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1.
Protein Sci ; 33(10): e5121, 2024 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-39276019

RESUMEN

Bispecific antibodies (BsAbs) have emerged as a major class of antibody therapeutics owing to their substantial potential in disease treatment. While several BsAbs have been successfully approved in recent years, ongoing development efforts continue to focus on optimizing various BsAbs tailored to particular antigens and action mechanisms, aiming to achieve favorable physicochemical properties. BsAbs generally encounter challenges due to their unfavorable physicochemical characteristics and poor manufacturing efficiencies, highlighting the need for optimization to achieve reliable productivity and developability. Herein, we describe the development of a novel symmetric BsAb, REGULGENT™ (N-term/C-term), comprising two Fab domains, using a common light chain. The heavy chain fragment encoded two antigen-binding determinants in one chain. The design and production of REGULGENT™ (N-term/C-term) are simple owing to the use of the same light chain, which does not induce heavy and light chain mispairing, frequently observed with the asymmetric BsAb format. REGULGENT™ (N-term/C-term) exhibited high expression and low aggregation characteristics during cell culture and stress treatment under low pH conditions. Differential scanning calorimetric data indicated that REGULGENT™ molecules had high conformational stability, similar to that of stabilized monoclonal antibodies. Surface plasmon resonance data showed that REGULGENT™ (N-term/C-term) could bind to two antigens simultaneously and exhibited a high affinity for two antigens. In summary, the symmetric BsAb format of REGULGENT™ confers its desirable IgG-like physicochemical properties, thus making it an excellent candidate for commercial development. The findings demonstrate a novel BsAb with substantial development potential for clinical applications.


Asunto(s)
Anticuerpos Biespecíficos , Ingeniería de Proteínas , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/genética , Humanos , Ingeniería de Proteínas/métodos , Estabilidad Proteica , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , Animales
2.
J Biol Chem ; 300(9): 107705, 2024 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-39178948

RESUMEN

The cell signaling molecules nitric oxide (NO) and Ca2+ regulate diverse biological processes through their closely coordinated activities directed by signaling protein complexes. However, it remains unclear how dynamically the multicomponent protein assemblies behave within the signaling complexes upon the interplay between NO and Ca2+ signals. Here we demonstrate that TRPC5 channels activated by the stimulation of G-protein-coupled ATP receptors mediate Ca2+ influx, that triggers NO production from endothelial NO synthase (eNOS), inducing secondary activation of TRPC5 via cysteine S-nitrosylation and eNOS in vascular endothelial cells. Mutations in the caveolin-1-binding domains of TRPC5 disrupt its association with caveolin-1 and impair Ca2+ influx and NO production, suggesting that caveolin-1 serves primarily as the scaffold for TRPC5 and eNOS to assemble into the signal complex. Interestingly, during ATP receptor activation, eNOS is dissociated from caveolin-1 and in turn directly associates with TRPC5, which accumulates at the plasma membrane dependently on Ca2+ influx and calmodulin. This protein reassembly likely results in a relief of eNOS from the inhibitory action of caveolin-1 and an enhanced TRPC5 S-nitrosylation by eNOS localized in the proximity, thereby facilitating the secondary activation of Ca2+ influx and NO production. In isolated rat aorta, vasodilation induced by acetylcholine was significantly suppressed by the TRPC5 inhibitor AC1903. Thus, our study provides evidence that dynamic remodeling of the protein assemblies among TRPC5, eNOS, caveolin-1, and calmodulin determines the ensemble of Ca2+ mobilization and NO production in vascular endothelial cells.

