The natural product withaferin A (WFA) exhibits antitumor and antiangiogenesis activity in vivo, which results from this drug's potent growth inhibitory activities. Here, we show that WFA binds to the intermediate filament (IF) protein, vimentin, by covalently modifying its cysteine residue, which is present in the highly conserved alpha-helical coiled coil 2B domain. WFA induces vimentin filaments to aggregate in vitro, an activity manifested in vivo as punctate cytoplasmic aggregates that colocalize vimentin and F-actin. WFA's potent dominant-negative effect on F-actin requires vimentin expression and induces apoptosis. Finally, we show that WFA-induced inhibition of capillary growth in a mouse model of corneal neovascularization is compromised in vimentin-deficient mice. These findings identify WFA as a chemical genetic probe of IF functions, and illuminate a potential molecular target for withanolide-based therapeutics for treating angioproliferative and malignant diseases.

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The ubiquitin-proteasome pathway has emerged as a central player in the regulation of several diverse cellular processes. Here, we describe the important components of this complex biochemical machinery as well as several important cellular substrates targeted by this pathway and examples of human diseases resulting from defects in various components of the ubiquitin-proteasome pathway. In addition, this review covers the chemistry of synthetic and natural proteasome inhibitors, emphasizing their mode of actions toward the 20S proteasome. Given the importance of proteasomemediated protein degradation in various intracellular processes, inhibitors of this pathway will continue to serve as both molecular probes of major cellular networks as well as potential therapeutic agents for various human diseases.

Geographic atrophy is a blinding form of age-related macular degeneration characterized by death of the retinal pigmented epithelium (RPE). In this disease, the RPE displays evidence of DICER1 deficiency, resultant accumulation of endogenous Alu retroelement RNA, and NLRP3 inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human cell culture and in mouse models is driven by a non-canonical inflammasome pathway that results in activation of caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-β (IFN-β) production and gasdermin D-dependent interleukin-18 (IL-18) secretion. Reduction of DICER1 levelsor accumulation of Alu RNA triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, IFN-β, and cGAS levels are elevated in the RPE of human eyes with geographic atrophy. Collectively, these data highlight an unexpected role for cGAS in responding to mobile element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial damage-induced inflammasome activation, expand the immune sensing repertoire of cGAS and caspase-4 to non-infectious human disease, and identify new potential targets for treatment of a major cause of blindness.

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The chymotrypsin-like (CT-L) activity of the proteasome is downregulated by substrates of the peptidyl-glutamyl peptide hydrolyzing (PGPH) activity. To investigate the nature of such interactions, we synthesized selective alpha',beta'-epoxyketone inhibitors of the PGPH activity. In cellular proliferation and protein degradation assays, these inhibitors revealed that selective PGPH inhibition was insufficient to inhibit protein degradation, indicating that the CT-L and PGPH sites function independently. We also demonstrated that CT-L inhibition by a PGPH substrate does not require the occupancy of the PGPH site or hydrolysis of the PGPH substrate. Thus, these results support a model in which a substrate of one subunit regulates the activity of another via binding to a noncatalytic site(s) rather than through binding to an active site.

The initial enthusiasm following the discovery of a pharmacologically active natural product is often fleeting due to the poor prospects for its ultimate clinical application. Despite this, the ever-changing landscape of modern biology has a constant need for molecular probes that can aid in our understanding of biological processes. After its initial discovery by Bristol-Myers Squibb as a microbial anti-tumor natural product, epoxomicin was deemed unfit for development due to its peptide structure and potentially labile epoxyketone pharmacophore. Despite its drawbacks, epoxomicin’s pharmacophore was found to provide unprecedented selectivity for the proteasome. Epoxomicin also served as a scaffold for the generation of a synthetic tetrapeptide epoxyketone with improved activity, YU-101, which became the parent lead compound of carfilzomib (Kyprolis™), the recently approved therapeutic agent for multiple myeloma. In this era of rational drug design and high-throughput screening, the prospects for turning an active natural product into an approved therapy are often slim. However, by understanding the journey that began with the discovery of epoxomicin and ended with the successful use of carfilzomib in the clinic, we may find new insights into the keys for success in natural product-based drug discovery.

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.