High-Affinity Fluorogenic Substrate for Tissue Transglutaminase Reveals Enzymatic Hysteresis.

Transglutaminases (TGases) are a family of calcium-dependent enzymes primarily known for their ability to cross-link proteins. Transglutaminase 2 (TG2) is one isozyme in this family whose role is multifaceted. TG2 can act not only as a typical transamidase through its catalytic core but also as a G-protein via its GTP binding site. These two discrete activities are tightly regulated by both environmental stimuli and redox reactions. Ubiquitously expressed in humans, TG2 has been implicated in numerous disease pathologies that require extensive investigation. The catalytic activity of TG2 can be monitored through various mechanisms, including hydrolysis, transamidation, or cleavage of isopeptide bonds. Activity assays are required to monitor the activity of this isozyme not only for studying its transamidation reaction but also for validation of therapeutics designed to abolish this activity. Herein, we present the design, synthesis, and evaluation of a new TG2 activity substrate based on a previously optimized inhibitor scaffold. The substrate APH7 exhibits excellent affinity, selectivity, and reactivity with TG2 (KM = 3.0 µM). Furthermore, its application also allowed the discovery of unique hysteresis at play within the catalytic activity and inhibition reactivity of TG2.

The genetic authentication of Panax ginseng and Panax quinquefolius based on using single nucleotide polymorphism (SNP) conducted in a nucleic acid test chip.

Panax ginseng and Panax quinquefolius, which are commonly called Chinese ginseng and American ginseng respectively, have different medicinal properties and market values; however, these samples can be difficult to differentiate from one another based on physical appearances of the samples especially when they are in powdery or granular forms. A molecular technique is thus needed to overcome this difficulty; this technique is based on the nucleic acid test (NAT) conducted on the microfluidic chip surface. Three single nucleotide polymorphism (SNP) sites (i.e. N1, N2, N3) on the Panax genome that differ between P. ginseng (G) and P. quinquefolius (Q) have been selected to design probes for the NAT. Primers were designed to amplify the antisense strands by asymmetric PCR. We have developed three different NAT methodologies involving surface immobilization and subsequent (stop flow or dynamic) hybridization of probes (i.e. N1G, N1Q, N2G, N2Q, N3Q) to the antisense strands. These NAT methods consist of two steps, namely immobilization and hybridization, and each method is distinguished by what is immobilized on the microfluidic chip surface in the first step (i.e. probe, target or capture strand). These three NATs developed are called probe-target method 1, target-probe method 2 and three-strand complex method 3. Out of the three methods, it was found that the capture strand-target-probe method 3 provided the best differentiation of the ginseng species, in which a 3' NH2 capture strand is first immobilized and the antisense PCR strand is then bound, while N2G and N3Q probes are used for detection of P. ginseng (G) and P. quinquefolius (Q) respectively.

Enhanced Immunoassay Using a Rotating Paper Platform for Quantitative Determination of Low Abundance Protein Biomarkers.

The changing concentrations of circulating protein biomarkers have been correlated with a variety of diseases. Quantitative bioassays capable of sensitive and specific determination of protein biomarkers at low levels can be essential for therapeutic treatments that can improve outcomes for patients. Herein, we describe the investigation of a rotating paper device (RPD) for quantitative determination of targeted proteins at the fM concentration level. The RPD consists of two circular papers each separately supported with a plastic disc. Protein detection is conducted via enhanced immunoassay using amplification in a sequential workflow, which includes a sandwich immunoassay in the upper paper and a signal amplification reaction in the lower paper. The sandwich immunoassay is conducted using biobarcode nanoparticles (BNPs) and results in the release of reporter oligonucleotides from BNPs. These oligonucleotides are transferred to the bottom paper, where they engage in a target recycling methodology that leads to the production of a colorimetric signal. The assay was evaluated for quantitation of interleukin-6 (IL-6), a cytokine biomarker in serum. A limit of detection of 63 fM and a dynamic range of 200 fM-8 pM was observed for the assay. The specificity of the assay was successfully verified against several common protein biomarkers.

Inorganic Nanoparticles as Donors in Resonance Energy Transfer for Solid-Phase Bioassays and Biosensors.

