Cysteine-Based Coupling: Challenges and Solutions.

Antibody-drug conjugates (ADCs) have attracted great attention in recent years in the wake of an accelerated FDA approval rate and several large-scale acquisitions. To date, there are ten ADC drugs on the market and more than 70 in various stages of clinical trials. Yet, due to the complicated nature of ADC molecules, considerations need to cover many aspects for the success of ADCs, including target specificity, linker-payload stability, tumor permeability, and clearance rate. This topical review summarizes and discusses current methods used to increase stability and homogeneity of ADCs of cysteine conjugation. We believe that they will lead to improvement of efficacy and pharmacokinetics (PK) of ADC drugs.

A Pretargeted Imaging Strategy for EGFR-Positive Colorectal Carcinoma via Modulation of Tz-Radioligand Pharmacokinetics.

PURPOSE: Previously, we successfully developed a pretargeted imaging strategy (atezolizumab-TCO/[99mTc]HYNIC-PEG11-Tz) for evaluating programmed cell death ligand-1 (PD-L1) expression in xenograft mice. However, the surplus unclicked [99mTc]HYNIC-PEG11-Tz is cleared somewhat sluggishly through the intestines, which is not ideal for colorectal cancer (CRC) imaging. To shift the excretion of the Tz-radioligand to the renal system, we developed a novel Tz-radioligand by adding a polypeptide linker between HYNIC and PEG11. PROCEDURES: Pretargeted molecular probes [99mTc]HYNIC-polypeptide-PEG11-Tz and cetuximab-TCO were synthesized. [99mTc]HYNIC-polypeptide-PEG11-Tz was evaluated for in vitro stability and in vivo blood pharmacokinetics. In vitro ligation reactivity of [99mTc]HYNIC-polypeptide-PEG11-Tz towards cetuximab-TCO was also tested. Biodistribution assay and imaging of [99mTc]HYNIC-polypeptide-PEG11-Tz were performed to observe its excretion pathway. Pretargeted biodistribution was measured at three different accumulation intervals to determine the optimal pretargeted interval time. Pretargeted (cetuximab-TCO 48 h/[99mTc]HYNIC-PEG11-Tz 6 h) and (cetuximab-TCO 48 h/[99mTc]HYNIC-Polypeptide-PEG11-Tz 6 h) imagings were compared to examine the effect of the excretion pathway on tumor imaging. RESULTS: [99mTc]HYNIC-polypeptide-PEG11-Tz showed favorable in vitro stability and rapid blood clearance in mice. SEC-HPLC revealed almost complete reaction between cetuximab-TCO and [99mTc]HYNIC-polypeptide-PEG11-Tz in vitro, with the 8:1 Tz-to-mAb reaction providing a conversion yield of 87.83 ± 3.27 %. Biodistribution and imaging analyses showed that the Tz-radioligand was cleared through the kidneys. After 24, 48, and 72 h of accumulation in HCT116 tumor, the tumor-to-blood ratio of cetuximab-TCO was 0.83 ± 0.13, 1.40 ± 0.31, and 1.15 ± 0.21, respectively. Both pretargeted (cetuximab-TCO 48 h/[99mTc]HYNIC-PEG11-Tz 6 h) and (cetuximab-TCO 48 h/[99mTc]HYNIC-polypeptide-PEG11-Tz 6 h) clearly delineated HCT116 tumor. Pretargeted imaging strategy using cetuximab-TCO/[99mTc]HYNIC-polypeptide-PEG11-Tz could be used for diagnosing CRC, as the surplus unclicked [99mTc]HYNIC-polypeptide-PEG11-Tz was cleared through the urinary system, leading to low abdominal uptake background. CONCLUSION: Our novel pretargeted imaging strategy (cetuximab-TCO/[99mTc]HYNIC-polypeptide-PEG11-Tz) was useful for imaging CRC, broadening the application scope of pretargeted imaging strategy. The pretargeted imaging strategy clearly delineated HCT116 tumor, showing that its use could be extended to selection of internalizing antibodies.

Ameliorate effects of soybean soluble polysaccharide on adenine-induced chronic renal failure in mice.

In the present study, the kidney protection effects of soluble soybean polysaccharide (SSPS) were evaluated. To address the issues, a mice model of Chronic renal failure (CRF) was established by gavage 0.2% (w/w) adenine for 3 weeks. In vivo results showed that SSPS could change the concentrations of blood urea nitrogen (BUN), creatinine (CRE), total protein (TP) and albumin (ALB), thereby affecting kidney function. In addition, Masson histopathology analysis indicated that SSPS could decrease the area of collagen fiber in the kidney tissues of CRF mice. Moreover, the results of mRNA expression and western experiment suggested that SSPS treatment could increase the expression of transforming growth factor-ß (TGF-ß), Smad3 and P-Smad3, while reduce the expression of α smooth muscle actin (α-SMA) when compared with the model group. These results indicated that SSPS potentially improve kidney function through TGF/Smad pathway in CRF mice.

