Discovery of HDAC6-Selective Inhibitor NN-390 with in Vitro Efficacy in Group 3 Medulloblastoma.

Histone deacetylase 6 (HDAC6) has been targeted in clinical studies for anticancer effects due to its role in oncogenic transformation and metastasis. Through a second-generation structure-activity relationship (SAR) study, the design, and biological evaluation of the selective HDAC6 inhibitor NN-390 is reported. With nanomolar HDAC6 potency, >200-550-fold selectivity for HDAC6 in analogous HDAC isoform functional assays, potent intracellular target engagement, and robust cellular efficacy in cancer cell lines, NN-390 is the first HDAC6-selective inhibitor to show therapeutic potential in metastatic Group 3 medulloblastoma (MB), an aggressive pediatric brain tumor often associated with leptomeningeal metastases and therapy resistance. MB stem cells contribute to these patients' poor clinical outcomes. NN-390 selectively targets this cell population with a 44.3-fold therapeutic margin between patient-derived Group 3 MB cells in comparison to healthy neural stem cells. NN-390 demonstrated a 45-fold increased potency over HDAC6-selective clinical candidate citarinostat. In summary, HDAC6-selective molecules demonstrated in vitro therapeutic potential against Group 3 MB.

Optical chemosensors for the detection of proximally phosphorylated peptides and proteins.

Proximal multi-site phosphorylation is a critical post-translational modification in protein biology. The additive effects of multiple phosphosite clusters in close spatial proximity triggers integrative and cooperative effects on protein conformation and activity. Proximal phosphorylation has been shown to modulate signal transduction pathways and gene expression, and as a result, is implicated in a broad range of disease states through altered protein function and/or localization including enzyme overactivation or protein aggregation. The role of proximal multi-phosphorylation events is becoming increasingly recognized as mechanistically important, although breakthroughs are limited due to a lack of detection technologies. To date, there is a limited selection of facile and robust sensing tools for proximal phosphorylation. Nonetheless, there have been considerable efforts in developing optical chemosensors for the detection of proximal phosphorylation motifs on peptides and proteins in recent years. This review provides a comprehensive overview of optical chemosensors for proximal phosphorylation, with the majority of work being reported in the past two decades. Optical sensors, in the form of fluorescent and luminescent chemosensors, hybrid biosensors, and inorganic nanoparticles, are described. Emphasis is placed on the rationale behind sensor scaffolds, relevant protein motifs, and applications in protein biology.

Unique Molecular Interaction with the Histone Deacetylase 6 Catalytic Tunnel: Crystallographic and Biological Characterization of a Model Chemotype.

Histone deacetylase 6 (HDAC6) is involved in multiple regulatory processes, ranging from cellular stress to intracellular transport. Inhibition of aberrant HDAC6 activity in several cancers and neurological diseases has been shown to be efficacious in both preclinical and clinical studies. While selective HDAC6 targeting has been pursued as an alternative to pan-HDAC drugs, identifying truly selective molecular templates has not been trivial. Herein, we report a structure-activity relationship study yielding TO-317, which potently binds HDAC6 catalytic domain 2 (Ki = 0.7 nM) and inhibits the enzyme function (IC50 = 2 nM). TO-317 exhibits 158-fold selectivity for HDAC6 over other HDAC isozymes by binding the catalytic Zn2+ and, uniquely, making a never seen before direct hydrogen bond with the Zn2+ coordinating residue, His614. This novel structural motif targeting the second-sphere His614 interaction, observed in a 1.84 Å resolution crystal structure with drHDAC6 from zebrafish, can provide new pharmacophores for identifying enthalpically driven, high-affinity, HDAC6-selective inhibitors.

Sensitive Detection of Broad-Spectrum Bacteria with Small-Molecule Fluorescent Excimer Chemosensors.

