Structure-Activity Relationship of USP5 Inhibitors.

USP5 is a deubiquitinase that has been implicated in a range of diseases, including cancer, but no USP5-targeting chemical probe has been reported to date. Here, we present the progression of a chemical series that occupies the C-terminal ubiquitin-binding site of a poorly characterized zinc-finger ubiquitin binding domain (ZnF-UBD) of USP5 and competitively inhibits the catalytic activity of the enzyme. Exploration of the structure-activity relationship, complemented with crystallographic characterization of the ZnF-UBD bound to multiple ligands, led to the identification of 64, which binds to the USP5 ZnF-UBD with a KD of 2.8 µM and is selective over nine proteins containing structurally similar ZnF-UBD domains. 64 inhibits the USP5 catalytic cleavage of a di-ubiquitin substrate in an in vitro assay. This study provides a chemical and structural framework for the discovery of a chemical probe to delineate USP5 function in cells.

Leveraging Open Science Drug Development for PET: Preliminary Neuroimaging of 11C-Labeled ALK2 Inhibitors.

Mutations in the gene encoding activin receptor-like kinase 2 (ALK2) are implicated in the pathophysiology of a pediatric brainstem cancer, diffuse intrinsic pontine glioma (DIPG). Inhibitors of ALK2 that cross the blood-brain barrier have been proposed as a method of treatment for DIPG. As part of an open science approach to radiopharmaceutical and drug discovery, we developed 11C-labeled radiotracers from potent and selective lead ALK2 inhibitors to investigate their brain permeability through positron emission tomography (PET) neuroimaging. Four radiotracers were synthesized by 11C-methylation and assessed by dynamic PET imaging in healthy Sprague-Dawley rats. One of the compounds, [ 11 C]M4K2127, showed high initial brain uptake (SUV ∼ 2), including in the region of interest (pons). This data supports the use of this chemotype as a brain penetrant ALK2 inhibitor that permeates evenly into the pons with potential application for the treatment of DIPG.

PRMT5 inhibition disrupts splicing and stemness in glioblastoma.

Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.

Preclinical evaluation of taxane-binding peptide-modified polymeric micelles loaded with docetaxel in an orthotopic breast cancer mouse model.

We developed a novel taxane-binding peptide (TBP) modified, biodegradable polymeric micelle that overcomes limitations of drug loading and poor serum stability typically seen with particle delivery, leading to enhanced pharmacokinetics and tumor distribution of docetaxel (DTX). The use of the taxane-binding peptide to increase docetaxel loading is particularly compelling as it takes advantage of a known intracellular binding mechanism in a new way. Docetaxel is a potent chemotherapeutic with a therapeutic index often limited by the toxicity of the excipients that are necessary to enhance its solubility for intravenous delivery. Our polymeric micelle has terminal furan groups that enable facile antibody Fab conjugation by Diels-Alder chemistry for targeted delivery. Compared to the conventional ethanolic polysorbate 80 formulation (Free DTX), our nanoparticle (NP DTX) formulation exhibited a two-fold increase in exposure and tumor accumulation. Notably, the reduced toxicity of the NP DTX formulation increased the therapeutic index and allowed for higher dosing regimens, with a maximum tolerated dose (MTD) 1.6-fold higher than that of the Free DTX formulation, which is significant and similar to enhancements observed in clinical products for docetaxel and other drugs. These improved properties led to enhanced mouse survival in an orthotopic model of breast cancer; however, the targeted formulation of Fab-NP DTX did not further improve efficacy. Together, these results clearly demonstrate the benefits of the TBP-modified polymeric micelles as promising carriers for docetaxel.

Genotoxic and carcinogenic products arising from reductive transformations of the azo dye, Disperse Yellow 7.

