Rainer Metcalf

Mixing Properties of Sphingomyelin Ceramide Bilayers: A Simulation Study

Ceramide is the simplest molecule in the class of glycosphingolipids composed of a sphingosine backbone and acyl moiety. It plays significant roles in cell signaling; apoptosis; binding of hormones, toxins, and viruses; and many other biologically important functions. Sphingomyelin, ceramide with a phosphotidylcholine headgroup, is another biologically vital lipid present in the myelin sheath of nerve cell axons. Regions with high concentrations of ceramide can be formed in biological membranes composed of sphingomyelin by enzymatic catalysis with sphingomyelinase. To better understand the biophysical and thermodynamic properties of these molecules and their mixtures, we have preformed NPT molecular dynamics simulations of hydrated 16:0 sphingomyelin bilayers with increasing concentrations of 16:0 ceramide at 323, 332, 340, and 358 K. From analyses of electron densities, hydrogen bonding, NMR order parameters, partial molecular volume, and partial molecular area, we have identified possible structural changes corresponding to liquid ordered and liquid disordered phases. These structural changes are the results of changes in intra- and intermolecular hydrogen bonds between SM and Cer molecules. Our results correspond to DSC experiments for sphingomyelin bilayer concentrations up to 50% Cer. Above 50% concentration, we observe conformational changes in the SM headgroup similar to that of the umbrella model for lipid cholesterol mixtures.

Recent advances in proteasome inhibitor discovery

Introduction: Proteasome inhibition is a quickly advancing subject of research and has a significant potential to become a potent therapeutic modality for many diseases and disorders. The aim of this review is to present the reader with the variety of approaches to the proteasome inhibitor discovery as well as highlight the diversity of scaffolds being considered for this task. Areas covered: This review focuses on current developments in proteasome inhibitor discovery, including an account of research efforts covered in the literature from the years 2009 – 2012, although some of the earlier work is also mentioned. Specifically, presented are the type of experiments performed, the compounds and compound families investigated along with their activities and assessment for potential therapeutic value. In particular, authors highlight different paths to discovery of the proteasome inhibitors such as screening of large libraries, repurposing of existing therapeutics, development of compounds with known proteasome inhibitory activities as well as utilizing novel scaffolds. Expert opinion: Discovery of therapeutically successful proteasome inhibitors depends on a number of factors and demands a multipronged approach. Screening protocols, choice of assays, desired mode of action, selection of a binding pocket, targeting and delivery strategy, all require careful consideration when attempting to target the proteasome.

Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1–CUL4A–Roc1–RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBNs activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBNs nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBNs E3 ubiquitin–conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBNs substrate-recruiting function.

Proteasome inhibitor patents (2010 – present)

Introduction: Over the past 3 years, numerous patents and patent applications have been submitted and published involving compounds designed to inhibit the proteasome. Proteasome inhibition has been of great interest in cancer research since disruption of proteolysis leads to a significant buildup of cytotoxic proteins and activation of apoptotic pathways, particularly in rapidly proliferating cells. The current standards in proteasome inhibition are the only FDA-approved inhibitors, bortezomib and carfilzomib. Although these drugs are quite effective in treating multiple myeloma and other blood tumors, there are shortcomings, including toxicities and resistance. Most of the current patents attempt to improve on existing compounds, by increasing bioavailability and selectivity, while attempting to reduce toxicity. A general categorization of similar compounds was employed to evaluate and compare drug design strategies. Areas covered: This review focuses on novel compounds and subsequent analogs developed for proteasome inhibition, used in preventing and treating human cancers. A comprehensive description and categorization of patents related to each type of compound and its derivatives, as well as their uses and efficacies as anticancer agents is included. A review of combination therapy patents has also been included. Expert opinion: Although there are many diverse chemical scaffolds being published, there are few patented proteasome inhibitors whose method of inhibition is genuinely novel. Most patents utilize a destructive chemical warhead to attack the catalytic threonine residue of the proteasome active sites. Few patents try to depart from this, emphasizing the need for developing new mechanisms of action and specific targeting.

Cupriphilic compounds to aid in proteasome inhibition.

It has been found that tumor cells and tissues, compared to normal cells, have higher levels of copper and possibly other metal ions. This presents a potential vulnerability of tumor cells that can serve as a physiological difference between cancer cells and normal cells and allows design of compounds that selectively target tumor cells while sparing normal cells. Recently we have identified compounds that have potential to inhibit the proteasome in tumor cells and induce cell death by mobilizing endogenous tumor copper resulting in in cellulo activation of the compound. These compounds hence act as pro-drugs, becoming active drugs in tumor cells with high copper content but remaining essentially inactive in normal cells, thereby greatly reducing adverse effects in patients. Such use would be of significant benefit in early detection and treatment of cancers, in particular, aggressive cancers such as pancreatic cancer which is usually not detected until it has reached an advanced stage. Six compounds were identified following virtual screening of the NCI Diversity Set with our proteasome computer model followed by confirmation with a biochemical assay that showed significant inhibition of the proteasome by the compounds in the presence of copper ions. In a dose response assay, NSC 37408 (6,7-dihydroxy-1-benzofuran-3-one), our best compound, exhibited an IC50 of 3μM in the presence of 100nM copper.