Bioassays for the rapid detection and quantification of specific nucleic acids, proteins, and peptides are fundamental tools in many clinical settings. Traditional optical emission methods have focused on the use of molecular dyes as labels to track selective binding interactions and as probes that are sensitive to environmental changes. Such dyes can offer good detection limits based on brightness but typically have broad emission bands and suffer from time-dependent photobleaching. Inorganic nanoparticles such as quantum dots and upconversion nanoparticles are photostable over prolonged exposure to excitation radiation and tend to offer narrow emission bands, providing a greater opportunity for multiwavelength multiplexing. Importantly, in contrast to molecular dyes, nanoparticles offer substantial surface area and can serve as platforms to carry a large number of conjugated molecules. The surface chemistry of inorganic nanoparticles offers both challenges and opportunities for the control of solubility and functionality for selective molecular interactions by the assembly of coatings through coordination chemistry. This report reviews advances in the compositional design and methods of conjugation of inorganic quantum dots and upconversion nanoparticles and the assembly of combinations of nanoparticles to achieve energy exchange. Our interest is the exploration of configurations where the modified nanoparticles can be immobilized to solid substrates for the development of bioassays and biosensors that operate by resonance energy transfer (RET).

Rapid Immobilization of Oligonucleotides at High Density on Semiconductor Quantum Dots and Gold Nanoparticles.

Oligonucleotide-coated nanoparticles (NPs) have been used in numerous applications such as bioassays, as intracellular probes, and for drug delivery. One challenge that is confronted in the preparation of oligonucleotide-NP conjugates is derived from surface charge because nanoparticles are often stabilized and made water-soluble with a coating of negatively charged capping ligands. Therefore, an electrostatic repulsion is present when attempting to conjugate oligonucleotides. The result is that the conjugation can be a slow process, sometimes requiring 1 to 2 days to equilibrate at the highest surface density. The effect is compounded by electrostatic repulsion between neighboring oligonucleotide strands on the NP surfaces, which tends to lower the surface density. Herein, we report a novel method that enables conjugation in less than 1 min with a surface density of oligonucleotides up to the theoretical physical limit of occupancy. Negatively charged NPs are first adsorbed onto the surface of positively charged magnetic beads (MBs) to create MB-NP conjugates. Oligonucleotides are subsequently electrostatically adsorbed onto the MB surfaces when added to a suspension of MB-NP conjugates. This creates an oligonucleotide concentration 105 to 106 greater than in bulk solution in the vicinity of the nanoparticles, resulting in the promotion of the kinetics by over 1000-fold and achieving the maximum density possible for the conjugation reaction.

Kras gene codon 12 mutation detection enabled by gold nanoparticles conducted in a nanobioarray chip.

This study employs a nanobioarray (NBA) chip for multiple biodetection of single base pair mutations at the Kras gene codon 12. To distinguish between the mutant and wild-type target DNAs, current bioarray methods use high-temperature hybridization of the targets to the allele-specific probes. However, these techniques need prior temperature optimization and become harder to implement in the case of the detection of multiple mutations. We aimed to detect these mutations at a single temperature (room temperature), enabled by the use of gold nanoparticles (AuNPs) on the bioarray created within nanofluidic channels. In this method, a low amount of target oligonucleotides (5fmol) and polymerase chain reaction (PCR) products (300pg) were first loaded on the AuNP surface, and then these AuNP-bound targets were introduced into the channels of a polydimethylsiloxane (PDMS) glass chip. The targets hybridized to their complementary probes at the intersection of the target channels to the pre-printed oligonucleotide probe lines on the glass surface, creating a bioarray. Using this technique, fast and high-throughput multiple discrimination of the Kras gene codon 12 were achieved at room temperature using the NBA chip, and the specificity of the method was proved to be as high as that with the temperature stringency method.

De novo GTP synthesis is a metabolic vulnerability for the interception of brain metastases.

