IL-1β stimulates a novel axis within the NFκB pathway in endothelial cells regulated by IKKα and TAK-1.

In this study we examined the activation of the non-canonical NFκB signalling pathway in endothelial cells. In HUVECs, LIGHT stimulated a delayed induction of serine 866/870 p100 phosphorylation linked to p52 NFκB formation. Surprisingly, the canonical ligand, IL-1β, stimulated a rapid phosphorylation or p100 which was not associated with p52 formation.

Design and Synthesis of Novel Aminoindazole-pyrrolo[2,3-]pyridine Inhibitors of IKKα That Selectively Perturb Cellular Non-Canonical NF-κB Signalling.

The inhibitory-kappaB kinases (IKKs) IKKα and IKKβ play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKβ-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances.

Targeting the Non-Canonical NF-κB Pathway in Chronic Lymphocytic Leukemia and Multiple Myeloma.

In this study, we evaluated an NF-κB inducing kinase (NIK) inhibitor, CW15337, in primary chronic lymphocytic leukemia (CLL) cells, CLL and multiple myeloma (MM) cell lines and normal B- and T-lymphocytes. Basal NF-κB subunit activity was characterized using an enzyme linked immunosorbent assay (ELISA), and the effects of NIK inhibition were then assessed in terms of cytotoxicity and the expression of nuclear NF-κB subunits following monoculture and co-culture with CD40L-expressing fibroblasts, as a model of the lymphoid niche. CW15337 induced a dose-dependent increase in apoptosis, and nuclear expression of the non-canonical NF-κB subunit, p52, was correlated with sensitivity to CW15337 ( = 0.

Mitokyne: A Ratiometric Raman Probe for Mitochondrial pH.

Mitochondrial pH (pH) is intimately related to mitochondrial function, and aberrant values for pH are linked to several disease states. We report the design, synthesis, and application of mitokyne -the first small molecule pH sensor for stimulated Raman scattering (SRS) microscopy. This ratiometric probe can determine subtle changes in pH in response to external stimuli and the inhibition of both the electron transport chain and ATP synthase with small molecule inhibitors.

Determining the prognostic significance of IKKα in prostate cancer.

Background: As the survival of castration-resistant prostate cancer (CRPC) remains poor, and the nuclear factor-κB (NF-κB) pathways play key roles in prostate cancer (PC) progression, several studies have focused on inhibiting the NF-κB pathway through generating inhibitory κB kinase subunit α (IKKα) small molecule inhibitors. However, the identification of prognostic markers able to discriminate which patients could benefit from IKKα inhibitors is urgently required. The present study investigated the prognostic value of IKKα, IKKα phosphorylated at serine 180 (p-IKKα S180) and threonine 23 (p-IKKα T23), and their relationship with the androgen receptor (AR) and Ki67 proliferation index to predict patient outcome.

A new class of ratiometric small molecule intracellular pH sensors for Raman microscopy.

Intracellular pH (pH) homeostasis is intertwined with a myriad of normal cellular behaviors as well as pathological processes. As such, small molecule probes for the measurement of pH are invaluable tools for chemical biology, facilitating the study of the role of pH in cellular function and disease. The field of small molecule pH sensors has traditionally been dominated with probes based on fluorescent scaffolds.

Is the Fate of Clinical Candidate Arry-520 Already Sealed? Predicting Resistance in Eg5-Inhibitor Complexes.

Arry-520 is an advanced drug candidate from the Eg5 inhibitor class undergoing clinical evaluation in patients with relapsed or refractory multiple myeloma. Here, we show by structural analysis that Arry-520 binds stoichiometrically to the motor domain of Eg5 in the conventional allosteric loop L5 pocket in a complex that suggests the same structural mechanism as other Eg5 inhibitors. We have previously shown that acquired resistance through mutations in the allosteric-binding site located at loop L5 in the Eg5 structure appears to be independent of the inhibitors' scaffold, which suggests that Arry-520 will ultimately have the same fate.

