Strategy, Design, and Fabrication of Electrochemical Biosensors: A Tutorial.

Advanced healthcare requires novel technologies capable of real-time sensing to monitor acute and long-term health. The challenge relies on converting a real-time quantitative biological and chemical signal into a desired measurable output. Given the success in detecting glucose and the commercialization of glucometers, electrochemical biosensors continue to be a mainstay of academic and industrial research activities. Despite the wealth of literature on electrochemical biosensors, reports are often specific to a particular application (e.g., pathogens, cancer markers, glucose, etc.), and most fail to convey the underlying strategy and design, and if it is transferable to detection of a different analyte. Here we present a tutorial review for those entering this research area that summarizes the basic electrochemical techniques utilized as well as discusses the designs and optimization strategies employed to improve sensitivity and maximize signal output.

Discovery of pyrazolopyrrolidinones as potent, broad-spectrum inhibitors of Leishmania infection.

Introduction: Leishmaniasis is a parasitic disease that affects more than 1 million people worldwide annually, predominantly in resource-limited settings. The challenge in compound development is to exhibit potent activity against the intracellular stage of the parasite (the stage present in the mammalian host) without harming the infected host cells. We have identified a compound series (pyrazolopyrrolidinones) active against the intracellular parasites of Leishmania donovani and L. major; the causative agents of visceral and cutaneous leishmaniasis in the Old World, respectively. Methods: In this study, we performed medicinal chemistry on a newly discovered antileishmanial chemotype, with over 100 analogs tested. Studies included assessments of antileishmanial potency, toxicity towards host cells, and in vitro ADME screening of key drug properties. Results and discussion: Members of the series showed high potency against the deadliest form, visceral leishmaniasis (approximate EC50 ≥ 0.01 µM without harming the host macrophage up to 10.0 µM). In comparison, the most efficient monotherapy treatment for visceral leishmaniasis is amphotericin B, which presents similar activity in the same assay (EC50 = 0.2 µM) while being cytotoxic to the host cell at 5.0 µM. Continued development of this compound series with the Discovery Partnership with Academia (DPAc) program at the GlaxoSmithKline Diseases of the Developing World (GSK DDW) laboratories found that the compounds passed all of GSK's criteria to be defined as a potential lead drug series for leishmaniasis. Conclusion: Here, we describe preliminary structure-activity relationships for antileishmanial pyrazolopyrrolidinones, and our progress towards the identification of candidates for future in vivo assays in models of visceral and cutaneous leishmaniasis.

Identification of Small Molecules with Improved Potency against Orthopoxviruses from Vaccinia to Smallpox.

The genus Orthopoxvirus contains several human pathogens, including vaccinia, monkeypox, cowpox, and variola virus, the causative agent of smallpox. Although there are a few effective vaccines, widespread prophylactic vaccination has ceased and is unlikely to resume, making therapeutics increasingly important to treat poxvirus disease. Here, we described efforts to improve the potency of the anti-poxvirus small molecule CMLDBU6128. This class of small molecules, referred to as pyridopyrimidinones (PDPMs), showed a wide range of biological activities. Through the synthesis and testing of several exploratory chemical libraries based on this molecule, we identified several compounds that had increased potency from the micromolar into the nanomolar range. Two compounds, designated (12) and (16), showed inhibitory concentrations of 326 nM and 101 nM, respectively, which was more than a 10-fold increase in potency to CMLDBU6128 with an inhibitory concentration of around 6 µM. We also expanded our investigation of the breadth of action of these molecules and showed that they can inhibit the replication of variola virus, a related orthopoxvirus. Together, these findings highlighted the promise of this new class of antipoxviral agents as broad-spectrum small molecules with significant potential to be developed as antiviral therapy. This would add a small molecule option for therapy of spreading diseases, including monkeypox and cowpox viruses, that would also be expected to have efficacy against smallpox.

Pharmacologic Manipulation of Late SV40 Factor Suppresses Wnt Signaling and Inhibits Growth of Allogeneic and Syngeneic Colon Cancer Xenografts.

