Electrochemical Resistive DNA Biosensor for the Detection of HPV Type 16.

In this work, a low-cost and rapid electrochemical resistive DNA biosensor based on the current relaxation method is described. A DNA probe, complementary to the specific human papillomavirus type 16 (HPV-16) sequence, was immobilized onto a screen-printed gold electrode. DNA hybridization was detected by applying a potential step of 30 mV to the system, composed of an external capacitor and the modified electrode DNA/gold, for 750 µs and then relaxed back to the OCP, at which point the voltage and current discharging curves are registered for 25 ms. From the discharging curves, the potential and current relaxation were evaluated, and by using Ohm's law, the charge transfer resistance through the DNA-modified electrode was calculated. The presence of a complementary sequence was detected by the change in resistance when the ssDNA is transformed in dsDNA due to the hybridization event. The target DNA concentration was detected in the range of 5 to 20 nM. The results showed a good fit to the regression equation ΔRtotal(Ω)=2.99 × [DNA]+81.55, and a detection limit of 2.39 nM was obtained. As the sensing approach uses a direct current, the electronic architecture of the biosensor is simple and allows for the separation of faradic and nonfaradaic contributions. The simple electrochemical resistive biosensor reported here is a good candidate for the point-of-care diagnosis of HPV at a low cost and in a short detection time.

DNA Biosensor Based on Double-Layer Discharge for the Detection of HPV Type 16.

DNA electrochemical biosensors represent a feasible alternative for the diagnosis of different pathologies. In this work, the development of an electrochemical method for Human Papillomavirus-16 (HPV-16) sensing is reported based on potential relaxation measurements related to the discharge of a complex double layer of a DNA-modified gold electrode. The method used allows us to propose an equivalent circuit (EC) for a DNA/Au electrode, which was corroborated by electrochemical impedance spectroscopy (EIS) measurement. This model differs from the Randles circuit that is commonly used in double-layer simulations. The change in the potential relaxation and associated charge transfer resistance were used for sensing the DNA hybridization by using the redox pair Fe(CN)64-/Fe(CN)63+ as an electrochemical indicator. In order to determinate only the potential relaxation of the composed double layer, the faradic and double-layer current contributions were separated using a rectifier diode arrangement. A detection limit of 0.38 nM was obtained for the target HPV-16 DNA sequences. The biosensor showed a qualitative discrimination between a single-base mismatched sequence and the fully complementary HPV-16 DNA target. The results indicate that the discharge of the double-layer detection method can be used to develop an HPV DNA biosensor.

Design of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pK a Polar Moiety.

Signaling pathways intersecting with the p21-activated kinases (PAKs) play important roles in tumorigenesis and cancer progression. By recognizing that the limitations of FRAX1036 (1) were chiefly associated with the highly basic amine it contained, we devised a mitigation strategy to address several issues such as hERG activity. The 5-amino-1,3-dioxanyl moiety was identified as an effective means of reducing pK a and logP simultaneously. When positioned properly within the scaffold, this group conferred several benefits including potency, pharmacokinetics, and selectivity. Mouse xenograft PK/PD studies were carried out using an advanced compound, G-5555 (12), derived from this approach. These studies concluded that dose-dependent pathway modulation was achievable and paves the way for further in vivo investigations of PAK1 function in cancer and other diseases.

Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents.

INTRODUCTION: Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis. METHODS: PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n=980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting. RESULTS: We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P=1.29×10(-4) and P=0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis. CONCLUSIONS: Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.

Group I Paks as therapeutic targets in NF2-deficient meningioma.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by the development of multiple tumors in the central nervous system, most notably schwannomas and meningiomas. Mutational inactivation of NF2 is found in 40-60% of sporadic meningiomas, but the molecular mechanisms underlying malignant changes of meningioma cells remain unclear. Because group I p21-activated kinases (Paks) bind to and are inhibited by the NF2-encoded protein Merlin, we assessed the signaling and anti-tumor effects of three group-I specific Pak inhibitors - Frax597, 716 and 1036 - in NF2-/- meningiomas in vitro and in an orthotopic mouse model. We found that these Pak inhibitors suppressed the proliferation and motility of both benign (Ben-Men1) and malignant (KT21-MG1) meningiomas cells. In addition, we found a strong reduction in phosphorylation of Mek and S6, and decreased cyclin D1 expression in both cell lines after treatment with Pak inhibitors. Using intracranial xenografts of luciferase-expressing KT21-MG1 cells, we found that treated mice showed significant tumor suppression for all three Pak inhibitors. Similar effects were observed in Ben-Men1 cells. Tumors dissected from treated animals exhibited an increase in apoptosis without notable change in proliferation. Collectively, these results suggest that Pak inhibitors might be useful agents in treating NF2-deficient meningiomas.

PAKs inhibitors ameliorate schizophrenia-associated dendritic spine deterioration in vitro and in vivo during late adolescence.