Topographical Mapping of Isoform-Selectivity Determinants for J-Channel-Binding Inhibitors of Sphingosine Kinases 1 and 2.

Sphingosine kinase enzymes (SK1 and SK2) catalyze the conversion of sphingosine into sphingosine 1-phosphate and play a key role in lipid signaling and cellular responses. Mapping of isoform amino acid sequence differences for SK2 onto the recently available crystal structures of SK1 suggests that subtle structural differences exist in the foot of the lipid-binding "J-channel" in SK2, the structure of which has yet to be defined by structural biology techniques. We have probed these isoform differences with a ligand series derived from the potent SK1-selective inhibitor, PF-543.

Inhibitory Kappa B Kinase α (IKKα) Inhibitors That Recapitulate Their Selectivity in Cells against Isoform-Related Biomarkers.

IKKβ plays a central role in the canonical NF-kB pathway, which has been extensively characterized. The role of IKKα in the noncanonical NF-kB pathway, and indeed in the canonical pathway as a complex with IKKβ, is less well understood. One major reason for this is the absence of chemical tools designed as selective inhibitors for IKKα over IKKβ.

Synthetic biological approaches for RNA labelling and imaging: design principles and future opportunities.

RNA is the most mercurial of all biomacromolecules. In contrast to DNA, where the predominant role is the storage of genetic information, the biological role of RNA varies; ranging from a template-based intermediary in gene expression to playing a direct role in catalysis. Their high turnover and metabolic lability makes the detection of specific sequences particularly challenging.

Pyrrole-Imidazole Polyamides: Manual Solid-Phase Synthesis.

Pyrrole-imidazole polyamides (PAs) are a family of DNA-binding peptides that bind in the minor groove of double-stranded DNA (dsDNA) in a sequence-selective, programmable fashion. This protocol describes a detailed manual procedure for the solid-phase synthesis of this family of compounds. The protocol entails solution-phase synthesis of the Boc-protected pyrrole (Py) and imidazole (Im) carboxylic acid building blocks.

Resveratrol and its oligomers: modulation of sphingolipid metabolism and signaling in disease.

Resveratrol, a natural compound endowed with multiple health-promoting effects, has received much attention given its potential for the treatment of cardiovascular, inflammatory, neurodegenerative, metabolic and age-related diseases. However, the translational potential of resveratrol has been limited by its specificity, poor bioavailability and uncertain toxicity. In recent years, there has been an accumulation of evidence demonstrating that resveratrol modulates sphingolipid metabolism.

Recognition of the DNA minor groove by thiazotropsin analogues.

Solution-phase self-association characteristics and DNA molecular-recognition properties are reported for three close analogues of minor-groove-binding ligands from the thiazotropsin class of lexitropsin molecules; they incorporate isopropyl thiazole as a lipophilic building block. Thiazotropsin B (AcImPy(iPr) ThDp) shows similar self-assembly characteristics to thiazotropsin A (FoPyPy(iPr) ThDp), although it is engineered, by incorporation of imidazole in place of N-methyl pyrrole, to swap its DNA recognition target from 5'-ACTAGT-3' to 5'-ACGCGT-3'. Replacement of the formamide head group in thiazotropsin A by nicotinamide in AIK-18/51 results in a measureable difference in solution-phase self-assembly character and substantially enhanced DNA association characteristics.

Stereoselective synthesis of carbocyclic analogues of the nucleoside Q precursor (PreQ0).

A convergent and stereoselective synthesis of chiral cyclopentyl- and cyclohexylamine derivatives of nucleoside Q precursor (PreQ0) has been accomplished. This synthetic route allows for an efficient preparation of 4-substituted analogues with interesting three-dimensional character, including chiral cyclopentane-1,2-diol and -1,2,3-triol derivatives. This unusual substitution pattern provides a useful starting point for the discovery of novel bioactive molecules.

Small-molecule inhibitors of IκB kinase (IKK) and IKK-related kinases.