Antibiotic resistance is a major problem for world health, triggered by the unnecessary usage of broad-spectrum antibiotics on purportedly infected patients. Current clinical standards require lengthy protocols for the detection of bacterial species in sterile physiological fluids. In this work, a class of small-molecule fluorescent chemosensors termed ProxyPhos was shown to be capable of rapid, sensitive, and facile detection of broad-spectrum bacteria. The sensors act via a turn-on fluorescent excimer mechanism, where close-proximity binding of multiple sensor units amplifies a red shift emission signal. ProxyPhos sensors were able to detect down to 10 CFUs of model strains by flow cytometry assays and showed selectivity over mammalian cells in a bacterial coculture through fluorescence microscopy. The studies reveal that the zinc(II)-chelates cyclen and cyclam are novel and effective binding units for the detection of both Gram-negative and Gram-positive bacterial strains. Mode of action studies revealed that the chemosensors detect Gram-negative and Gram-positive strains with two distinct mechanisms. Preliminary studies applying ProxyPhos sensors to sterile physiological fluids (cerebrospinal fluid) in flow cytometry assays were successful. The results suggest that ProxyPhos sensors can be developed as a rapid, inexpensive, and robust tool for the "yes-no" detection of broad-spectrum bacteria in sterile fluids.

Characterization of Conformationally Constrained Benzanilide Scaffolds for Potent and Selective HDAC8 Targeting.

Histone deacetylases (HDACs) are an attractive therapeutic target for a variety of human diseases. Currently, all four FDA-approved HDAC-targeting drugs are nonselective, pan-HDAC inhibitors, exhibiting adverse side effects at therapeutic doses. Although selective HDAC inhibition has been proposed to mitigate toxicity, the targeted catalytic domains are highly conserved. Herein, we describe a series of rationally designed, conformationally constrained, benzanilide foldamers which selectively bind the catalytic tunnel of HDAC8. The series includes benzanilides, MMH371, MMH409, and MMH410, which exhibit potent in vitro HDAC8 activity (IC50 = 66, 23, and 66 nM, respectively) and up to 410-fold selectivity for HDAC8 over the next targeted HDAC. Experimental and computational analyses of the benzanilide structure docked with human HDAC8 enzyme showed the adoption of a low-energy L-shaped conformer that favors HDAC8 selectivity. The conformationally constrained HDAC8 inhibitors present an alternative biological probe for further determining the clinical utility and safety of pharmacological knockdown of HDAC8 in diseased cells.

PTG-0861: A novel HDAC6-selective inhibitor as a therapeutic strategy in acute myeloid leukaemia.

Dysregulated Histone Deacetylase (HDAC) activity across multiple human pathologies have highlighted this family of epigenetic enzymes as critical druggable targets, amenable to small molecule intervention. While efficacious, current approaches using non-selective HDAC inhibitors (HDACi) have been shown to cause a range of undesirable clinical toxicities. To circumvent this, recent efforts have focused on the design of highly selective HDACi as a novel therapeutic strategy. Beyond roles in regulating transcription, the unique HDAC6 (with two catalytic domains) regulates the deacetylation of α-tubulin; promoting growth factor-controlled cell motility, cell division, and metastatic hallmarks. Recent studies have linked aberrant HDAC6 function in various hematological cancers including acute myeloid leukaemia and multiple myeloma. Herein, we report the discovery, in vitro characterization, and biological evaluation of PTG-0861 (JG-265), a novel HDAC6-selective inhibitor with strong isozyme-selectivity (∼36× ) and low nanomolar potency (IC50 = 5.92 nM) against HDAC6. This selectivity profile was rationalized via in silico docking studies and also observed in cellulo through cellular target engagement. Moreover, PTG-0861 achieved relevant potency against several blood cancer cell lines (e.g. MV4-11, MM1S), whilst showing limited cytotoxicity against non-malignant cells (e.g. NHF, HUVEC) and CD-1 mice. In examining compound stability and cellular permeability, PTG-0861 revealed a promising in vitro pharmacokinetic (PK) profile. Altogether, in this study we identified a novel and potent HDAC6-selective inhibitor (∼4× more selective than current clinical standards - citarinostat, ricolinostat), which achieves cellular target engagement, efficacy in hematological cancer cells with a promising safety profile and in vitro PK.

