Design and Synthesis of Inhibitors of the E3 Ligase SMAD Specific E3 Ubiquitin Protein Ligase 1 as a Treatment for Lung Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a devastating rare disease, which despite currently available treatments, still represents a high unmet medical need. Specific E3 ubiquitin protein ligase 1 (SMURF1) is a HECT E3 ligase that ubiquitinates key signaling molecules from the TGFß/BMP pathways, which are of great relevance in the pathophysiology of PAH. Herein, the design and synthesis of novel potent small-molecule SMURF1 ligase inhibitors are described. Lead molecule 38 has demonstrated good oral pharmacokinetics in rats and significant efficacy in a rodent model of pulmonary hypertension.

A strategy to assess the cellular activity of E3 ligase components against neo-substrates using electrophilic probes.

While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.

Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo.

Peptides constitute an important class of drugs for the treatment of multiple metabolic, oncological, and neurodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability. Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide-based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an inability to provide kinetic information on the internalization of a compound or distinguish between internalized and membrane-bound compounds. In addition, many of these methods are based on end point assays and require multiple sample manipulation steps. Herein, we report a novel "Split Luciferin Peptide" (SLP) assay that enables the real-time noninvasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a d-cysteine tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening peptide libraries and can thus become an important tool for preclinical peptide drug development.

Evaluating Cellular Drug Uptake with Fluorescent Sensor Proteins.

We are introducing a new approach to evaluate cellular uptake of drugs and drug candidates into living cells. The approach is based on converting the protein target of a given class of compounds into a fluorescent biosensor. By measuring the binding of different compounds to their cognate biosensor in live cells and comparing these values to those measured in vitro, their cellular uptake and concentrations can be ranked. We demonstrate that our strategy enables the evaluation of the cellular uptake into the cytosol of 2 classes of inhibitors using two different sensor designs; first, sensors comprising the self-labeling protein SNAP conjugated with a chemically modified inhibitor shown for inhibitors of the enzyme human carbonic anhydrase II; and a label-free sensor for inhibitors of protein-protein interactions demonstrated for the protein pair p53-HDM2.

Bioorthogonal Probes for the Study of MDM2-p53 Inhibitors in Cells and Development of High-Content Screening Assays for Drug Discovery.

To study the behavior of MDM2-p53 inhibitors in a disease-relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high-content screening assays. By using automated image analysis with single-cell resolution, we could visualize the intracellular target binding of compounds by co-localization and quantify target upregulation upon MDM2-p53 inhibition in an osteosarcoma model. Additionally, we developed a high-throughput assay to quantify target occupancy of non-tagged MDM2-p53 inhibitors by competition and to identify novel chemical matter. This approach could be expanded to other targets for lead discovery applications.