Aberrant hyperactivation of Wnt signaling, driven by nuclear ß-catenin in the colonic epithelium, represents the seminal event in the initiation and progression of colorectal cancer (CRC). Despite its established role in CRC tumorigenesis, clinical translation of Wnt inhibitors remains unsuccessful. Late SV40 factor (LSF; encoded by TFCP2) is a transcription factor and a potent oncogene. The current study identified a chemotype, named factor quinolinone inhibitors (FQIs), that specifically inhibits LSF DNA-binding, partner protein-binding, and transactivation activities. The role of LSF and FQIs in CRC tumor growth was examined. Herein, the study showed that LSF and ß-catenin interacted in several CRC cell lines irrespective of their mutational profile, which was disrupted by FQI2-34. FQI2-34 suppressed Wnt activity in CRC cells in a dose-dependent manner. Leveraging both allogeneic and syngeneic xenograft models showed that FQI2-34 suppressed CRC tumor growth, significantly reduced nuclear ß-catenin, and down-regulated Wnt targets such as axis inhibition protein 2 (AXIN-2) and SRY-box transcription factor 9, in the xenograft cells. FQI2-34 suppressed the proliferation of xenograft cells. Adenocarcinomas from a series of stage IV CRC patients revealed a positive correlation between LSF expression and Wnt targets (AXIN-2 and SRY-box transcription factor 9) within the CRC cells. Collectively, this study uncovers the Wnt inhibitory and CRC growth-suppressive effects of these LSF inhibitors in CRC cells, revealing a novel target in CRC therapeutics.

Factor quinolinone inhibitors disrupt spindles and multiple LSF (TFCP2)-protein interactions in mitosis, including with microtubule-associated proteins.

Factor quinolinone inhibitors (FQIs), a first-in-class set of small molecule inhibitors targeted to the transcription factor LSF (TFCP2), exhibit promising cancer chemotherapeutic properties. FQI1, the initial lead compound identified, unexpectedly induced a concentration-dependent delay in mitotic progression. Here, we show that FQI1 can rapidly and reversibly lead to mitotic arrest, even when added directly to mitotic cells, implying that FQI1-mediated mitotic defects are not transcriptionally based. Furthermore, treatment with FQIs resulted in a striking, concentration-dependent diminishment of spindle microtubules, accompanied by a concentration-dependent increase in multi-aster formation. Aberrant γ-tubulin localization was also observed. These phenotypes suggest that perturbation of spindle microtubules is the primary event leading to the mitotic delays upon FQI1 treatment. Previously, FQIs were shown to specifically inhibit not only LSF DNA-binding activity, which requires LSF oligomerization to tetramers, but also other specific LSF-protein interactions. Other transcription factors participate in mitosis through non-transcriptional means, and we recently reported that LSF directly binds α-tubulin and is present in purified cellular tubulin preparations. Consistent with a microtubule role for LSF, here we show that LSF enhanced the rate of tubulin polymerization in vitro, and FQI1 inhibited such polymerization. To probe whether the FQI1-mediated spindle abnormalities could result from inhibition of mitotic LSF-protein interactions, mass spectrometry was performed using as bait an inducible, tagged form of LSF that is biotinylated by endogenous enzymes. The global proteomics analysis yielded expected associations for a transcription factor, notably with RNA processing machinery, but also to nontranscriptional components. In particular, and consistent with spindle disruption due to FQI treatment, mitotic, FQI1-sensitive interactions were identified between the biotinylated LSF and microtubule-associated proteins that regulate spindle assembly, positioning, and dynamics, as well as centrosome-associated proteins. Probing the mitotic LSF interactome using small molecule inhibitors therefore supported a non-transcriptional role for LSF in mediating progression through mitosis.

Factor quinolinone inhibitors alter cell morphology and motility by destabilizing interphase microtubules.

