RESUMO
Metastatic castration-resistant prostate cancer (mCRPC) is an advanced disease in which patients ultimately fail standard of care androgen-deprivation therapies and exhibit poor survival rates. The prostate-specific membrane antigen (PSMA) has been validated as a mCRPC tumor antigen with over-expression in tumors and low expression in healthy tissues. Using our proprietary technology for incorporating synthetic amino acids (SAAs) into proteins at selected sites, we have developed ARX517, an antibody drug conjugate (ADC) which is composed of a humanized anti-PSMA antibody site-specifically conjugated to a tubulin inhibitor at a drug-to-antibody ratio of 2. After binding PSMA, ARX517 is internalized and catabolized, leading to cytotoxic payload delivery and apoptosis. To minimize premature payload release and maximize delivery to tumor cells, ARX517 employs a non-cleavable PEG linker and stable oxime conjugation enabled via SAA protein incorporation to ensure its overall stability. In vitro studies demonstrate that ARX517 selectively induces cytotoxicity of PSMA-expressing tumor cell lines. ARX517 exhibited a long terminal half-life and high serum exposure in mice, and dose-dependent anti-tumor activity in both enzalutamide-sensitive and -resistant CDX and PDX prostate cancer models. Repeat dose toxicokinetic studies in non-human primates demonstrated ARX517 was tolerated at exposures well above therapeutic exposures in mouse pharmacology studies, indicating a wide therapeutic index. In summary, ARX517 inhibited tumor growth in diverse mCRPC models, demonstrated a tolerable safety profile in monkeys, and had a wide therapeutic index based on preclinical exposure data. Based on the encouraging preclinical data, ARX517 is currently being evaluated in a Phase 1 clinical trial ([NCT04662580]).
RESUMO
The innate immune system employs a variety of antimicrobial oxidants to control and kill host-associated bacteria. Hypothiocyanite/hypothiocyanous acid (-OSCN/HOSCN) is one such antimicrobial oxidant that is synthesized by lactoperoxidase, myeloperoxidase, and eosinophil peroxidase at sites throughout the human body. HOSCN has potent antibacterial activity while being largely non-toxic toward human cells. The molecular mechanisms by which bacteria sense and defend themselves against HOSCN have only recently begun to be elaborated, notably by the discovery of bacterial HOSCN reductase (RclA), an HOSCN-degrading enzyme widely conserved among bacteria that live on epithelial surfaces. In this paper, I show that Ni2+ sensitizes Escherichia coli to HOSCN by inhibiting glutathione reductase and that inorganic polyphosphate protects E. coli against this effect, probably by chelating Ni2+ ions. I also found that RclA is very sensitive to inhibition by Cu2+ and Zn2+, metals that are accumulated to high levels by innate immune cells, and that, surprisingly, thioredoxin and thioredoxin reductase are not involved in HOSCN stress resistance in E. coli. These results advance our understanding of the contribution of different oxidative stress responses and redox buffering pathways to HOSCN resistance in E. coli and illustrate important interactions between metal ions and the enzymes bacteria use to defend themselves against oxidative stress. IMPORTANCE: Hypothiocyanite (HOSCN) is an antimicrobial oxidant produced by the innate immune system. The molecular mechanisms by which host-associated bacteria defend themselves against HOSCN have only recently begun to be understood. The results in this paper are significant because they show that the low molecular weight thiol glutathione and enzyme glutathione reductase are critical components of the Escherichia coli HOSCN response, working by a mechanism distinct from that of the HOSCN-specific defenses provided by the RclA, RclB, and RclC proteins and that metal ions (including nickel, copper, and zinc) may impact the ability of bacteria to resist HOSCN by inhibiting specific defensive enzymes (e.g., glutathione reductase or RclA).