Patients with brain metastases (BM) face a 90% mortality rate within one year of diagnosis and the current standard of care is palliative. Targeting BM-initiating cells (BMICs) is a feasible strategy to treat BM, but druggable targets are limited. Here, we apply Connectivity Map analysis to lung-, breast-, and melanoma-pre-metastatic BMIC gene expression signatures and identify inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo GTP synthesis pathway, as a target for BM. We show that pharmacological and genetic perturbation of IMPDH attenuates BMIC proliferation in vitro and the formation of BM in vivo. Metabolomic analyses and CRISPR knockout studies confirm that de novo GTP synthesis is a potent metabolic vulnerability in BM. Overall, our work employs a phenotype-guided therapeutic strategy to uncover IMPDH as a relevant target for attenuating BM outgrowth, which may provide an alternative treatment strategy for patients who are otherwise limited to palliation.

Evaluation of Small-Molecule HDAC Inhibitors Through In Vitro and In Cellulo Approaches.

The aberrant activity of histone deacetylases (HDACs) across a broad range of cancers and other disease indications has led to the development of small-molecule inhibitors that target one or more members of the HDAC protein family. Emerging HDAC inhibitors that show promise in drug discovery programs must be assessed across a range of in vitro assays to establish an inhibitor profile for potency and cellular selectivity towards target HDAC(s) as well as preliminary absorption, distribution, metabolism, and excretion (ADME) features. Here we provide an overview of methods to determine a subset of pivotal in vitro drug-like parameters for HDAC inhibitors (HDACi). We initially describe protocols for parallel artificial membrane permeability assays (PAMPA) to evaluate the passive permeability of small molecules across lipid membranes. Subsequently, we elaborate on cytotoxicity assays using CellTiter-Blue to determine HDACi-induced cell death in healthy/diseased cellular models. We next focus on assessing the target engagement of inhibitors with the appropriate HDAC isoforms in a cellular environment via Western blotting of acetylated HDAC substrates. Finally, we provide detailed guidelines on how to assess the metabolic stability of HDACi through whole blood stability assays. Collectively, these assays provide an overview of the permeability, selectivity, and stability of the HDAC inhibitor under development.

Discovery of YSR734: A Covalent HDAC Inhibitor with Cellular Activity in Acute Myeloid Leukemia and Duchenne Muscular Dystrophy.

Histone deacetylases (HDACs) have emerged as powerful epigenetic modifiers of histone/non-histone proteins via catalyzing the deacetylation of ε-N-acetyl lysines. The dysregulated activity of these Zn2+-dependent hydrolases has been broadly implicated in disease, notably cancer. Clinically, the recurring dose-limiting toxicities of first-generation HDACi sparked a paradigm shift toward safer isoform-specific molecules. With pervasive roles in aggressive diseases, there remains a need for novel approaches to target these enzymes. Herein, we report the discovery of YSR734, a first-in-class covalent HDACi, with a 2-aminobenzanilide Zn2+ chelate and a pentafluorobenzenesulfonamide electrophile. This class I selective proof of concept modified HDAC2Cys274 (catalytic domain), with nM potency against HDAC1-3, sub-µM activity in MV4-11 cells, and limited cytotoxicity in MRC-9 fibroblasts. In C2C12 myoblasts, YSR734 activated muscle-specific biomarkers myogenin/Cav3, causing potent differentiation into myotubes (applications in Duchenne Muscular Dystrophy). Current efforts are focused on improving in vivo ADME toward a preclinical covalent HDACi.

Phenotypic Screening of Histone Deacetylase (HDAC) Inhibitors against Schistosoma mansoni.

Schistosomiasis is a prevalent yet neglected tropical parasitic disease caused by the Schistosoma genus of blood flukes. Praziquantel is the only currently available treatment, hence drug resistance poses a major threat. Recently, histone deacetylase 8 (HDAC8) selective inhibitors have been proposed as a viable treatment for schistosomiasis. Herein, we report the phenotypic screening of a focused library of small molecules of varying HDAC isozyme-inhibition profiles, including eight HDAC8 inhibitors with >10-fold selectivity in comparable functional inhibition assays and IC50 values against HDAC8<100 nM. HDAC8-selective inhibitors showed the lowest potency against Schistosoma mansoni newly transformed schistosomula (NTS). Pan-HDAC inhibitors MMH258, MMH259, and MMH373, as assessed by functional inhibition assays, with minimal or no-observed hHDAC8 and SmHDAC8 activities, were active against both NTS (MMH258, IC50 =1.5 µM; MMH259, IC50 =2.3 µM) and adult S. mansoni (MMH258, IC50 =2.1 µM; MMH373, IC50 =3.4 µM). Our results indicate that neither hHDAC8 nor SmHDAC8 activity were directly correlated to their NTS and adult S. mansoni activities.