Drug discovery in psychiatry has been limited to chemical modifications of compounds originally discovered serendipitously. Therefore, more mechanism-oriented strategies of drug discovery for mental disorders are awaited. Schizophrenia is a devastating mental disorder with synaptic disconnectivity involved in its pathophysiology. Reduction in the dendritic spine density is a major alteration that has been reproducibly reported in the cerebral cortex of patients with schizophrenia. Disrupted-in-Schizophrenia-1 (DISC1), a factor that influences endophenotypes underlying schizophrenia and several other neuropsychiatric disorders, has a regulatory role in the postsynaptic density in association with the NMDA-type glutamate receptor, Kalirin-7, and Rac1. Prolonged knockdown of DISC1 leads to synaptic deterioration, reminiscent of the synaptic pathology of schizophrenia. Thus, we tested the effects of novel inhibitors to p21-activated kinases (PAKs), major targets of Rac1, on synaptic deterioration elicited by knockdown expression of DISC1. These compounds not only significantly ameliorated the synaptic deterioration triggered by DISC1 knockdown but also partially reversed the size of deteriorated synapses in culture. One of these PAK inhibitors prevented progressive synaptic deterioration in adolescence as shown by in vivo two-photon imaging and ameliorated a behavioral deficit in prepulse inhibition in adulthood in a DISC1 knockdown mouse model. The efficacy of PAK inhibitors may have implications in drug discovery for schizophrenia and related neuropsychiatric disorders in general.

FRAX597, a small molecule inhibitor of the p21-activated kinases, inhibits tumorigenesis of neurofibromatosis type 2 (NF2)-associated Schwannomas.

The p21-activated kinases (PAKs) are immediate downstream effectors of the Rac/Cdc42 small G-proteins and implicated in promoting tumorigenesis in various types of cancer including breast and lung carcinomas. Recent studies have established a requirement for the PAKs in the pathogenesis of Neurofibromatosis type 2 (NF2), a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate signaling through the PAKs and the tumor suppressive functions of Merlin are mediated, at least in part, through inhibition of the PAKs. Knockdown of PAK1 and PAK2 expression, through RNAi-based approaches, impairs the proliferation of NF2-null schwannoma cells in culture and inhibits their ability to form tumors in vivo. These data implicate the PAKs as potential therapeutic targets. High-throughput screening of a library of small molecules combined with a structure-activity relationship approach resulted in the identification of FRAX597, a small-molecule pyridopyrimidinone, as a potent inhibitor of the group I PAKs. Crystallographic characterization of the FRAX597/PAK1 complex identifies a phenyl ring that traverses the gatekeeper residue and positions the thiazole in the back cavity of the ATP binding site, a site rarely targeted by kinase inhibitors. FRAX597 inhibits the proliferation of NF2-deficient schwannoma cells in culture and displayed potent anti-tumor activity in vivo, impairing schwannoma development in an orthotopic model of NF2. These studies identify a novel class of orally available ATP-competitive Group I PAK inhibitors with significant potential for the treatment of NF2 and other cancers.

Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by the small-molecule PAK inhibitor FRAX486.

Fragile X syndrome (FXS) is the most common inherited form of autism and intellectual disability and is caused by the silencing of a single gene, fragile X mental retardation 1 (Fmr1). The Fmr1 KO mouse displays phenotypes similar to symptoms in the human condition--including hyperactivity, repetitive behaviors, and seizures--as well as analogous abnormalities in the density of dendritic spines. Here we take a hypothesis-driven, mechanism-based approach to the search for an effective therapy for FXS. We hypothesize that a treatment that rescues the dendritic spine defect in Fmr1 KO mice may also ameliorate autism-like behavioral symptoms. Thus, we targeted a protein that regulates spines through modulation of actin cytoskeleton dynamics: p21-activated kinase (PAK). Our results demonstrate that a potent small molecule inhibitor of group I PAKs reverses dendritic spine phenotypes in Fmr1 KO mice. Moreover, this PAK inhibitor--which we call FRAX486--also rescues seizures and behavioral abnormalities such as hyperactivity and repetitive movements, thereby supporting the hypothesis that a drug treatment that reverses the spine abnormalities can also treat neurological and behavioral symptoms. Finally, a single administration of FRAX486 is sufficient to rescue all of these phenotypes in adult Fmr1 KO mice, demonstrating the potential for rapid, postdiagnostic therapy in adults with FXS.

p21-Activated kinase 1 is required for efficient tumor formation and progression in a Ras-mediated skin cancer model.

The RAS genes are the most commonly mutated oncogenes in human cancer and present a particular therapeutic dilemma, as direct targeting of Ras proteins by small molecules has proved difficult. Signaling pathways downstream of Ras, in particular Raf/Mek/Erk and PI3K/Akt/mTOR, are dominated by lipid and protein kinases that provide attractive alternate targets in Ras-driven tumors. As p21-activated kinase 1 (Pak1) has been shown to regulate both these signaling pathways and is itself upregulated in many human cancers, we assessed the role of Pak1 in Ras-driven skin cancer. In human squamous cell carcinoma (SCC), we found a strong positive correlation between advanced stage and grade and PAK1 expression. Using a mouse model of Kras-driven SCC, we showed that deletion of the mouse Pak1 gene led to markedly decreased tumorigenesis and progression, accompanied by near total loss of Erk and Akt activity. Treatment of Kras(G12D) mice with either of two distinct small molecule Pak inhibitors (PF3758309 and FRAX597) caused tumor regression and loss of Erk and Akt activity. Tumor regression was also seen in mice treated with a specific Mek inhibitor, but not with an Akt inhibitor. These findings establish Pak1 as a new target in KRAS-driven tumors and suggest a mechanism of action through the Erk, but not the Akt, signaling pathway.

