptors are prepared from pre-synthesized, purified constituents to ensure increased consistency over standard synthetic methods. These nanoparticles are then tested both in vitro for uptake to cancer cells and in vivo for accumulation at the tumor site and uptake to tumor cells. Finally, drug-loaded nanoparticles are tested for long-term efficacy in a month-long in vivo study by tracking tumor size and mouse health. RESULTS: The targeted nanoparticles are first optimized in vitro and show increased uptake and cytotoxicity over nontargeted nanoparticles, with CD138-targeting showing superior enhancement over CD38-targeted nanoparticles. However, biodistribution and tumor suppression studies established CD38-targeted nanoparticles to have significantly increased in vivo tumor accumulation, tumor cell uptake, and tumor suppression over both nontargeted and CD138-targeted nanoparticles due to the latter's poor selectivity. CONCLUSION: These results both highlight a promising cancer treatment option in CD38-targeted nanoparticles and emphasize that targeting success in vitro does not necessarily translate to success in vivo.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Lipossomos/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Sindecana-1/metabolismo , ADP-Ribosil Ciclase 1/química , Animais , Antibióticos Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Doxorrubicina/farmacocinética , Humanos , Lipossomos/química , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Simulação de Acoplamento Molecular , Mieloma Múltiplo/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Sindecana-1/química , Distribuição TecidualRESUMO
A promising protein target for computational drug development, the human cluster of differentiation 38 (CD38), plays a crucial role in many physiological and pathological processes, primarily through the upstream regulation of factors that control cytoplasmic Ca2+ concentrations. Recently, a small-molecule inhibitor of CD38 was shown to slow down pathways relating to aging and DNA damage. We examined the performance of seven docking programs for their ability to model protein-ligand interactions with CD38. A test set of twelve CD38 crystal structures, containing crystallized biologically relevant substrates, were used to assess pose prediction. The rankings for each program based on the median RMSD between the native and predicted were Vina, AD4 > PLANTS, Gold, Glide, Molegro > rDock. Forty-two compounds with known affinities were docked to assess the accuracy of the programs at affinity/ranking predictions. The rankings based on scoring power were: Vina, PLANTS > Glide, Gold > Molegro >> AutoDock 4 >> rDock. Out of the top four performing programs, Glide had the only scoring function that did not appear to show bias towards overpredicting the affinity of the ligand-based on its size. Factors that affect the reliability of pose prediction and scoring are discussed. General limitations and known biases of scoring functions are examined, aided in part by using molecular fingerprints and Random Forest classifiers. This machine learning approach may be used to systematically diagnose molecular features that are correlated with poor scoring accuracy.
Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , ADP-Ribosil Ciclase 1/química , Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/química , Simulação de Acoplamento Molecular/métodos , Algoritmos , Sítios de Ligação , Bases de Dados de Proteínas , Ligantes , Aprendizado de Máquina , Conformação Proteica , SoftwareRESUMO
CD38 is a transmembrane glycoprotein with ectoenzymatic activity involved in regulation of migration, signal transduction, and receptor-mediated adhesion. CD38 is highly expressed on various malignant cells, including multiple myeloma (MM), and at relatively low levels in other tissues, making it a suitable target for therapeutic antibodies. Several anti-CD38 therapies have been, or are being, developed for the treatment of MM, including daratumumab and isatuximab (SAR650984), respectively. Studies have shown that anti-CD38 therapies are effective in the treatment of relapsed/refractory MM and are well tolerated, with infusion reactions being the most common side effects. They can be used as monotherapy or in combination with immunomodulatory agents, such as pomalidomide, or proteasome inhibitors to potentiate their activity. Here we examine isatuximab and several anti-CD38 agents in development that were generated using new antibody engineering techniques and that may lead to more effective CD38 targeting. We also summarize trials assessing these antibodies in MM, other malignancies, and solid organ transplantation. Finally, we propose that further research on the mechanisms of resistance to anti-CD38 therapy and the development of biomarkers and new backbone regimens with CD38 antibodies will be important steps in building more personalized treatment for patients with MM.
Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , ADP-Ribosil Ciclase 1/química , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/uso terapêutico , Humanos , Glicoproteínas de Membrana/química , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two structurally distinct messengers that mobilize the endoplasmic and endolysosomal Ca2+ stores, respectively. Both are synthesized by the CD38 molecule (CD38), which has long been thought to be a type II membrane protein whose catalytic domain, intriguingly, faces to the outside of the cell. Accordingly, for more than 20 years, it has remained unresolved how CD38 can use cytosolic substrates such as NAD and NADP to produce messengers that target intracellular Ca2+ stores. The discovery of type III CD38, whose catalytic domain faces the cytosol, has now begun to clarify this topological conundrum. This article reviews the ideas and clues leading to the discovery of the type III CD38; highlights an innovative approach for uncovering its natural existence; and discusses the regulators of its activity, folding, and degradation. We also review the compartmentalization of cADPR and NAADP biogenesis. We further discuss the possible mechanisms that promote type III CD38 expression and appraise a proposal of a Ca2+-signaling mechanism based on substrate limitation and product translocation. The surprising finding of another enzyme that produces cADPR and NAADP, sterile α and TIR motif-containing 1 (SARM1), is described. SARM1 regulates axonal degeneration and has no sequence similarity with CD38 but can catalyze the same set of multireactions and has the same cytosolic orientation as the type III CD38. The intriguing finding that SARM1 is activated by nicotinamide mononucleotide to produce cADPR and NAADP suggests that it may function as a regulated Ca2+-signaling enzyme like CD38.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Sinalização do Cálcio , ADP-Ribose Cíclica/metabolismo , NADP/análogos & derivados , ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/genética , Animais , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Citoesqueleto/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , NADP/metabolismo , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Leukemia develops as the result of intrinsic features of the transformed cell, such as gene mutations and derived oncogenic signaling, and extrinsic factors, such as a tumor-friendly, immunosuppressed microenvironment, predominantly in the lymph nodes and the bone marrow. There, high extracellular levels of nucleotides, mainly NAD+ and ATP, are catabolized by different ectonucleotidases, which can be divided in two families according to substrate specificity: on one side those that metabolize NAD+, including CD38, CD157, and CD203a; on the other, those that convert ATP, namely CD39 (and other ENTPDases) and CD73. They generate products that modulate intracellular calcium levels and that activate purinergic receptors. They can also converge on adenosine generation with profound effects, both on leukemic cells, enhancing chemoresistance and homing, and on non-malignant immune cells, polarizing them toward tolerance. This review will first provide an overview of ectonucleotidases expression within the immune system, in physiological and pathological conditions. We will then focus on different hematological malignancies, discussing their role as disease markers and possibly pathogenic agents. Lastly, we will describe current efforts aimed at therapeutic targeting of this family of enzymes.
Assuntos
Trifosfato de Adenosina/metabolismo , Neoplasias Hematológicas/enzimologia , NAD/metabolismo , Nucleotidases/fisiologia , 5'-Nucleotidase/química , 5'-Nucleotidase/fisiologia , ADP-Ribosil Ciclase/química , ADP-Ribosil Ciclase/fisiologia , ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/fisiologia , Animais , Antígenos CD/química , Antígenos CD/fisiologia , Apirase/química , Apirase/fisiologia , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/fisiologia , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Nucleotidases/antagonistas & inibidoresRESUMO
The NAD-glycohydrolase/ADP-ribosyl cyclase CD38 catalyzes the metabolism of nicotinamide adenine dinucleotide (NAD) to the Ca2+ mobilizing second messengers ADP-ribose (ADPR), 2'-deoxy-ADPR, and cyclic ADP-ribose (cADPR). In the present study, we investigated binding and metabolism of NAD by a soluble fragment of human CD38, sCD38, and its catalytically inactive mutant by monitoring changes in endogenous tryptophan (Trp) fluorescence. Addition of NAD resulted in a concentration-dependent decrease in sCD38 fluorescence that is mainly caused by the Trp residue W189. Amplitude of the fluorescence decrease was fitted as one-site binding curve revealing a dissociation constant for NAD of 29⯵M. A comparable dissociation constant was found with the catalytically inactive sCD38 mutant (KD 37⯵M NAD) indicating that binding of NAD is not significantly affected by the mutation. The NAD-induced decrease in Trp fluorescence completely recovered in case of sCD38. Kinetics of recovery was slowed down with decreasing temperature and sCD38 concentration and increasing NAD concentration demonstrating that recovery in fluorescence is proportional to the enzymatic activity of sCD38. Accordingly, recovery in fluorescence was not observed with the catalytically inactive mutant. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Assuntos
ADP-Ribosil Ciclase 1/química , Glicoproteínas de Membrana/química , NAD/química , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , NAD/metabolismo , Triptofano/química , Triptofano/genética , Triptofano/metabolismoRESUMO