3.
Nihon Yakurigaku Zasshi ; 159(3): 165-168, 2024.
Artículo en Japonés | MEDLINE | ID: mdl-38692881

RESUMEN

Molecular oxygen suffices the ATP production required for the survival of us aerobic organisms. But it is also true that oxygen acts as a source of reactive oxygen species that elicit a spectrum of damages in living organisms. To cope with such intrinsic ambiguity of biological activity oxygen exerts, aerobic mechanisms are equipped with an exquisite adaptive system, which sensitively detects partial pressure of oxygen within the body and controls appropriate oxygen supply to the tissues. Physiological responses to hypoxia are comprised of the acute and chronic phases, in the former of which the oxygen-sensing remains controversial particularly from mechanistic points of view. Recently, we have revealed that the prominently redox-sensitive cation channel TRPA1 plays key roles in oxygen-sensing mechanisms identified in the peripheral tissues and the central nervous system. In this review, we summarize recent development of researches on oxygen-sensing mechanisms including that in the carotid body, which has been recognized as the oxygen receptor organ central to acute oxygen-sensing. We also discuss how ubiquitously the TRPA1 contributes to the mechanisms underlying the acute phase of adaptation to hypoxia.


Asunto(s)
Oxígeno , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio , Canal Catiónico TRPA1/metabolismo , Humanos , Oxígeno/metabolismo , Animales , Canales de Potencial de Receptor Transitorio/metabolismo , Hipoxia/metabolismo , Canales de Calcio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Cuerpo Carotídeo/metabolismo
4.
J Antibiot (Tokyo) ; 77(6): 353-364, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38523145

RESUMEN

The antimicrobial activity of tumescenamide C against the scab-forming S. scabiei NBRC13768 was confirmed with a potent IC50 value (1.5 µg/mL). Three tumescenamide C-resistant S. scabiei strains were generated to compare their gene variants. All three resistant strains contained nonsynonymous variants in genes related to cellobiose/cellotriose transport system components; cebF1, cebF2, and cebG2, which are responsible for the production of the phytotoxin thaxtomin A. Decrease in thaxtomin A production and the virulence of the three resistant strains were revealed by the LC/MS analysis and necrosis assay, respectively. Although the nonsynonymous variants were insufficient for identifying the molecular target of tumescenamide C, the cell wall component wall teichoic acid (WTA) was observed to bind significantly to tumescenamide C. Moreover, changes in the WTA contents were detected in the tumescenamide C-resistant strains. These results imply that tumescenamide C targets the cell wall system to exert antimicrobial effects on S. scabiei.


Asunto(s)
Antibacterianos , Depsipéptidos , Péptidos Cíclicos , Streptomyces , Antibacterianos/farmacología , Antibacterianos/química , Pared Celular/efectos de los fármacos , Depsipéptidos/farmacología , Depsipéptidos/química , Depsipéptidos/aislamiento & purificación , Farmacorresistencia Bacteriana , Indoles , Pruebas de Sensibilidad Microbiana , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificación , Piperazinas , Streptomyces/química , Streptomyces/efectos de los fármacos , Streptomyces/genética , Ácidos Teicoicos/metabolismo
5.
JID Innov ; 4(2): 100258, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38375189

RESUMEN

Inhibition of IL-4/IL-13 signaling has dramatically improved the treatment of atopic dermatitis (AD). However, in many patients, clinical responses are slow to develop and remain modest. Indeed, some symptoms of AD are dependent on IL-31, which is only partially reduced by IL-4/IL-13 inhibition. Thus, there is an unmet need for AD treatments that concomitantly block IL-4/IL-13 and IL-31 pathways. We engineered NM26-2198, a bispecific tetravalent antibody designed to accomplish this task. In reporter cell lines, NM26-2198 concomitantly inhibited IL-4/IL-13 and IL-31 signaling with a potency comparable with that of the combination of an anti-IL-4Rα antibody (dupilumab) and an anti-IL-31 antibody (BMS-981164). In human PBMCs, NM26-2198 inhibited IL-4-induced upregulation of CD23, demonstrating functional binding to FcγRII (CD32). NM26-2198 also inhibited the secretion of the AD biomarker thymus and activation-regulated chemokine (TARC) in blood samples from healthy human donors. In male cynomolgus monkeys, NM26-2198 exhibited favorable pharmacokinetics and significantly inhibited IL-31-induced scratching at a dose of 30 mg/kg. In a repeat-dose, good laboratory practice toxicology study in cynomolgus monkeys, no adverse effects of NM26-2198 were observed at a weekly dose of 125 mg/kg. Together, these results justify the clinical investigation of NM26-2198 as a treatment for moderate-to-severe AD.

