Discovery of novel 1H-benzo[d]imidazole-4,7-dione based transglutaminase 2 inhibitors as p53 stabilizing anticancer agents in renal cell carcinoma.

Overexpression of transglutaminase 2 (TGase 2; TG2) has been implicated in the progression of renal cell carcinoma (RCC) through the inactivation of p53 by forming a protein complex. Because most p53 in RCC has no mutations, apoptosis can be increased by inhibiting the binding between TG2 and p53 to increase the stability of p53. In the present study, a novel TG2 inhibitor was discovered by investigating the structure of 1H-benzo[d]imidazole-4,7-dione as a simpler chemotype based on the amino-1,4-benzoquinone moiety of streptonigrin, a previously reported inhibitor. Through structure-activity relationship (SAR) studies, compound 8j (MD102) was discovered as a potent TG2 inhibitor with an IC50 value of 0.35 µM, p53 stabilization effect and anticancer effects in the ACHN and Caki-1 RCC cell lines with sulforhodamine B (SRB) GI50 values of 2.15 µM and 1.98 µM, respectively. The binding property of compound 8j (MD102) with TG2 was confirmed to be reversible in a competitive enzyme assay, and the binding interaction was expected to be formed at the ß-sandwich domain, a p53 binding site, in the SPR binding assay with mutant proteins. The mode of binding of compound 8j (MD102) to the ß-sandwich domain of TG2 was analyzed by molecular docking using the crystal structure of the active conformation of human TG2. Compound 8j (MD102) induced a decrease in the downstream signaling of p-AKT and p-mTOR through the stabilization of p53 by TG2 inhibition, resulting in tumor cell apoptosis. In a xenograft animal model using ACHN cancer cells, oral administration and intraperitoneal injection of compound 8j (MD102) showed an inhibitory effect on tumor growth, confirming increased levels of p53 and decreased levels of Ki-67 in tumor tissues through immunohistochemical (IHC) tissue staining. These results indicated that the inhibition of TG2 by compound 8j (MD102) could enhance p53 stabilization, thereby ultimately showing anticancer effects in RCC. Compound 8j (MD102), a novel TG2 inhibitor, can be further applied for the development of an anticancer candidate drug targeting RCC.

Lecithin nano-liposomal particle as a CRISPR/Cas9 complex delivery system for treating type 2 diabetes.

BACKGROUND: Protein-based Cas9 in vivo gene editing therapeutics have practical limitations owing to their instability and low efficacy. To overcome these obstacles and improve stability, we designed a nanocarrier primarily consisting of lecithin that can efficiently target liver disease and encapsulate complexes of Cas9 with a single-stranded guide RNA (sgRNA) ribonucleoprotein (Cas9-RNP) through polymer fusion self-assembly. RESULTS: In this study, we optimized an sgRNA sequence specifically for dipeptidyl peptidase-4 gene (DPP-4) to modulate the function of glucagon-like peptide 1. We then injected our nanocarrier Cas9-RNP complexes directly into type 2 diabetes mellitus (T2DM) db/db mice, which disrupted the expression of DPP-4 gene in T2DM mice with remarkable efficacy. The decline in DPP-4 enzyme activity was also accompanied by normalized blood glucose levels, insulin response, and reduced liver and kidney damage. These outcomes were found to be similar to those of sitagliptin, the current chemical DPP-4 inhibition therapy drug which requires recurrent doses. CONCLUSIONS: Our results demonstrate that a nano-liposomal carrier system with therapeutic Cas9-RNP has great potential as a platform to improve genomic editing therapies for human liver diseases.

Effects of phospholipids on the functional regulation of tBID in membranes.

The functional interplay between tBID and phospholipids was investigated in this study. The binding of tBID to model membranes was increased by an incorporation of phosphatidylserine (PS) into the liposomes. Using limited proteolysis and mass spectrometry, two peptide regions, which correspond to Ser(100)-Arg(114) and His(89)-Arg(114) in BID, revealed the specific PS-binding site. tBID also decreased the light scattering values of PS-containing liposomes and increased the leakage of fluorescent dye encapsulated in vesicles, which suggest that tBID reduces membrane integrity by fragmentation. The membrane fragmentation by tBID was also observed using confocal and transmission electron microscopy. The activity of tBID paralleled results that were obtained with cardiolipin (CL)-containing membranes. However, other anionic phospholipids had little effect. CL- and PS-induced conformational changes of tBID were observed by circular dichroism and intrinsic fluorescence. CL and PS also stimulated the insertion of BID into lipid monolayers. tBID stimulated the leakage of Ca(2+) from purified microsomes and mitochondria in a protein concentration-dependent manner. In contrast, BID showed significantly reduced effects when compared to tBID in all of the experiments performed. These results suggest that tBID specifically interacts with PS as well as CL and decreases membrane integrity without the aid of other pro-apoptotic proteins.