Optimization of a high-throughput fluorescence polarization assay for STAT5B DNA binding domain-targeting inhibitors.

Signal transducer and activator of transcription 5B (STAT5B) is constitutively activated in multiple cancers as a result of hyperactivating mutations or dysregulation of upstream effectors. Therapeutic strategies have predominantly targeted the Src homology 2 (SH2) domain to inhibit STAT phosphorylation, a prerequisite for STAT5B transcriptional activation. An alternative approach for STAT5B pharmacologic inhibition involves targeting the DNA-binding domain (DBD). However, this strategy remains relatively unexplored and is further hindered by the lack of a high-throughput in vitro engagement assay. Herein, we present the development and optimization of a STAT5B DBD fluorescence polarization (FP) assay, which facilitates rapid screening of small molecules targeting the STAT5B DBD though displacement of a fluorescently labelled oligonucleotide. The assay can generate a complete DNA-binding profile in 10 min, with signal stability up to 2 h, and minimal changes under a range of conditions including 10 % (v/v) glycerol, 15 % (v/v) DMSO, 1 mM NaCl, 0.02 % (w/v) BSA, and 1 mM EDTA. This assay is compatible with both unphosphorylated and phosphorylated STAT5B and demonstrates suitability for high-throughput screening with a Z' factor of 0.68 ±â€¯0.07 and a signal to noise ratio of 6.7 ±â€¯0.84.

Class I/IIb-Selective HDAC Inhibitor Exhibits Oral Bioavailability and Therapeutic Efficacy in Acute Myeloid Leukemia.

The HDAC inhibitor 4-tert-butyl-N-(4-(hydroxycarbamoyl)phenyl)benzamide (AES-350, 51) was identified as a promising preclinical candidate for the treatment of acute myeloid leukemia (AML), an aggressive malignancy with a meagre 24% 5-year survival rate. Through screening of low-molecular-weight analogues derived from the previously discovered novel HDAC inhibitor, AES-135, compound 51 demonstrated greater HDAC isoform selectivity, higher cytotoxicity in MV4-11 cells, an improved therapeutic window, and more efficient absorption through cellular and lipid membranes. Compound 51 also demonstrated improved oral bioavailability compared to SAHA in mouse models. A broad spectrum of experiments, including FACS, ELISA, and Western blotting, were performed to support our hypothesis that 51 dose-dependently triggers apoptosis in AML cells through HDAC inhibition.

ProxyPhos sensors for the detection of negatively charged membranes.

Membrane-embedded negatively charged phospholipids (MENCP) can be used as biomarkers for a range of biological processes, including early detection of apoptosis in animal cells, drug-induced phospholipidosis, and selective detection of bacterial over animal cells. Currently, several technologies for the detection of apoptosis and bacterial cells are based on the recognition of MENCPs, including the AnnexinV stain and PSVue™ probes. As probes, these technologies have limitations, the most significant of which is the need for washing the unbound probe away to achieve optimal signal. In contrast, a turn-on chemosensor selective for MENCP would address this shortcoming, and allow for a more rapid protocol for the detection of apoptosis, bacteria and for other relevant applications. In this work, the aim was to explore whether ProxyPhos chemosensors, previously reported by our group for the detection of proximally phosphorylated peptides and proteins, could be re-purposed for the detection of MENCPs. Six lead ProxyPhos sensors were screened against synthetic vesicles containing biologically relevant negatively charged phospholipids including phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS). Through these screens, ProxyPhos sensors exhibiting high selectivity for the detection of MENCPs over zwitterionic lipids were identified. Particular selectivity was observed for PA and CL. Sensitivity of the lead sensors for MENCPs was suitable for the detection of apoptosis: ProxyPhos detected vesicles containing as little as 2.5% PS and detected camptothecin-induced apoptosis in mammalian cells in flow cytometry experiments. The results suggest that ProxyPhos sensors can be used for the detection of MENCPs in synthetic vesicles and live mammalian cells.