Factor quinolinone inhibitors are promising anti-cancer compounds, initially characterized as specific inhibitors of the oncogenic transcription factor LSF (TFCP2). These compounds exert anti-proliferative activity at least in part by disrupting mitotic spindles. Herein, we report additional interphase consequences of the initial lead compound, FQI1, in two telomerase immortalized cell lines. Within minutes of FQI1 addition, the microtubule network is disrupted, resulting in a substantial, although not complete, depletion of microtubules as evidenced both by microtubule sedimentation assays and microscopy. Surprisingly, this microtubule breakdown is quickly followed by an increase in tubulin acetylation in the remaining microtubules. The sudden breakdown and partial depolymerization of the microtubule network precedes FQI1-induced morphological changes. These involve rapid reduction of cell spreading of interphase fetal hepatocytes and increase in circularity of retinal pigment epithelial cells. Microtubule depolymerization gives rise to FH-B cell compaction, as pretreatment with taxol prevents this morphological change. Finally, FQI1 decreases the rate and range of locomotion of interphase cells, supporting an impact of FQI1-induced microtubule breakdown on cell motility. Taken together, our results show that FQI1 interferes with microtubule-associated functions in interphase, specifically cell morphology and motility.

Targeting the oncogene LSF with either the small molecule inhibitor FQI1 or siRNA causes mitotic delays with unaligned chromosomes, resulting in cell death or senescence.

BACKGROUND: The oncogene LSF (encoded by TFCP2) has been proposed as a novel therapeutic target for multiple cancers. LSF overexpression in patient tumors correlates with poor prognosis in particular for both hepatocellular carcinoma and colorectal cancer. The limited treatment outcomes for these diseases and disappointing clinical results, in particular, for hepatocellular carcinoma in molecularly targeted therapies targeting cellular receptors and kinases, underscore the need for molecularly targeting novel mechanisms. LSF small molecule inhibitors, Factor Quinolinone Inhibitors (FQIs), have exhibited robust anti-tumor activity in multiple pre-clinical models, with no observable toxicity. METHODS: To understand how the LSF inhibitors impact cancer cell proliferation, we characterized the cellular phenotypes that result from loss of LSF activity. Cell proliferation and cell cycle progression were analyzed, using HeLa cells as a model cancer cell line responsive to FQI1. Cell cycle progression was studied either by time lapse microscopy or by bulk synchronization of cell populations to ensure accuracy in interpretation of the outcomes. In order to test for biological specificity of targeting LSF by FQI1, results were compared after treatment with either FQI1 or siRNA targeting LSF. RESULTS: Highly similar cellular phenotypes are observed upon treatments with FQI1 and siRNA targeting LSF. Along with similar effects on two cellular biomarkers, inhibition of LSF activity by either mechanism induced a strong delay or arrest prior to metaphase as cells progressed through mitosis, with condensed, but unaligned, chromosomes. This mitotic disruption in both cases resulted in improper cellular division leading to multiple outcomes: multi-nucleation, apoptosis, and cellular senescence. CONCLUSIONS: These data strongly support that cellular phenotypes observed upon FQI1 treatment are due specifically to the loss of LSF activity. Specific inhibition of LSF by either small molecules or siRNA results in severe mitotic defects, leading to cell death or senescence - consequences that are desirable in combating cancer. Taken together, these findings confirm that LSF is a promising target for cancer treatment. Furthermore, this study provides further support for developing FQIs or other LSF inhibitory strategies as treatment for LSF-related cancers with high unmet medical needs.

The microtubule-associated histone methyltransferase SET8, facilitated by transcription factor LSF, methylates α-tubulin.

Microtubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics. Although many of these modifications have been investigated, only one prior study reports tubulin methylation and an enzyme responsible for this methylation. Here we used in vitro radiolabeling, MS, and immunoblotting approaches to monitor protein methylation and immunoprecipitation, immunofluorescence, and pulldown approaches to measure protein-protein interactions. We demonstrate that N-lysine methyltransferase 5A (KMT5A or SET8/PR-Set7), which methylates lysine 20 in histone H4, bound α-tubulin and methylated it at a specific lysine residue, Lys311 Furthermore, late SV40 factor (LSF)/CP2, a known transcription factor, bound both α-tubulin and SET8 and enhanced SET8-mediated α-tubulin methylation in vitro In addition, we found that the ability of LSF to facilitate this methylation is countered by factor quinolinone inhibitor 1 (FQI1), a specific small-molecule inhibitor of LSF. These findings suggest the general model that microtubule-associated proteins, including transcription factors, recruit or stimulate protein-modifying enzymes to target tubulins. Moreover, our results point to dual functions for SET8 and LSF not only in chromatin regulation but also in cytoskeletal modification.