Heteroaromatic-aminomethyl quinolones: potent and selective iNOS inhibitors.

The overproduction of nitric oxide during the biological response to inflammation by the nitric oxide synthase (NOS) enzymes have been implicated in the pathology of many diseases. By removal of the amide core from uHTS-derived quinolone 4, a new series highly potent heteroaromatic-aminomethyl quinolone iNOS inhibitors 8 were identified. SAR studies led to identification of piperazine 22 and pyrimidine 32, both of which reduced plasma nitrates following oral dosing in a mouse lipopolysaccharide challenge assay.

Annulation of thioimidates and vinyl carbodiimides to prepare 2-aminopyrimidines, competent nucleophiles for intramolecular alkyne hydroamination. Synthesis of (-)-crambidine.

A convergent synthesis of (-)-crambidine is reported. The sequence capitalizes on two novel key transformations, including a [4+2] annulation of thioimidates with vinyl carbodiimides and an alkyne hydroamination employing 2-aminopyrimidine nucleophiles.

Total synthesis of (+)-batzelladine A and (-)-batzelladine D via [4 + 2]-annulation of vinyl carbodiimides with N-alkyl imines.

A diastereoselective [4 + 2]-annulation of vinyl carbodiimides with chiral N-alkyl imines has been developed to access the stereochemically rich polycyclic guanidine cores of the batzelladine alkaloids. Application of this strategy, together with additional key steps such as long-range directed hydrogenation and diastereoselective intramolecular iodo-amination, led to highly convergent total syntheses of (-)-batzelladine D and (+)-batzelladine A with excellent stereocontrol.

Diastereoselective [4+2] annulation of vinyl carbodiimides with N-alkyl imines. Asymmetric synthetic access to the batzelladine alkaloids.

A diastereoselective [4+2] annulation of vinyl carbodiimides with chiral N-alkyl imines has been developed to access the stereochemically rich tricyclic core of the batzelladine alkaloids. Its application to the asymmetric synthesis of batzelladine D permitted the use of long-range, directed hydrogenation and stereoselective intramolecular iodoamination as additional key steps to establish the remaining stereocenters within the natural product with excellent stereocontrol.

Selectfluor: mechanistic insight and applications.

The replacement of hydrogen atoms with fluorine substituents in organic substrates is of great interest in synthetic chemistry because of the strong electronegativity of fluorine and relatively small steric footprint of fluorine atoms. Many sources of nucleophilic fluorine are available for the derivatization of organic molecules under acidic, basic, and neutral conditions. However, electrophilic fluorination has historically required molecular fluorine, whose notorious toxicity and explosive tendencies limit its application in research. The necessity for an electrophilic fluorination reagent that is safe, stable, highly reactive, and amenable to industrial production as an alternative to very hazardous molecular fluorine was the inspiration for the discovery of selectfluor. This reagent is not only one of the most reactive electrophilic fluorinating reagents available, but it is also safe, nontoxic, and easy to handle. In this Review we document the many applications of selectfluor and discuss possible mechanistic pathways for its reaction.

N-(phenylthio)-epsilon-caprolactam: a new promoter for the activation of thioglycosides.

N-(phenylthio)-epsilon-caprolactam (1) has been applied as a new promoter for the activation of thioglycosides. This proceeds by the reaction of 1 with trifluoromethansulfonic anhydride, which subsequently activates the thioglycoside for glycosidic bond formation. Notably, the reaction proceeds efficiently at room temperature and is adaptable to our reactivity-based one-pot oligosaccharide synthesis. [reaction: see text]

Reactivity-based one-pot total synthesis of fucose GM1 oligosaccharide: a sialylated antigenic epitope of small-cell lung cancer.

The total synthesis of the sialic acid-containing antigenic epitope fucose GM(1) (Fuc-GM(1)) by an improved reactivity-based one-pot synthetic strategy is reported. Based on a thioglycoside reactivity database, three saccharide building blocks, 3, 4, and 5, were designed and prepared to incorporate a descending order of reactivity toward thiophilic activation. Using the reactivity-based one-pot synthetic method, the fully protected Fuc-GM(1) glycoside 2 was furnished in a facile manner, which was globally deprotected to give the Fuc-GM(1) glycoside 1. In addition, using the promoter system 1-(benzensulfinyl)piperidinetrifluoromethanesulfonic anhydride, the product yield was improved and the reaction time was reduced in comparison with the N-iodosuccinimidetrifluoromethanesulfonic acid- and dimethyl (thiomethyl) sulfonium trifluoromethanesulfonate-promoted systems.