Wooin Lee

Consortium of fold-catalyzing proteins increases soluble expression of cyclohexanone monooxygenase in recombinant Escherichia coli.

Abstract. The cyclohexanone monooxygenase (CHMO) gene of Acinetobacter sp. NCIMBxa09871 was simultaneously expressed with the genes encoding molecular chaperones and foldases in Escherichia coli. While the expression of the CHMO gene alone resulted in the formation of inclusion bodies, coexpression of the chaperone or foldase genes remarkably increased the production of soluble CHMO enzyme in recombinant E. coli. Furthermore, it was found that molecular chaperones were more beneficial than foldases for enhancing active CHMO enzyme production. The recombinant E. coli strain simultaneously expressing the genes for CHMO, GroEL/GroES and DnaK/DnaJ/GrpE showed a specific CHMO activity of 111xa0units g−1 cell protein, corresponding to a 38-fold enhancement in CHMO activity compared with the control E. coli strain expressing the CHMO gene alone.

Proteasome inhibitors with pyrazole scaffolds from structure-based virtual screening.

We performed a virtual screen of ∼340u202f000 small molecules against the active site of proteasomes followed by in vitro assays and subsequent optimization, yielding a proteasome inhibitor with pyrazole scaffold. The pyrazole-scaffold compound displayed excellent metabolic stability and was highly effective in suppressing solid tumor growth in vivo. Furthermore, the effectiveness of this compound was not negatively impacted by resistance to bortezomib or carfilzomib.

Polymer Micelle Formulations of Proteasome Inhibitor Carfilzomib for Improved Metabolic Stability and Anticancer Efficacy in Human Multiple Myeloma and Lung Cancer Cell Lines

Carfilzomib (CFZ) is a second-generation proteasome inhibitor drug approved for the treatment of multiple myeloma. Contrary to its excellent antimyeloma activity, CFZ has shown only limited efficacy in patients with solid malignancies. This lack of efficacy has been attributed in part to rapid degradation of CFZ in the body, possibly hindering the ability of CFZ to access the proteasome target in solid tumors. We hypothesized that polymer micelles, a currently Food and Drug Administration–approved nanoparticle drug delivery formulation, may protect CFZ from metabolic degradation and thus expand the clinical utility of the drug as an anticancer agent. To test our hypothesis, we prepared CFZ-entrapped polymer micelle particles with various compositions and drug release profiles and examined the extent of the CFZ metabolism in vitro using mouse liver homogenates. We also assessed the cytotoxic activities of the CFZ-entrapped micelle formulations in human cancer cell lines derived from B lymphocytes (RPMI-8226) and the lung (H460). Our data indicated that polymer micelle-based formulations can improve metabolic stability and cytotoxic effects of CFZ compared with free CFZ in human cancer cell lines tested. Taken together, these results suggest that polymer micelles may have potential as a delivery system for CFZ with an extended therapeutic utility for nonhematologic malignancies in the future.

Elucidating the Catalytic Subunit Composition of Distinct Proteasome Subtypes: A Crosslinking Approach Employing Bifunctional Activity-Based Probes

In addition to two well‐recognized proteasome subtypes—constitutive proteasomes and immunoproteasomes—mounting evidence also suggests the existence of intermediate proteasome subtypes containing unconventional mixtures of catalytic subunits. Although they appear to play unique biological roles, the lack of practical methods for detecting distinct proteasome subtypes has limited functional investigations. Here, we report the development of activity‐based probes that crosslink two catalytic subunits within intact proteasome complexes. Identification of the crosslinked subunit pairs provides direct evidence of the catalytic subunit composition of proteasomes. Using these probes, we found that U266 multiple myeloma cells contain intermediate proteasomes comprising both β1i and β2, but not β1 and β2i, consistent with previous findings with other cell types. Our bifunctional probes can be utilized in functional investigations of distinct proteasome subtypes in various biological settings.

Comprehensive PBPK Model of Rifampicin for Quantitative Prediction of Complex Drug‐Drug Interactions: CYP3A/2C9 Induction and OATP Inhibition Effects

This study aimed to construct a physiologically based pharmacokinetic (PBPK) model of rifampicin that can accurately and quantitatively predict complex drug‐drug interactions (DDIs) involving its saturable hepatic uptake and auto‐induction. Using in silico and in vitro parameters, and reported clinical pharmacokinetic data, rifampicin PBPK model was built and relevant parameters for saturable hepatic uptake and UDP‐glucuronosyltransferase (UGT) auto‐induction were optimized by fitting. The parameters for cytochrome P450 (CYP) 3A and CYP2C9 induction by rifampicin were similarly optimized using clinical DDI data with midazolam and tolbutamide as probe substrates, respectively. For validation, our current PBPK model was applied to simulate complex DDIs with glibenclamide (a substrate of CYP3A/2C9 and hepatic organic anion transporting polypeptides (OATPs)). Simulated results were in quite good accordance with the observed data. Altogether, our constructed PBPK model of rifampicin demonstrates the robustness and utility in quantitatively predicting CYP3A/2C9 induction‐mediated and/or OATP inhibition‐mediated DDIs with victim drugs.