6.
Cell Death Discov ; 9(1): 467, 2023 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-38135680

RESUMEN

IFN-alpha have been reported to suppress hepatitis B virus (HBV) cccDNA via APOBEC3 cytidine deaminase activity through interferon signaling. To develop a novel anti-HBV drug for a functional cure, we performed in silico screening of the binding compounds fitting the steric structure of the IFN-alpha-binding pocket in IFNAR2. We identified 37 compounds and named them in silico cccDNA modulator (iCDM)-1-37. We found that iCDM-34, a new small molecule with a pyrazole moiety, showed anti-HCV and anti-HBV activities. We measured the anti-HBV activity of iCDM-34 dependent on or independent of entecavir (ETV). iCDM-34 suppressed HBV DNA, pgRNA, HBsAg, and HBeAg, and also clearly exhibited additive inhibitory effects on the suppression of HBV DNA with ETV. We confirmed metabolic stability of iCDM-34 was stable in human liver microsomal fraction. Furthermore, anti-HBV activity in human hepatocyte-chimeric mice revealed that iCDM-34 was not effective as a single reagent, but when combined with ETV, it suppressed HBV DNA compared to ETV alone. Phosphoproteome and Western blotting analysis showed that iCDM-34 did not activate IFN-signaling. The transcriptome analysis of interferon-stimulated genes revealed no increase in expression, whereas downstream factors of aryl hydrocarbon receptor (AhR) showed increased levels of the expression. CDK1/2 and phospho-SAMHD1 levels decreased under iCDM-34 treatment. In addition, AhR knockdown inhibited anti-HCV activity of iCDM-34 in HCV replicon cells. These results suggest that iCDM-34 decreases the phosphorylation of SAMHD1 through CDK1/2, and suppresses HCV replicon RNA, HBV DNA, and pgRNA formation.

7.
Nihon Yakurigaku Zasshi ; 158(6): 475-477, 2023.
Artículo en Japonés | MEDLINE | ID: mdl-37914327

RESUMEN

The unscheduled proliferation of cancer cells outside their natural niches subjects the cells to multiple insults, such as metabolic aberrations, detachment from the extracellular matrix (ECM), hypoxia, and immune cell attacks. Oxidative stress is a hallmark of cancer because these insults can all lead to the accumulation of reactive oxygen species (ROS). However, it remained largely elusive how cancer cells are able to adapt to harsh oxidative environments. Here, we provide evidence that cancer cells co-opt the neuronal ROS-sensing channel TRPA1 to tolerate highly oxidative environments. While TRPA1 is usually expressed at sensory neurons, we found that the channel is also overexpressed in various types of human cancer. TRPA1 does not affect canonical ROS-neutralizing programs but senses ROS and upregulates Ca2+-dependent anti-apoptotic programs that promotes oxidative-stress tolerance. These findings offer a significant advance in our understanding of adaptation mechanisms to oxidative stress, which represents a substantial hurdle that impedes tumor initiation and progression.


Asunto(s)
Neoplasias , Estrés Oxidativo , Humanos , Canal Catiónico TRPA1/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Oxidativo/fisiología , Neoplasias/metabolismo , Células Receptoras Sensoriales/metabolismo
8.
J Physiol ; 2023 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-37147468