FBXL20-mediated Vps34 ubiquitination as a p53 controlled checkpoint in regulating autophagy and receptor degradation.

Vacuolar protein-sorting 34 (Vps34), the catalytic subunit in the class III PtdIns3 (phosphatidylinositol 3) kinase complexes, mediates the production of PtdIns3P, a key intracellular lipid involved in regulating autophagy and receptor degradation. However, the signal transduction pathways by which extracellular signals regulate Vps34 complexes and the downstream cellular mechanisms are not well understood. Here we show that DNA damage-activated mitotic arrest and CDK activation lead to the phosphorylation of Vps34, which provides a signal to promote its ubiquitination and proteasomal degradation mediated by FBXL20 (an F-box protein) and the associated Skp1 (S-phase kinase-associated protein-1)-Cullin1 complex, leading to inhibition of autophagy and receptor endocytosis. Furthermore, we show that the expression of FBXL20 is regulated by p53-dependent transcription. Our study provides a molecular pathway by which DNA damage regulates Vps34 complexes and its downstream mechanisms, including autophagy and receptor endocytosis, through SCF (Skp1-Cul1-F-box)-mediated ubiquitination and degradation. Since the expression of FBXL20 is regulated by p53-dependent transcription, the control of Vps34 ubiquitination and proteasomal degradation by FBXL20 and the associated SCF complex expression provides a novel checkpoint for p53 to regulate autophagy and receptor degradation in DNA damage response.

Caspase-11 controls interleukin-1ß release through degradation of TRPC1.

Caspase-11 is a highly inducible caspase that controls both inflammatory responses and cell death. Caspase-11 controls interleukin 1ß (IL-1ß) secretion by potentiating caspase-1 activation and induces caspase-1-independent pyroptosis downstream of noncanonical NLRP3 inflammasome activators such as lipopolysaccharide (LPS) and Gram-negative bacteria. However, we still know very little about the downstream mechanism of caspase-11 in regulating inflammation because the known substrates of caspase-11 are only other caspases. Here, we identify the cationic channel subunit transient receptor potential channel 1 (TRPC1) as a substrate of caspase-11. TRPC1 deficiency increases the secretion of IL-1ß without modulating caspase-1 cleavage or cell death in cultured macrophages. Consistently, trpc1(-/-) mice show higher IL-1ß secretion in the sepsis model of intraperitoneal LPS injection. Altogether, our data suggest that caspase-11 modulates the cationic channel composition of the cell and thus regulates the unconventional secretion pathway in a manner independent of caspase-1.

Mitochondrial electron transport chain complex III is required for antimycin A to inhibit autophagy.

Autophagy is a cellular lysosome-dependent catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. We show that antimycin A, a known inhibitor of mETC complex III, can inhibit autophagy. A structural and functional study shows that four close analogs of antimycin A that have no effect on mitochondria inhibition also do not inhibit autophagy, whereas myxothiazol, another mETC complex III inhibitor with unrelated structure to antimycin A, inhibits autophagy. Additionally, antimycin A and myxothiazol cannot inhibit autophagy in mtDNA-depleted H4 and mtDNA-depleted HeLa cells. These data suggest that antimycin A inhibits autophagy through its inhibitory activity on mETC complex III. Our data suggest that mETC complex III may have a role in mediating autophagy induction.

Control of basal autophagy by calpain1 mediated cleavage of ATG5.

Autophagy functions as an important catabolic mechanism by mediating the turnover of intracellular organelles and protein complexes. Although the induction of autophagy by starvation has been extensively studied, we still understand very little about how autophagy is regulated under normal nutritional conditions. Here we describe a study using a small molecule autophagy inducer, fluspirilene, as a tool to explore the mechanism of autophagy induction in normal living cells. We confirm the activity of fluspirilene in inhibiting Ca(2+) flux. Furthermore, we show that reducing intracellular Ca(2+) prevents the cleavage of ATG5, which in turn increases the levels of full-length ATG5 and ATG12-ATG5 conjugate. Using siRNA mediated gene silencing, we demonstrate that inhibiting calpain1 is sufficient to induce autophagy in living cells. We conclude that calpain1 plays an important role in controlling the levels of autophagy in normal living cells by regulating the levels of a key signaling molecule, ATG12-ATG5 conjugate.