Physiologically Based Pharmacokinetic Modeling of Bosentan Identifies the Saturable Hepatic Uptake As a Major Contributor to Its Nonlinear Pharmacokinetics

Bosentan is a substrate of hepatic uptake transporter organic anion–transporting polypeptides (OATPs), and undergoes extensive hepatic metabolism by cytochrome P450 (P450), namely, CYP3A4 and CYP2C9. Several clinical investigations have reported a nonlinear relationship between bosentan doses and its systemic exposure, which likely involves the saturation of OATP-mediated uptake, P450-mediated metabolism, or both in the liver. Yet, the underlying causes for the nonlinear bosentan pharmacokinetics are not fully delineated. To address this, we performed physiologically based pharmacokinetic (PBPK) modeling analyses for bosentan after its intravenous administration at different doses. As a bottom-up approach, PBPK modeling analyses were performed using in vitro kinetic parameters, other relevant parameters, and scaling factors. As top-down approaches, three different types of PBPK models that incorporate the saturation of hepatic uptake, metabolism, or both were compared. The prediction from the bottom-up approach (models 1 and 2) yielded blood bosentan concentration-time profiles and their systemic clearance values that were not in good agreement with the clinically observed data. From top-down approaches (models 3, 4, 5-1, and 5-2), the prediction accuracy was best only with the incorporation of the saturable hepatic uptake for bosentan. Taken together, the PBPK models for bosentan were successfully established, and the comparison of different PBPK models identified the saturation of the hepatic uptake process as a major contributing factor for the nonlinear pharmacokinetics of bosentan.

Feasibility of the functional expression of the human organic anion transporting polypeptide 1B1 (OATP1B1) and its genetic variant 521T/C in the mouse liver

&NA; The objective of this study was to examine the feasibility of functional expression of the human organic anion transporting polypeptide 1B1 (hOATP1B1) forms in the liver of the mouse. After the mouse received the gene of interest (i.e., luciferase as the reporter or hOATP1B1) via hydrodynamic gene delivery (HGD) method, the expression was found to be liver‐specific while alterations in the serum biochemistry and hepatocyte histology were apparently transient and reversible. The reporter activity was also detected in the plasma, but not in the blood cell in mice that received HGD, suggesting that the protein is probably released due to transiently increased permeability in hepatocytes by HGD. Using this delivery condition, the expression of hOATP1B1 was readily detected in the liver, but not in other tissues, of the mice receiving HGD for the transporter gene. Compared with the sham control mice, the uptake of pravastatin into the liver increased significantly in mice receiving hOATP1B1 wild type; the uptake parameters decreased consistently in mice expressing the 521T>C variant compared with that of the wild type control. These observations suggest that the functional expression of human transporter gene in mice is feasible, further suggesting that this treatment is practically useful in the pharmacokinetic studies for hOATP1B1 substrates. Graphical abstract Figure. No caption available.

Effect of Enhancers on in vitro and in vivo Skin Permeation and Deposition of S-Methyl-L-Methionine

S-methyl-L-methionine (SMM), also known as vitamin U, is commercially available as skin care cosmetic products for its wound healing and photoprotective effects. However, the low skin permeation expected of SMM due to its hydrophilic nature with a log P value of -3.3, has not been thoroughly addressed. The purpose of this study thus was to evaluate the effect of skin permeation enhancers on the skin permeation/deposition of SMM. Among the enhancers tested for the in vitro skin permeation and deposition of SMM, oleic acid showed the most significant enhancing effect. Moreover, the combination of oleic acid and ethanol further enhanced in vitro permeation and deposition of SMM through hairless mouse skin. Furthermore, the combination of oleic acid and ethanol significantly increased the in vivo deposition of SMM in the epidermis/dermis for 12 hr, which was high enough to exert a therapeutic effect. Therefore, based on the in vitro and in vivo studies, the combination of oleic acid and ethanol was shown to be effective in improving the topical skin delivery of SMM, which may be applied in the cosmetic production process for SMM.

High‐Resolution Snapshots of Proteasome Inhibitors In Action Revise Inhibition Paradigms and Inspire Next‐Generation Inhibitor Design

New high-resolution crystal structures reported by Schrader and colleagues refine our understanding of how peptide epoxyketone anticancer drugs inactivate their target: the human proteasome. These findings provide important clues for the design of next-generation proteasome inhibitor drugs.

Next-generation proteasome inhibitors for cancer therapy

&NA; Over 2 decades ago, the proteasome was considered a risky or even untenable therapeutic target. Today, proteasome inhibitors are a mainstay in the treatment of multiple myeloma (MM) and have sales in excess of 3 billion US dollars annually. More importantly, the availability of proteasome inhibitors has greatly improved the survival and quality of life for patients with MM. Despite the remarkable success of proteasome inhibitor therapies to date, the potential for improvement remains, and the development and optimal use of proteasome inhibitors as anticancer agents continues to be an active area of research. In this review, we briefly discuss the features and limitations of the 3 proteasome inhibitor drugs currently used in the clinic and provide an update on current efforts to develop next‐generation proteasome inhibitors with the potential to overcome the limitations of existing proteasome inhibitor drugs.