RESUMEN

Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily of cation channels, is broadly expressed in sensory neural pathways, including the trigeminal neurons innervating the nasal cavity and vagal neurons innervating the trachea and the lung. TRPA1 acts as a detector of various irritant chemicals as well as hypoxia and hyperoxia. For the past 15 years, we have characterised its role in respiratory and behavioural modulation in vivo using Trpa1 knockout (KO) mice and wild-type (WT) littermates. Trpa1 KO mice failed to detect, wake up from sleeping, and escape from formalin vapour and a mild hypoxic (15% O2 ) environment. Respiratory augmentation induced by mild hypoxia was absent in either Trpa1 KO mice or WT mice treated with a TRPA1 antagonist. Irritant gas introduced into the nasal cavity inhibited respiratory responses in WT mice but not in the KO mice. The effect of TRPA1 on the olfactory system seemed minimal because olfactory bulbectomized WT mice reacted similarly to the intact mice. Immunohistological analyses using a cellar activation marker, the phosphorylated form of extracellular signal-regulated kinase, confirmed activation of trigeminal neurons in WT mice but not in Trpa1 KO mice in response to irritant chemicals and mild hypoxia. These data collectively show that TRPA1 is necessary for multiple chemical-induced protective responses in respiration and behaviour. We propose that TRPA1 channels in the airway may play a sentinel role for environmental threats and prevent incoming damage.

9.
RSC Med Chem ; 13(10): 1197-1204, 2022 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-36325399

RESUMEN

Transient receptor potential cation channel subfamily V member 1 (TRPV1)-targeted compounds were synthesized by modifying the structure of SB366791, a pharmaceutically representative TRPV1 antagonist. To avoid amide-iminol tautomerization, structurally supported N-methylated amides (i.e., 3-alkoxy-substitued N-meythylamide derivatives of SB366791) were evaluated using a Ca2+ influx assay, in which cells expressed recombinant TRPV1 in the presence of 1.0 µM capsaicin. The antagonistic activities of N-(3-methoxyphenyl)-N-methyl-4-chlorocinnamamide (2) (RLC-TV1004) and N-{3-(3-fluoropropoxy)phenyl}-N-methyl-4-chlorocinnamamide (4) (RLC-TV1006) were found to be approximately three-fold higher (IC50: 1.3 µM and 1.1 µM, respectively) than that of SB366791 (IC50: 3.7 µM). These results will help reinvigorate the potential of SB366791 in medicinal chemistry applications. The 3-methoxy and 3-fluoroalkoxy substituents were used to obtain radioactive [11C]methoxy- or [18F]fluoroalkoxy-incorporated tracers for in vivo positron emission tomography (PET). Using the 11C- or 18F-labeled derivatives, explorative PET imaging trials were performed in rats.

10.
Cancer Sci ; 113(6): 2118-2128, 2022 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-35348270

RESUMEN

Although esophageal cancer has a poor prognosis after recurrence, some patients have shown long-term survival despite recurrence. We hypothesized that induction of either antitumor Abs or antitumor-specific CTLs could play a role in long-term survival (5 years or longer) in patients with recurrence and/or distant metastases. Therefore, we aimed to obtain Abs that specifically bind to cancer cells by using serum samples from patients with a good prognosis. A phage library was prepared using PBMC mRNA of the patients, and cell panning was carried out using an esophageal cancer cell line. Results showed the presence of an epidermal growth factor receptor (EGFR) Ab, KT112, that specifically bound to the cancer cell line. Notably, KT112 bound to only EGFR-positive cancer cells but failed to bind to normal esophageal cells. Furthermore, KT112 was characterized by responses to EGFR expressed on cancer cells but not to the recombinant extracellular domain of EGFR. Immunohistochemical analysis showed that KT112 reacted with 17.4% of esophageal squamous cell carcinoma tissue but not with any other cancer or normal tissue, suggesting that the Ab recognizes cancer-specific forms of EGFR and might have contributed to tumor suppression in patients with esophageal cancer. Furthermore, because of its high cancer specificity, KT112 could be a promising therapeutic option (e.g., in Ab-drug conjugates) for esophageal cancer.


Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Receptores ErbB/genética , Neoplasias Esofágicas/patología , Humanos , Leucocitos Mononucleares/química
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