Expansile Nanoparticles Encapsulate Factor Quinolinone Inhibitor 1 and Accumulate in Murine Liver upon Intravenous Administration.

Expansile nanoparticles (eNPs) are a promising pH-responsive polymeric drug delivery vehicle, as demonstrated in multiple intraperitoneal cancer models. However, previous delivery routes were limited to intraperitoneal injection and to a single agent, paclitaxel. In this study, we preliminarily evaluate the biodistribution and in vivo toxicity of eNPs in mice after intravenous injection. The eNPs localize predominantly to the liver, without detectable acute toxicity in the liver or other key organs. On the basis of these results, we encapsulated FQI1, a promising lead compound for treatment of hepatocellular carcinoma, in eNPs. eNPs are taken up by cancerous and noncancerous human liver cells in vitro, although at different rates. FQI1-loaded eNPs release FQI1 in a pH-dependent manner and limit proliferation equivalently to unencapsulated FQI1 in immortalized hepatocytes in vitro. eNPs are a versatile platform delivery system for therapeutic compounds and have potential utility in the treatment of liver disease.

Asymmetric Synthesis of Griffipavixanthone Employing a Chiral Phosphoric Acid-Catalyzed Cycloaddition.

Asymmetric synthesis of the biologically active xanthone dimer griffipavixanthone is reported along with its absolute stereochemistry determination. Synthesis of the natural product is accomplished via dimerization of a p-quinone methide using a chiral phosphoric acid catalyst to afford a protected precursor in excellent diastereo- and enantioselectivity. Mechanistic studies, including an unbiased computational investigation of chiral ion-pairs using parallel tempering, were performed in order to probe the mode of asymmetric induction.

[4 + 2]-Cycloaddition and 1,4-Addition of ortho-Quinone Methides by a Chiral Crotyl Silane.

Anhydrous FeCl3 in the presence of 2,6-lutidine promotes the substrate-controlled enantioselective [4 + 2]-cycloaddition and crotylation reaction between an enantioenriched ( S, E)-crotyl silane and in situ generated ortho-quinone methides ( oQMs). The reaction produces both the chiral chroman and crotylation products in a ratio reflective of the electronic nature of the parent oQM with overall combined yields up to 96%. A ring-opening and elimination sequence was subsequently developed to provide direct access to the crotylation products, containing two contiguous tertiary carbon stereocenters, in good yields and enantioselectivities.

High-throughput Screening in Larval Zebrafish Identifies Novel Potent Sedative-hypnotics.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Many general anesthetics were discovered empirically, but primary screens to find new sedative-hypnotics in drug libraries have not used animals, limiting the types of drugs discovered. The authors hypothesized that a sedative-hypnotic screening approach using zebrafish larvae responses to sensory stimuli would perform comparably to standard assays, and efficiently identify new active compounds. METHODS: The authors developed a binary outcome photomotor response assay for zebrafish larvae using a computerized system that tracked individual motions of up to 96 animals simultaneously. The assay was validated against tadpole loss of righting reflexes, using sedative-hypnotics of widely varying potencies that affect various molecular targets. A total of 374 representative compounds from a larger library were screened in zebrafish larvae for hypnotic activity at 10 µM. Molecular mechanisms of hits were explored in anesthetic-sensitive ion channels using electrophysiology, or in zebrafish using a specific reversal agent. RESULTS: Zebrafish larvae assays required far less drug, time, and effort than tadpoles. In validation experiments, zebrafish and tadpole screening for hypnotic activity agreed 100% (n = 11; P = 0.002), and potencies were very similar (Pearson correlation, r > 0.999). Two reversible and potent sedative-hypnotics were discovered in the library subset. CMLD003237 (EC50, ~11 µM) weakly modulated γ-aminobutyric acid type A receptors and inhibited neuronal nicotinic receptors. CMLD006025 (EC50, ~13 µM) inhibited both N-methyl-D-aspartate and neuronal nicotinic receptors. CONCLUSIONS: Photomotor response assays in zebrafish larvae are a mechanism-independent platform for high-throughput screening to identify novel sedative-hypnotics. The variety of chemotypes producing hypnosis is likely much larger than currently known.