Assuntos
Escherichia coli , Tiocianatos , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Tiocianatos/farmacologia , Tiocianatos/metabolismo , Níquel/farmacologia , Níquel/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Farmacorresistência Bacteriana , Glutationa Redutase/metabolismo , Glutationa Redutase/genética , Antibacterianos/farmacologia , Zinco/metabolismo , Zinco/farmacologia , Cobre/metabolismo , Cobre/farmacologiaRESUMO
Gut microbiota influence anti-tumor immunity, often by producing immune-modulating metabolites. However, microbes consume a variety of metabolites that may also impact host immune responses. We show that tumors grow unchecked in the omenta of microbe-replete mice due to immunosuppressive Tregs. By contrast, omental tumors in germ-free, neomycin-treated mice or mice colonized with altered Schaedler's flora (ASF) are spontaneously eliminated by CD8+ T cells. These mice lack Proteobacteria capable of arginine catabolism, causing increases in serum arginine that activate the mammalian target of the rapamycin (mTOR) pathway in Tregs to reduce their suppressive capacity. Transfer of the Proteobacteria, Escherichia coli (E. coli), but not a mutant unable to catabolize arginine, to ASF mice reduces arginine levels, restores Treg suppression, and prevents tumor clearance. Supplementary arginine similarly decreases Treg suppressive capacity, increases CD8+ T cell effectiveness, and reduces tumor burden. Thus, microbial consumption of arginine alters anti-tumor immunity, offering potential therapeutic strategies for tumors in visceral adipose tissue.
Assuntos
Arginina , Linfócitos T CD8-Positivos , Microbioma Gastrointestinal , Camundongos Endogâmicos C57BL , Omento , Linfócitos T Reguladores , Animais , Arginina/metabolismo , Camundongos , Linfócitos T Reguladores/imunologia , Microbioma Gastrointestinal/imunologia , Linfócitos T CD8-Positivos/imunologia , Omento/imunologia , Serina-Treonina Quinases TOR/metabolismo , Proteobactérias , Escherichia coli/imunologia , Neoplasias/imunologia , FemininoRESUMO
In Escherichia coli, many environmental stressors trigger polyphosphate (polyP) synthesis by polyphosphate kinase (PPK1), including heat, nutrient restriction, toxic compounds, and osmotic imbalances. PPK1 is essential for virulence in many pathogens and has been the target of multiple screens for small molecule inhibitors that might serve as new anti-virulence drugs. However, the mechanisms by which PPK1 activity and polyP synthesis are regulated are poorly understood. Our previous attempts to uncover PPK1 regulatory elements resulted in the discovery of PPK1* mutants, which accumulate more polyP in vivo, but do not produce more in vitro. In attempting to further characterize these mutant enzymes, we discovered that the most commonly-used PPK1 purification method - Ni-affinity chromatography using a C-terminal poly-histidine tag - altered intrinsic aspects of the PPK1 enzyme, including specific activity, oligomeric state, and kinetic values. We developed an alternative purification strategy using a C-terminal C-tag which did not have these effects. Using this strategy, we were able to demonstrate major differences in the in vitro response of PPK1 to 5-aminosalicylic acid, a known PPK1 inhibitor, and observed several key differences between the wild-type and PPK1* enzymes, including changes in oligomeric distribution, increased enzymatic activity, and increased resistance to both product (ADP) and substrate (ATP) inhibition, that help to explain their in vivo effects. Importantly, our results indicate that the C-terminal poly-histidine tag is inappropriate for purification of PPK1, and that any in vitro studies or inhibitor screens performed with such tags need to be reconsidered in that light.
Assuntos
Escherichia coli , Histidina , Fosfotransferases (Aceptor do Grupo Fosfato) , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Histidina/metabolismo , Histidina/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Polifosfatos/metabolismo , CinéticaRESUMO
The innate immune system employs a variety of antimicrobial oxidants to control and kill host-associated bacteria. Hypothiocyanite/hypothiocyanous acid (-OSCN/HOSCN) is one such antimicrobial oxidant that is synthesized by lactoperoxidase, myeloperoxidase, and eosinophil peroxidase at sites throughout the human body. HOSCN has potent antibacterial activity while being largely non-toxic towards human cells. The molecular mechanisms by which bacteria sense and defend themselves against HOSCN have only recently begun to be elaborated, notably by the discovery of bacterial HOSCN reductase (RclA), an HOSCN-degrading enzyme widely conserved among bacteria that live on epithelial surfaces. In this paper, I show that Ni2+ sensitizes Escherichia coli to HOSCN by inhibiting glutathione reductase, and that inorganic polyphosphate protects E. coli against this effect, probably by chelating Ni2+ ions. I also found that RclA is very sensitive to inhibition by Cu2+ and Zn2+, metals that are accumulated to high levels by innate immune cells, and that, surprisingly, thioredoxin and thioredoxin reductase are not involved in HOSCN stress resistance in E. coli. These results advance our understanding of the contribution of different oxidative stress response and redox buffering pathways to HOSCN resistance in E. coli and illustrate important interactions between metal ions and the enzymes bacteria use to defend themselves against oxidative stress.