High Efficacy and Drug Synergy of HDAC6-Selective Inhibitor NN-429 in Natural Killer (NK)/T-Cell Lymphoma.

NK/T-cell lymphoma (NKTCL) and γδ T-cell non-Hodgkin lymphomas (γδ T-NHL) are highly aggressive lymphomas that lack rationally designed therapies and rely on repurposed chemotherapeutics from other hematological cancers. Histone deacetylases (HDACs) have been targeted in a range of malignancies, including T-cell lymphomas. This study represents exploratory findings of HDAC6 inhibition in NKTCL and γδ T-NHL through a second-generation inhibitor NN-429. With nanomolar in vitro HDAC6 potency and high in vitro and in cellulo selectivity for HDAC6, NN-429 also exhibited long residence time and improved pharmacokinetic properties in contrast to older generation inhibitors. Following unique selective cytotoxicity towards γδ T-NHL and NKTCL, NN-429 demonstrated a synergistic relationship with the clinical agent etoposide and potential synergies with doxorubicin, cytarabine, and SNS-032 in these disease models, opening an avenue for combination treatment strategies.

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.

Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma.

Leukemic cutaneous T-cell lymphomas (L-CTCL) are lymphoproliferative disorders of skin-homing mature T-cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L-CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L-CTCL, which correlated with the increased clonal T-cell count in the blood. Dual inhibition of STAT3/5 using small-molecule degraders and multi-kinase blockers abolished L-CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L-CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti-leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L-CTCL.

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.

Multifunctional Droplet Microfluidic Platform for Rapid Immobilization of Oligonucleotides on Semiconductor Quantum Dots.

Quantum dot-DNA oligonucleotide (QD-DNA) conjugates have been used in many fields such as nucleic acid bioassays, intracellular probes, and drug delivery systems. A typical solid-phase method that achieves rapid loading of oligonucleotides on surfaces of QDs involves a two-step reaction and is performed in a batch-based approach. In contrast, droplet microfluidics offers many advantages that are unavailable when using batch processing, providing rapid and dense immobilized DNA oligonucleotides on QDs. The presented droplet microfluidic approach allows high-quality QD-DNA conjugates to be produced using one single device, which is designed to have two droplet generators, one droplet merger, and one mixer. One of the droplet generators coencapsulates QDs and magnetic beads (MBs) into nanoliter-sized droplets for the production of QD-MB conjugates and the other encapsulates oligonucleotides in nanoliter-sized droplets. These two streams of droplets then merge at a one-to-one ratio in a chamber. The merged droplets travel along the mixer, which is a serpentine microchannel with 30 turns, resulting in QD-DNA conjugation structures of high quality. This multifunctional microfluidic device provides advantages such as higher degree of control over the reaction conditions, minimized cross-contamination and impurities, and reduction of reagent consumption while eliminating any need for external vortexing and pipetting. To evaluate the quality of the QD-DNA conjugates, they were used as Forster resonance energy transfer (FRET) probes to quantify oligonucleic targets.

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.

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.

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.

Enzymatic amplification of oligonucleotides in paper substrates.

Several solution-based methods have recently been adapted for use in paper substrates for enzymatic amplification to increase the number of copies of DNA sequences. There is limited information available about the impact of a paper matrix on DNA amplification by enzymatic processes, and about how to optimize conditions to maximize yields. The work reported herein provides insights about the impact of physicochemical properties of a paper matrix, using nuclease-assisted amplification by exonuclease III and nicking endonuclease Nt. Bbv, and a quantum dot (QD) - based Forster Resonance Energy Transfer (FRET) assay to monitor the extent of amplification. The influence of several properties of paper on amplification efficiency and kinetics were investigated, such as surface adsorption of reactants, and pore size. Additional factors that impact amplification processes such as target length and the packing density of oligonucleotide probes on the nanoparticle surfaces were also studied. The work provides guidance for development of more efficient enzymatic target-recycling DNA amplification methods in paper substrates.