Lipophilicity of the Cystic Fibrosis Drug, Ivacaftor (VX-770), and Its Destabilizing Effect on the Major CF-causing Mutation: F508del.

Deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cystic fibrosis (CF)-causing mutation. Recently, ORKAMBI, a combination therapy that includes a corrector of the processing defect of F508del-CFTR (lumacaftor or VX-809) and a potentiator of channel activity (ivacaftor or VX-770), was approved for CF patients homozygous for this mutation. However, clinical studies revealed that the effect of ORKAMBI on lung function is modest and it was proposed that this modest effect relates to a negative impact of VX-770 on the stability of F508del-CFTR. In the current studies, we showed that this negative effect of VX-770 at 10 µM correlated with its inhibitory effect on VX-809-mediated correction of the interface between the second membrane spanning domain and the first nucleotide binding domain bearing F508del. Interestingly, we found that VX-770 exerted a similar negative effect on the stability of other membrane localized solute carriers (SLC26A3, SLC26A9, and SLC6A14), suggesting that this negative effect is not specific for F508del-CFTR. We determined that the relative destabilizing effect of a panel of VX-770 derivatives on F508del-CFTR correlated with their predicted lipophilicity. Polarized total internal reflection fluorescence microscopy on a supported lipid bilayer model shows that VX-770, and not its less lipophilic derivative, increased the fluidity of and reorganized the membrane. In summary, our findings show that there is a potential for nonspecific effects of VX-770 on the lipid bilayer and suggest that this effect may account for its destabilizing effect on VX-809- rescued F508del-CFTR.

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.

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.

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.

Development of HDAC Inhibitors Exhibiting Therapeutic Potential in T-Cell Prolymphocytic Leukemia.

Epigenetic targeting has emerged as an efficacious therapy for hematological cancers. The rare and incurable T-cell prolymphocytic leukemia (T-PLL) is known for its aggressive clinical course. Current epigenetic agents such as histone deacetylase (HDAC) inhibitors are increasingly used for targeted therapy. Through a structure-activity relationship (SAR) study, we developed an HDAC6 inhibitor KT-531, which exhibited higher potency in T-PLL compared to other hematological cancers. KT-531 displayed strong HDAC6 inhibitory potency and selectivity, on-target biological activity, and a safe therapeutic window in nontransformed cell lines. In primary T-PLL patient cells, where HDAC6 was found to be overexpressed, KT-531 exhibited strong biological responses, and safety in healthy donor samples. Notably, combination studies in T-PLL patient samples demonstrated KT-531 synergizes with approved cancer drugs, bendamustine, idasanutlin, and venetoclax. Our work suggests HDAC inhibition in T-PLL could afford sufficient therapeutic windows to achieve durable remission either as stand-alone or in combination with targeted drugs.

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.

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.

A functional in vitro assay for screening inhibitors of STAT5B phosphorylation.

Inhibition of STAT phosphorylation is recognized as a viable therapeutic strategy for disrupting tumorigenesis. Constitutive STAT phosphorylation is found with high frequency in a number of primary tumor types, while non-cancer cells exhibit low basal activity, providing an exploitable therapeutic window. STAT activation involves phosphorylation of the SH2 domain by a number of tyrosine kinases followed by STAT dimerization and translocation to the nucleus. By blocking the cognate binding site, STAT SH2-domain inhibitors can impede kinase-mediated de novo STAT phosphorylation. Assessing for inhibitors of STAT phosphorylation has previously been conducted exclusively in cellulo using Western blot analysis. However, while providing useful in cellulo efficacy, it is not possible to conclude that inhibition is due to a direct blockade of STAT protein. Here we developed a functional assay that directly reports the blockade of phosphorylation as a result of inhibitor interaction with STAT proteins. We have optimized reaction conditions for the functional assay and validated the assay against known STAT5B ligands, including peptides and small molecule inhibitors. As part of the study, we have also identified several sites of STAT5B phosphorylation by Abl kinase. This assay will serve to delineate the functional mechanism of STAT binders in vitro and deconvolute the mechanism of phospho-STAT inhibition observed in Western blot analysis.