Selected aromatic azo and benzidine based dyes are priority compounds under the Government of Canada's Chemical Management Plan (CMP) for environmental risk assessments. Organic compounds undergo chemical and biological transformations when they interact with environmental matrices and biotic species; identifying the transformation products is thus a critical component of the risk assessment process. Here, we used zero valent iron (ZVI) to initiate the reduction of the diazo compound dye Disperse Yellow 7 (DY 7). Using state-of-the-art accurate mass Liquid Chromatography-Quadrupole Time of Flight-Mass Spectroscopy (LC-QToF-MS), four transformation products were conclusively identified, while a fifth product was tentatively ascertained. The conclusively established transformation products included p-phenylenediamine (p-PDA, a known genotoxin), 4-aminoazobenzene (4-AAB, a category 2 carcinogen) and 4-aminobiphenyl (4-ABP, a category 1 human carcinogen). 4-ABP is thought to form via a benzidine rearrangement; this is the first report of DY 7 undergoing a benzidine rearrangement. Given the importance of reduction processes in the metabolism of organic contaminants by aquatic species, we used LC-MS/MS to analyze sediment samples that had been generated previously upon exposure of Western clawed frogs (Silurana tropicalis) to DY 7 (at exposure levels where cellular stress was observed in S. tropicalis). We found p-PDA, 4-AAB, and 4-ABP were present in all exposures, but not in any of the sediment controls, demonstrating that upon release of DY 7 to the aquatic environment, sediment dwelling organisms will metabolize DY 7 to generate known (and suspected) human carcinogens, including through a previously unreported in vivo benzidine rearrangement to produce 4-ABP.

Potent Targeting of the STAT3 Protein in Brain Cancer Stem Cells: A Promising Route for Treating Glioblastoma.

The STAT3 gene is abnormally active in glioblastoma (GBM) and is a critically important mediator of tumor growth and therapeutic resistance in GBM. Thus, for poorly treated brain cancers such as gliomas, astrocytomas, and glioblastomas, which harbor constitutively activated STAT3, a STAT3-targeting therapeutic will be of significant importance. Herein, we report a most potent, small molecule, nonphosphorylated STAT3 inhibitor, 31 (SH-4-54) that strongly binds to STAT3 protein (K D = 300 nM). Inhibitor 31 potently kills glioblastoma brain cancer stem cells (BTSCs) and effectively suppresses STAT3 phosphorylation and its downstream transcriptional targets at low nM concentrations. Moreover, in vivo, 31 exhibited blood-brain barrier permeability, potently controlled glioma tumor growth, and inhibited pSTAT3 in vivo. This work, for the first time, demonstrates the power of STAT3 inhibitors for the treatment of BTSCs and validates the therapeutic efficacy of a STAT3 inhibitor for GBM clinical application.

Amphiphilic micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol) for anti-cancer drug delivery to solid tumours.

Drug delivery to solid tumours remains a challenge because both tumour physiology and drug solubility are unfavourable. Engineered materials can provide the basis for drug reformulation, incorporating active compounds and modulating their pharmacokinetic and biodistribution behaviour. To this end, we encapsulated docetaxel, a poorly soluble taxane drug, in a self-assembled polymeric nanoparticle micelle of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol) (poly(TMCC-co-LA)-g-PEG). This formulation was compared with its conventional ethanolic polysorbate 80 formulation in terms of plasma circulation and biodistribution in an orthotopic mouse model of breast cancer. Notably, the polymeric nanoparticle formulation achieved greater tumour retention, resulting in prolonged exposure of cancer cells to the active drug. This behaviour was unique to the tumour tissue. The active drug was eliminated at equal or greater rates in all other tissues assayed when delivered in the polymeric nanoparticles vs. the free drug formulation. Thus, these polymeric nanoparticles are promising vehicles for solid tumour drug delivery applications, offering greater tumour exposure while eliminating the need for toxic solvents and surfactants in the dosing formulation.

Investigation of the noncovalent interactions between anti-amyloid agents and amyloid beta peptides by ESI-MS.

This paper describes an efficient and reproducible screening method for identifying low molecular weight compounds that bind to amyloid beta peptides (Abeta) peptides using electrospray ionization mass spectrometry (ESI-MS). Low molecular weight compounds capable of interacting with soluble Abeta may be able to modulate/inhibit the Abeta aggregation process and serve as potential disease-modifying agents for AD. The present approach was used to rank the binding affinity of a library of compounds to Abeta1-40 peptide. The results obtained show that low molecular weight compounds bind similarly to Abeta1-42, Abeta1-40, as well as Abeta1-28 peptides and they underline the critical role of Abeta peptide charge motif in binding at physiological pH. Finally, some elements of structure-activity relationship (SAR) involved in the binding affinity of homotaurine to soluble Abeta peptides are discussed.