PURPOSE: Phenytoin is a major anticonvulsant drug that is effective to improve arrhythmia and neuropathic pain. According to early works, phenytoin affected cell membrane depolarization by sodium channel blocking, guanylyl and adenylyl cyclase suppression that cause to intracellular Na+ and Ca2+ downregulation. This study was aimed to clarify some ambiguities in pathophysiological action of phenytoin by in vitro and molecular docking analyses. METHODS: In this study intracellular free Ca2+ of primary culture of embryonic mouse hippocampus evaluated via Fura 2 as fluorescent probe. The effects of phenytoin on ADP ribosyl cyclase activity was assessed by recently developed fluorometric assay. Molecular docking simulation was also implemented to investigate the possible interaction between phenytoin and CD38. RESULTS: Our results confirmed phenytoin competitively inhibits cyclase activity of CD38 (IC50 = 8.1 µM) and reduces cADPR content. cADPR is a Ca2+-mobilising second messenger which binds to L-type calcium channel and ryanodine receptors in cell and ER membrane and increases cytosolic free Ca2+. Ca2+ content of cells decreased significantly in the presence of phenytoin in a dose dependent manner (IC50 = 12.74 µM). Based on molecular docking analysis, phenytoin binds to deeper site of CD38 active site, mainly via hydrophobic interactions and consequently inhibits proper contact of substrate with catalytic residues specially Glu 226, Trp 186, Thr221. CONCLUSION: Taken together, one of the anticonvulsant mechanisms of phenytoin is Ca2+ inhibition from CD38 pathway, therefore could be used in disorders that accompanied by CD38 over production or activation such as heart disease, depression, brain sepsis, airway disease, oxidative stress and inflammation. Graphical abstract á .
Assuntos
ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/metabolismo , Cálcio/metabolismo , Hipocampo/citologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Fenitoína/farmacologia , Animais , Domínio Catalítico/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , ADP-Ribose Cíclica/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/embriologia , Hipocampo/metabolismo , Homeostase , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Cultura Primária de Células , Ligação Proteica/efeitos dos fármacos , Conformação ProteicaRESUMO
BACKGROUND: CD38 is a surface membrane antigen highly expressed in malignant blood cells, such as multiple myeloma (MM). A soluble form of CD38 (sCD38) is also present in the plasma, deriving likely from the shedding from the cells. The plasma levels of sCD38 should thus correlate closely with the proliferation of the MM cells, allowing the development of a simple diagnostic blood test for monitoring the progress of the disease. However, the plasma sCD38 levels are extremely low, requiring the design of a highly sensitive and specific assay. RESULTS: In this study, we developed an ultra-sensitive assay, based on two nanobodies (Nbs) targeting two distinct epitopes of sCD38. One Nb acts as a capturer, and the other is fused with the firefly luciferase serving as a reporter to ensure sensitivity. We showed that this Dual epitopes protein IDentification (DepID) assay has sensitivity reaching 10â¯pg/mL, which is 10 times higher than that of a commercial ELISA kit. By this method, we were able to precisely quantify the levels of sCD38 in the plasma of MM patients, which were significantly higher than those from healthy donors. We further showed that the increase plasma levels of sCD38 correlated with the progress of MM. CONCLUSION: We have developed a Nb-based luminescence sandwich assay, named as DepID, for quantification of the soluble CD38 in MM patients' plasma and showed the potency of this method as a tool for general diagnosis of MM or companion diagnosis of the CD38-targeted therapies.
Assuntos
ADP-Ribosil Ciclase 1/sangue , Análise Química do Sangue/métodos , Epitopos/imunologia , Mieloma Múltiplo/sangue , Anticorpos de Domínio Único/imunologia , ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/imunologia , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Limite de Detecção , Modelos Moleculares , Mieloma Múltiplo/patologia , Conformação Proteica , Anticorpos de Domínio Único/química , SolubilidadeRESUMO
X-box binding protein 1 (XBP1), CD138 (Syndecan-1) and CS1 (SLAMF7) are highly expressed antigens in cancers including multiple myeloma (MM). Here, we identify and characterize immunogenic HLA-A24 peptides derived from these antigens for potential vaccination therapy of HLA-A24+ patients with MM. The identified immunogenic HLA-A24-specific XBP1 unspliced (UN)185-193 (I S P W I L A V L), XBP1 spliced (SP)223-231 (V Y P E G P S S L), CD138265-273 (I F A V C L V G F) and CS1240-248 (L F V L G L F L W) peptides induced antigen-specific CTL with anti-MM activity in an HLA-A24 restricted manner. Furthermore, a cocktail containing the four HLA-A24 peptides evoked MM-specific CTL with distinct phenotypic profiles (CD28, CD40L, 41BB, CD38, CD69) and anti-tumor activities, evidenced by perforin upregulation, CD107a degranulation (cytotoxicity) and Th1-type cytokines (IFN-γ/IL-2/TNF-α) production in response to HLA-A24+ MM cells. The multipeptide-specific CTL included antigen-specific memory CD8+ T cells expressing both T-cell activation (CD38, CD69) and immune checkpoints antigens (CTLA, PD-1, LAG-3, TIM-3). These results provide the framework for a multipeptide vaccination therapy to induce tumor-specific CTL in HLA-A24-positive patients with myeloma and other cancers expressing these antigens.