Fluorescent Dendritic Micro-Hydrogels: Synthesis, Analysis and Use in Single-Cell Detection.

Hydrogels are of keen interest for a wide range of medical and biotechnological applications including as 3D substrate structures for the detection of proteins, nucleic acids, and cells. Hydrogel parameters such as polymer wt % and crosslink density are typically altered for a specific application; now, fluorescence can be incorporated into such criteria by specific macromonomer selection. Intrinsic fluorescence was observed at λmax 445 nm from hydrogels polymerized from lysine and aldehyde- terminated poly(ethylene glycol) macromonomers upon excitation with visible light. The hydrogel's photochemical properties are consistent with formation of a nitrone functionality. Printed hydrogels of 150 µm were used to detect individual cell adherence via a decreased in fluorescence. The use of such intrinsically fluorescent hydrogels as a platform for cell sorting and detection expands the current repertoire of tools available.

Asymmetric Traceless Petasis Borono-Mannich Reactions of Enals: Reductive Transposition of Allylic Diazenes.

The traceless Petasis borono-Mannich reaction of enals, sulfonylhydrazines, and allylboronates, catalyzed by chiral biphenols, results in an asymmetric reductive transposition of the in situ generated allylic diazene. Acyclic 1,4-diene products bearing either alkyl- or aryl-substituted benzylic stereocenters are afforded in excellent yields and enantiomeric ratios of up to 99:1. The use of crotylboronates in the reaction results in concomitant formation of two stereocenters in either a 1,4-syn or anti relationship from the corresponding E- or Z-crotylboronate used in the reaction. The use of ß-monosubstituted enals in the asymmetric traceless Petasis borono-Mannich reaction of crotylboronates installs tertiary methyl-bearing stereocenters in good yields and high enantioselectivities.

Dihydropyrimidine-Thiones and Clioquinol Synergize To Target ß-Amyloid Cellular Pathologies through a Metal-Dependent Mechanism.

The lack of therapies for neurodegenerative diseases arises from our incomplete understanding of their underlying cellular toxicities and the limited number of predictive model systems. It is critical that we develop approaches to identify novel targets and lead compounds. Here, a phenotypic screen of yeast proteinopathy models identified dihydropyrimidine-thiones (DHPM-thiones) that selectively rescued the toxicity caused by ß-amyloid (Aß), the peptide implicated in Alzheimer's disease. Rescue of Aß toxicity by DHPM-thiones occurred through a metal-dependent mechanism of action. The bioactivity was distinct, however, from that of the 8-hydroxyquinoline clioquinol (CQ). These structurally dissimilar compounds strongly synergized at concentrations otherwise not competent to reduce toxicity. Cotreatment ameliorated Aß toxicity by reducing Aß levels and restoring functional vesicle trafficking. Notably, these low doses significantly reduced deleterious off-target effects caused by CQ on mitochondria at higher concentrations. Both single and combinatorial treatments also reduced death of neurons expressing Aß in a nematode, indicating that DHPM-thiones target a conserved protective mechanism. Furthermore, this conserved activity suggests that expression of the Aß peptide causes similar cellular pathologies from yeast to neurons. Our identification of a new cytoprotective scaffold that requires metal-binding underscores the critical role of metal phenomenology in mediating Aß toxicity. Additionally, our findings demonstrate the valuable potential of synergistic compounds to enhance on-target activities, while mitigating deleterious off-target effects. The identification and prosecution of synergistic compounds could prove useful for developing AD therapeutics where combination therapies may be required to antagonize diverse pathologies.

Multicomponent Condensation Reactions via ortho-Quinone Methides.

Iron(III) salts promote the condensation of aldehydes or acetals with electron-rich phenols to generate ortho-quinone methides that undergo Diels-Alder condensations with alkenes. The reaction sequence occurs in a single vessel to afford benzopyrans in up to 95% yield. The reaction was discovered while investigating a two-component strategy using 2-(hydroxy(phenyl)methyl)phenols to access the desired ortho-quinone methide in a Diels-Alder condensation. The two-component condensation also afforded the corresponding benzopyran products in yields up to 97%. Taken together, the two- and three-component strategies using ortho-quinone methide intermediates provide efficient access to benzopyrans in good yields and selectivities.

