Using protein turnover assay to explore the drug mechanism of Carfilzomib

Abstract

Carfilzomib (CFZ) is the second-generation proteasome inhibitor that is approved by Food and Drug Administration (FDA) of USA for the treatment of relapsed and refractory multiple myeloma. Although the preclinical and clinical efficacy of CFZ is obvious, the mechanism by which CFZ leads to cell death has not been fully elucidated. Since CFZ primarily functions as a proteasome inhibitor, profiling CFZ-induced changes in protein turnover at the systematic level is sufficient and necessary. In this study, we characterize the effects of CFZ on the stability of 15,000 human proteins using Protein Turnover Assay (ProTA). CFZ affects fundamental cellular glycolysis, nitric oxide production and proteasome subunit homeostasis in multiple myeloma cells. In addition, LY294002 or KU-0063794 has synergistic effects with CFZ in multiple myeloma treatment. A profound understanding of how cells respond to chemotherapeutic agents provides insights into the basic mechanism of drug function and the rationale for CFZ combination therapy.

1. Introduction

Protein degradation by the ubiquitin proteasome system (UPS) is tightly regulated and plays important roles in a wide range of basic cellular processes [1,2]. The proteasome, which functions as a principal regulator of intracellular protein degradation, has attracted the attention of researchers historically and has recently been regarded as a promising drug target [3,4]. Mechanistic and structural studies have accelerated the development of highly efficient and specific cell-permeable proteasome inhibitors [5]. A number of proteasome inhibitors have been synthesized, and several of them are in preclinical trials or are clinically used [6,7].

Multiple myeloma is an incurable plasma cell malignant neoplasm and is the second most common hematological malignancy in the USA [8,9]. Multiple myeloma cells produce high amounts of monoclonal antibodies, making it crucial to maintain protein homeostasis from synthesis through folding to degradation [10]. Protein ubiquitination and organized degradation are widely recognized as essential for cellular health. An emerging strategy is to inhibit these processes to induce cell death in disease-state cells that are characterized by protein overproduction [11]. Bortezomib (BTZ) was the first Food and Drug Administration (FDA)-approved proteasome inhibitor for treating multiple myeloma and mantle cell lymphoma. Although BTZ combination therapy substantially improves the outcome of myeloma patients [12–14], it has noteworthy side effects, such as peripheral neuropathy and cyclical thrombocytopenia [12,15,16]. In addition, many patients exhibit primary or secondary BTZ resistance [17]. Thus, both academia and the pharmaceutical industry have been working on developing new proteasome inhibitors.

Carfilzomib (CFZ; also known as PR-171) is a second-generation proteasome inhibitor that is used for the treatment of relapsed and refractory multiple myeloma. Unlike BTZ, it is a tetrapeptide derived from the natural product epoxomicin and can irreversibly inhibit the proteasome [18]. Clinical trials have shown that CFZ is less toxic and more effective than BTZ for treating relapsed and refractory myeloma [19–24]. These two compounds appear to have different specificities for the different catalytic sites of the 20S complex [3], and their mechanisms of action are somewhat different. It is possible that the repertoire of proteins affected by CFZ differs from that affected by BTZ to some extent [5].