Assuntos
ADP-Ribosil Ciclase 1/imunologia , Antígeno HLA-A24/imunologia , Mieloma Múltiplo/imunologia , Peptídeos/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Proteína 1 de Ligação a X-Box/imunologia , ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/metabolismo , Sequência de Aminoácidos , Biomarcadores , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Antígeno HLA-A24/genética , Antígeno HLA-A24/metabolismo , Humanos , Memória Imunológica , Peptídeos e Proteínas de Sinalização Intercelular , Ativação Linfocitária/imunologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Fenótipo , Ligação Proteica , Linfócitos T Citotóxicos/metabolismo , Proteína 1 de Ligação a X-Box/química , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca2+-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca2+ signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca2+ entry and, consequently, apoptosis. To date, adenosine 5'-diphosphoribose (ADPR) has been assumed to be the main agonist for TRPM2. Here we show that 2'-deoxy-ADPR was a significantly better TRPM2 agonist, inducing 10.4-fold higher whole-cell currents at saturation. Mechanistically, this increased activity was caused by a decreased rate of inactivation and higher average open probability. Using high-performance liquid chromatography (HPLC) and mass spectrometry, we detected endogenous 2'-deoxy-ADPR in Jurkat T lymphocytes. Consistently, cytosolic nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) and nicotinamide adenine dinucleotide (NAD)-glycohydrolase CD38 sequentially catalyzed the synthesis of 2'-deoxy-ADPR from nicotinamide mononucleotide (NMN) and 2'-deoxy-ATP in vitro. Thus, 2'-deoxy-ADPR is an endogenous TRPM2 superagonist that may act as a cell signaling molecule.
Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Clusterina/agonistas , ADP-Ribosil Ciclase 1/química , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/farmacologia , Cromatografia Líquida de Alta Pressão , Humanos , Peróxido de Hidrogênio/química , Células Jurkat , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacosRESUMO
Chimeric antigen receptors (CARs) can effectively redirect cytotoxic T cells toward highly expressed surface antigens on tumor cells. The low expression of several tumor-associated antigens (TAAs) on normal tissues, however, hinders their safe targeting by CAR T cells due to on-target/off-tumor effects. Using the multiple myeloma (MM)-associated CD38 antigen as a model system, here, we present a rational approach for effective and tumor-selective targeting of such TAAs. Using "light-chain exchange" technology, we combined the heavy chains of two high-affinity CD38 antibodies with 176 germline light chains and generated â¼124 new antibodies with 10- to >1,000-fold lower affinities to CD38. After categorizing them into three distinct affinity classes, we incorporated the single-chain variable fragments of eight antibodies from each class into new CARs. T cells carrying these CD38-CARs were extensively evaluated for their on-tumor/off-tumor cytotoxicity as well as CD38-dependent proliferation and cytokine production. We identified CD38-CAR T cells of â¼1,000- fold reduced affinity, which optimally proliferated, produced Th1-like cytokines, and effectively lysed CD382+ MM cells, but spared CD38+ healthy hematopoietic cells in vitro and in vivo. Thus, this systematic approach is highly suitable for the generation of optimal CARs for effective and selective targeting of TAAs.
Assuntos
ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão , ADP-Ribosil Ciclase 1/metabolismo , Animais , Afinidade de Anticorpos/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Imunoterapia Adotiva , Ativação Linfocitária/imunologia , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/terapia , Ligação Proteica/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC) analysis. Standard electrospray ionization (ESI)-mass spectrometry does not provide a direct solution as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, we introduce a native tandem MS-based approach, enabling charge-state resolution and charge assignment of protein ions including those that escape mass analysis under standard MS conditions. Using this method, we determined the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody.