Enantioselective Synthesis of Allenes by Catalytic Traceless Petasis Reactions.

Allenes are useful functional groups in synthesis as a result of their inherent chemical properties and established reactivity patterns. One property of chemical bonding renders 1,3-substituted allenes chiral, making them attractive targets for asymmetric synthesis. While there are many enantioselective methods to synthesize chiral allenes from chiral starting materials, fewer methods exist to directly synthesize enantioenriched chiral allenes from achiral precursors. We report here an asymmetric boronate addition to sulfonyl hydrazones catalyzed by chiral biphenols to access enantioenriched allenes in a traceless Petasis reaction. The resulting Mannich product from nucleophilic addition eliminates sulfinic acid, yielding a propargylic diazene that performs an alkyne walk to afford the allene. Two enantioselective approaches have been developed; alkynyl boronates add to glycolaldehyde imine to afford allylic hydroxyl allenes, and allyl boronates add to alkynyl imines to form 1,3-alkenyl allenes. In both cases, the products are obtained in high yields and enantioselectivities.

Asymmetric Petasis Borono-Mannich Allylation Reactions Catalyzed by Chiral Biphenols.

Chiral biphenols catalyze the asymmetric Petasis borono-Mannich allylation of aldehydes and amines through the use of a bench-stable allyldioxaborolane. The reaction proceeds via a two-step, one-pot process and requires 2-8 mole % of 3,3'-Ph2 -BINOL as the optimal catalyst. Under microwave heating the reaction affords chiral homoallylic amines in excellent yields (up to 99 %) and high enantioselectivies (er up to 99:1). The catalytic reaction is a true multicomponent condensation reaction whereas both the aldehyde and the amine can possess a wide range of structural and electronic properties. Use of crotyldioxaborolane in the reaction results in stereodivergent products with anti- and syn-diastereomers both in good diastereoselectivities and enantioselectivities from the corresponding E- and Z-borolane stereoisomers.

Transcription factor LSF-DNMT1 complex dissociation by FQI1 leads to aberrant DNA methylation and gene expression.

The transcription factor LSF is highly expressed in hepatocellular carcinoma (HCC) and promotes oncogenesis. Factor quinolinone inhibitor 1 (FQI1), inhibits LSF DNA-binding activity and exerts anti-proliferative activity. Here, we show that LSF binds directly to the maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1) and its accessory protein UHRF1 both in vivo and in vitro. Binding of LSF to DNMT1 stimulated DNMT1 activity and FQI1 negated the methyltransferase activation. Addition of FQI1 to the cell culture disrupted LSF bound DNMT1 and UHRF1 complexes, resulting in global aberrant CpG methylation. Differentially methylated regions (DMR) containing at least 3 CpGs, were significantly altered by FQI1 compared to control cells. The DMRs were mostly concentrated in CpG islands, proximal to transcription start sites, and in introns and known genes. These DMRs represented both hypo and hypermethylation, correlating with altered gene expression. FQI1 treatment elicits a cascade of effects promoting altered cell cycle progression. These findings demonstrate a novel mechanism of FQI1 mediated alteration of the epigenome by DNMT1-LSF complex disruption, leading to aberrant DNA methylation and gene expression.

Enantioselective Multicomponent Condensation Reactions of Phenols, Aldehydes, and Boronates Catalyzed by Chiral Biphenols.

Chiral diols and biphenols catalyze the multicomponent condensation reaction of phenols, aldehydes, and alkenyl or aryl boronates. The condensation products are formed in good yields and enantioselectivities. The reaction proceeds via an initial Friedel-Crafts alkylation of the aldehyde and phenol to yield an ortho-quinone methide that undergoes an enantioselective boronate addition. A cyclization pathway was discovered while exploring the scope of the reaction that provides access to chiral 2,4-diaryl chroman products, the core of which is a structural motif found in natural products.