Conformational change of Escherichia coli signal recognition particle Ffh is affected by the functionality of signal peptides of ribose-binding protein.

We examined the effects of synthetic signal peptides, wild-type (WT) and export-defective mutant (MT) of ribose-binding protein, on the conformational changes of signal recognition particle 54 homologue (Ffh) in Escherichia coli. Upon interaction of Ffh with WT peptide, the intrinsic Tyr fluorescence, the transition temperature of thermal unfolding, and the GTPase activity of Ffh decreased in a peptide concentration-dependent manner, while the emission intensity of 8-anilinonaphthalene-1-sulfonic acid increased. In contrast, the secondary structure of the protein was not affected. Additionally, polarization of fluorescein-labeled WT increased upon association with Ffh. These results suggest that WT peptide induces the unfolded states of Ffh. The WT-mediated conformational change of Ffh was also revealed to be important in the interaction between SecA and Ffh. However, MT had marginal effect on these conformational changes suggesting that the in vivo functionality of signal peptide is important in the interaction with Ffh and concomitant structural change of the protein.

Bax inhibitor 1 regulates ER-stress-induced ROS accumulation through the regulation of cytochrome P450 2E1.

This study investigated the molecular mechanism by which Bax inhibitor 1 (BI1) abrogates the accumulation of reactive oxygen species (ROS) in the endoplasmic reticulum (ER). Electron uncoupling between NADPH-dependent cytochrome P450 reductase (NPR) and cytochrome P450 2E1 (P450 2E1) is a major source of ROS on the ER membrane. ER stress produced ROS accumulation and lipid peroxidation of the ER membrane, but BI1 reduced this accumulation. Under ER stress, expression of P450 2E1 in control cells was upregulated more than in BI1-overexpressing cells. In control cells, inhibiting P450 2E1 through chemical or siRNA approaches suppressed ROS accumulation, ER membrane lipid peroxidation and the resultant cell death after ER stress. However, it had little effect in BI1-overexpressing cells. In addition, BI1 knock down also increased ROS accumulation and expression of P450 2E1. In a reconstituted phospholipid membrane containing purified BI1, NPR and P450 2E1, BI1 dose-dependently decreased the production of ROS. BI1 bound to NPR with higher affinity than P450 2E1. Furthermore, BI1 overexpression reduced the interaction of NPR and P450 2E1, and decreased the catalytic activity of P450 2E1, suggesting that the flow of electrons from NPR to P450 2E1 can be modulated by BI1. In summary, BI1 reduces the accumulation of ROS and the resultant cell death through regulating P450 2E1.

Lysophosphatidylserine-induced functional switch of human cytochrome P450 1A2 and 2E1 from monooxygenase to phospholipase D.

Interaction of human cytochrome P450 1A2 (CYP1A2) and 2E1 (CYP2E1) with phospholipid, lysophosphatidylserine (LysoPS) in the context of a PC matrix specifically stimulated the PLD activity of both enzymes in a LysoPS concentration-dependent manner. However, other anionic lysophospholipids as well as the neutral lysophospholipids, lysophosphatidylcholine and lysophosphatidylethanolamine, had no effect. LysoPS also accompanied conformational changes in both CYPs when assayed by circular dichroism. Although the PLD activity was decreased in the presence of components required for the monooxygenase (MMO) activity, including 100% PC membranes, NADPH-cytochrome P450 reductase and NADPH, as compared to the activity in the absence of the reducing system, LysoPS recovered the PLD activity in a concentration-dependent manner. Considering that LysoPS induced a decrease in the MMO activities of both CYPs, the results suggest that the functional roles of CYP1A2 and 2E1 can be switched by interaction with a specific anionic lysophospholipid in vivo.

Anionic phospholipid-induced regulation of reactive oxygen species production by human cytochrome P450 2E1.

We suggest that the cytochrome P450 2E1 (CYP2E1)-induced formation of reactive oxygen species (ROS) can be regulated by anionic phospholipids and the presence of the N-terminal region of the enzyme. When the content of cardiolipin (CL) in membranes at the expense of phosphatidylcholine matrix was increased, the ROS produced by recombinant human CYP2E1 was decreased as a function of CL concentration. On the contrary, the N-terminally truncated CYP2E1 had a decreased effect on the lipid-induced reduction of ROS formation. These results suggest that specific phospholipids can regulate the function of CYP2E1 by interaction with the enzyme including the N-terminal region(s).