Assuntos
ADP-Ribosil Ciclase 1/química , Receptores ErbB/química , Confiabilidade dos Dados , Peso Molecular , Espectrometria de Massas em Tandem/métodosRESUMO
In this research, molecular docking and 3D-QSAR studies were carried out on a series of 79 thiazoloquin(az)olin(on)es as CD38 inhibitors. Based on docking results, four interactions including hydrogen bonding with main chain of GLU-226 (H-M-GLU-226), Van der Waals interactions with side chain of TRP-125 (V-S-TRP-125), TRP-189 (V-S-TRP-189), and THR-221 (V-S-THR-221) were considered as pharmacological interactions. Active conformation of each ligand was extracted from docking studies and was used for carrying out 3D-QSAR modeling. Comparative molecular field analysis (CoMFA) was performed on CD38 inhibitory activities of these compounds on human and mouse. We developed CoMFA models with five components as optimum models for both data-sets. For human data-set, a model with high predictive power was developed. R2, RMSE, and F-test values for training set of this model were .94, .24, and 179.58, respectively, and R2 and RMSE for its test set were .92 and .32, respectively. The q2 and RMSE values for leave-one-out cross validation test on training set were .78 and .46, respectively, that demonstrate created model is robust. Based on extracted steric and electrostatic contour maps for this model, three inhibitors with pIC50 larger than 8.85 were designed.
Assuntos
ADP-Ribosil Ciclase 1/química , Conformação Proteica/efeitos dos fármacos , Tiazóis/química , ADP-Ribosil Ciclase 1/antagonistas & inibidores , Humanos , Ligação de Hidrogênio/efeitos dos fármacos , Ligantes , Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-Atividade , Eletricidade Estática , Tiazóis/farmacologiaRESUMO
Cyclic ADP-ribose (cADPR) mobilizes intracellular Ca(2+) stores and activates Ca(2+) influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD(+) by the multifunctional CD38/ADP-ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD(+) can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD(+) to form linear ADPR while Aplysia ADP-ribosyl cyclase prefers cyclizing NAD(+) to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD(+) cyclase activity producing cADPR. We also determined the X-ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD(+) reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , NAD/metabolismo , ADP-Ribosil Ciclase/química , ADP-Ribosil Ciclase/metabolismo , ADP-Ribosil Ciclase 1/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , ADP-Ribose Cíclica/metabolismo , Humanos , Modelos Moleculares , Domínios Proteicos , SuínosRESUMO
The goal of this review is to provide historical, recent preclinical, and current clinical summaries of efforts to understand the CD38 molecule and to develop monoclonal antibodies that target it. We focus particularly on efforts involving multiple myeloma, a malignancy of terminally differentiated B cells that remains incurable despite many advances. An era of anti-CD38 monoclonal antibody therapy for myeloma is approaching, one that, we hope, will enable patients to live longer and better lives.
Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Ensaios Clínicos como Assunto , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Humanos , Terapia de Alvo Molecular , Mieloma Múltiplo/etiologia , Mieloma Múltiplo/metabolismo , Resultado do TratamentoRESUMO
In the past decade, the discovery, synthesis, and evaluation for hundreds of CD38 covalent and non-covalent inhibitors has been reported sequentially by our group and partners; however, a systematic structure-based guidance is still lacking for rational design of CD38 inhibitor. Here, we carried out a comparative analysis of pharmacophore features and quantitative structure-activity relationships for CD38 inhibitors. The results uncover that the essential interactions between key residues and covalent/non-covalent CD38 inhibitors include (i) hydrogen bond and hydrophobic interactions with residues Glu226 and Trp125, (ii) electrostatic or hydrogen bond interaction with the positively charged residue Arg127 region, and (iii) the hydrophobic interaction with residue Trp189. For covalent inhibitors, besides the covalent effect with residue Glu226, the electrostatic interaction with residue Arg127 is also necessary, while another hydrogen/non-bonded interaction with residues Trp125 and Trp189 can also be detected. By means of the SYBYL multifit alignment function, the best CoMFA and CoMSIA with CD38 covalent inhibitors presented cross-validated correlation coefficient values (q(2)) of 0.564 and 0.571, and non-cross-validated values (r(2)) of 0.967 and 0.971, respectively. The CD38 non-covalent inhibitors can be classified into five groups according to their chemical scaffolds, and the residues Glu226, Trp189, and Trp125 are indispensable for those non-covalent inhibitors binding to CD38, while the residues Ser126, Arg127, Asp155, Thr221, and Phe222 are also important. The best CoMFA and CoMSIA with the F12 analogues presented cross-validated correlation coefficient values (q(2)) of 0.469 and 0.454, and non-cross-validated values (r(2)) of 0.814 and 0.819, respectively.