Discovery of Monovalent Direct Degraders of BRD4 that Act via the Recruitment of DCAF11.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity in vivo.

Discovery of Monovalent Direct Degraders of BRD4 That Act Via the Recruitment of DCAF11.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extra-terminal (BET) protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent anti-tumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent anti-tumor activity in vivo.

Rational Design of Surface-State Controlled Multicolor Cross-Linked Carbon Dots with Distinct Photoluminescence and Cellular Uptake Properties.

We disclose for the first time a facile synthetic methodology for the preparation of multicolor carbon dots (CDs) from a single source barring any chromatographic separations. This was achieved via sequential intraparticle cross-linking of surface abundant carboxylic acid groups on the CDs synthesized from a precursor to control their photoluminescence (PL) spectra as well as affect their degree of cellular internalization in cancer cells. The change in PL spectra with sequential cross-linking was projected by theoretical density functional theory (DFT) studies and validated by multiple characterization tools such as X-ray photoelectron spectroscopy (XPS), PL spectroscopy, ninhydrin assay, etc. The variation in cellular internalization of these cross-linked CDs was demonstrated using inhibitor assays, confocal microscopy, and flow cytometry. We supplemented our findings with high-resolution dark-field imaging to visualize and confirm the colocalization of these CDs into distinct intracellular compartments. Finally, to prove the surface-state controlled PL mechanisms of these cross-linked CDs, we fabricated a triple-channel sensor array for the identification of different analytes including metal ions and biologically relevant proteins.

Intratumoral generation of photothermal gold nanoparticles through a vectorized biomineralization of ionic gold.

Various cancer cells have been demonstrated to have the capacity to form plasmonic gold nanoparticles when chloroauric acid is introduced to their cellular microenvironment. But their biomedical applications are limited, particularly considering the millimolar concentrations and longer incubation period of ionic gold. Here, we describe a simplistic method of intracellular biomineralization to produce plasmonic gold nanoparticles at micromolar concentrations within 30 min of application utilizing polyethylene glycol as delivery vector for ionic gold. We have characterized this process for intracellular gold nanoparticle formation, which progressively accumulates proteins as the ionic gold clusters migrate to the nucleus. This nano-vectorized application of ionic gold emphasizes its potential biomedical opportunities while reducing the quantity of ionic gold and required incubation time. To demonstrate its biomedical potential, we further induce in-situ biosynthesis of gold nanoparticles within MCF7 tumor mouse xenografts which is followed by its photothermal remediation.

Nano-Assembly of Pamitoyl-Bioconjugated Coenzyme-A for Combinatorial Chemo-Biologics in Transcriptional Therapy.

Pathogenesis, the biological mechanism that leads to the diseased state, of many cancers is driven by interruptions to the role of Myc oncoprotein, a regulator protein that codes for a transcription factor. One of the most significant biological interruptions to Myc protein is noted as its dimerization with Max protein, another important factor of family of transcription factors. Binding of this heterodimer to E-Boxes, enhancer boxes as DNA response element found in some eukaryotes that act as a protein-binding site and have been found to regulate gene expression, are interrupted to regulate cancer pathogenesis. The systemic effectiveness of potent small molecule inhibitors of Myc-Max dimerization has been limited by poor bioavailability, rapid metabolism, and inadequate target site penetration. The potential of gene therapy for targeting Myc can be fully realized by successful synthesis of a smart cargo. We developed a "nuclein" type nanoparticle "siNozyme" (45 ± 5 nm) from nanoassembly of pamitoyl-bioconjugated acetyl coenzyme-A for stable incorporation of chemotherapeutics and biologics to achieve remarkable growth inhibition of human melanoma. Results indicated that targeting transcriptional gene cMyc with siRNA with codelivery of a topoisomerase inhibitor, amonafide caused ∼90% growth inhibition and 95% protein inhibition.

Phenotypically Screened Carbon Nanoparticles for Enhanced Combinatorial Therapy in Triple Negative Breast Cancer.

INTRODUCTION: Triple negative breast cancer (TNBC) is a highly aggressive type of breast cancer with high resistance to current standard therapies. We demonstrate that phenotypically stratified carbon nanoparticle is highly effective in delivering a novel combinatorial triple drug formulation for synergistic regression of TNBC in vitro and in vivo. METHOD: The combinatorial formulation is comprised of repurposed inhibitors of STAT3 (nifuroxazide), topoisomerase-II-activation-pathway (amonafide) and NFκb (pentoxifylline). Synergistic effect of drug combination was established in a panel of TNBC-lines comprising mesenchymal-stem-like, mesenchymal and basal-like cells along with non-TNBC-cells. The delivery of combinatorial drug formulation was achieved using a phenotypically screened carbon nanoparticles for TNBC cell lines. RESULTS: Results indicated a remarkable five-fold improvement (IC50-6.75 µM) from the parent drugs with a combinatorial index <1 in majority of the TNBC cells. Multi-compartmental carbon nanoparticles were then parametrically assessed based on size, charge (positive/negative/neutral) and chemistry (functionalities) to study their likelihood of crossing endocytic barriers from phenotypical standpoint in TNBC lines. Interestingly, a combination of clathrin mediated, energy and dynamin dependent pathways were predominant for sulfonated nanoparticles, whereas pristine and phospholipid particles followed all the investigated endocytic pathways. CONCLUSIONS: An exactitude 'omics' approach helps to predict that phospholipid encapsulated-particles will predominantly accumulate in TNBC comprising the drug-'cocktail'. We investigated the protein expression effects inducing synergistic effect and simultaneously suppressing drug resistance through distinct mechanisms of action.