The transcription factors NF-κB and IFN control important signaling cascades and mediate the expression of a number of important pro-inflammatory cytokines, adhesion molecules, growth factors and anti-apoptotic survival proteins. IκB kinase (IKK) and IKK-related kinases (IKKε and TBK1) are key regulators of these biological pathways and, as such, modulators of these enzymes may be useful in the treatment of inflammatory diseases and cancer. We have reviewed the most recent IKK patent literature (2008-2012), added publications of interest overlooked in previous patent reviews and identified all the players involved in small-molecule inhibitors of the IKKs.

Structure-activity relationships and molecular modeling of sphingosine kinase inhibitors.

The design, synthesis, and evaluation of the potency of new isoform-selective inhibitors of sphingosine kinases 1 and 2 (SK1 and SK2), the enzyme that catalyzes the phosphorylation of d-erythro-sphingosine to produce the key signaling lipid, sphingosine 1-phosphate, are described. Recently, we reported that 1-(4-octylphenethyl)piperidin-4-ol (RB-005) is a selective inhibitor of SK1. Here we report the synthesis of 43 new analogues of RB-005, in which the lipophilic tail, polar headgroup, and linker region were modified to extend the structure-activity relationship profile for this lead compound, which we explain using modeling studies with the recently published crystal structure of SK1.

Mitotic kinesin Eg5 overcomes inhibition to the phase I/II clinical candidate SB743921 by an allosteric resistance mechanism.

Development of drug resistance during cancer chemotherapy is one of the major causes of chemotherapeutic failure for the majority of clinical agents. The aim of this study was to investigate the underlying molecular mechanism of resistance developed by the mitotic kinesin Eg5 against the potent second-generation ispinesib analogue SB743921 (1), a phase I/II clinical candidate. Biochemical and biophysical data demonstrate that point mutations in the inhibitor-binding pocket decrease the efficacy of 1 by several 1000-fold.

Flavanoids induce expression of the suppressor of cytokine signalling 3 (SOCS3) gene and suppress IL-6-activated signal transducer and activator of transcription 3 (STAT3) activation in vascular endothelial cells.

The atherogenic cytokine IL-6 (interleukin-6) induces pro-inflammatory gene expression in VECs (vascular endothelial cells) by activating the JAK (Janus kinase)/STAT3 (signal transducer and activator of transcription 3) signalling pathway, which is normally down-regulated by the STAT3-dependent induction of the E3 ubiquitin ligase component SOCS3 (suppressor of cytokine signalling 3). Novel treatments based on the regulation of SOCS3 protein levels could therefore have value in the treatment of diseases with an inflammatory component, such as atherosclerosis. To this end we carried out a screen of 1031 existing medicinal compounds to identify inducers of SOCS3 gene expression and identified the flavanoids naringenin and flavone as effective inducers of SOCS3 protein, mRNA and promoter activity.

Thiazotropsin aggregation and its relationship to molecular recognition in the DNA minor groove.

Aggregated states have been alluded to for many DNA minor groove binders but details of the molecule-on-molecule relationship have either been under-reported or ignored. Here we report our findings from ITC and NMR measurements carried out with AIK-18/51, a compound representative of the thiazotropsin class of DNA minor groove binders. The free aqueous form of AIK-18/51 is compared with that found in its complex with cognate DNA duplex d(CGACTAGTCG)2.

Optimized S-trityl-L-cysteine-based inhibitors of kinesin spindle protein with potent in vivo antitumor activity in lung cancer xenograft models.

The mitotic kinesin Eg5 is critical for the assembly of the mitotic spindle and is a promising chemotherapy target. Previously, we identified S-trityl-L-cysteine as a selective inhibitor of Eg5 and developed triphenylbutanamine analogues with improved potency, favorable drug-like properties, but moderate in vivo activity. We report here their further optimization to produce extremely potent inhibitors of Eg5 (K(i)(app) < 10 nM) with broad-spectrum activity against cancer cell lines comparable to the Phase II drug candidates ispinesib and SB-743921.

Confined compression of collagen hydrogels.

Reconstituted collagen hydrogels are often used for in vitro studies of cell-matrix interaction and as scaffolds for tissue engineering. Understanding the mechanical and transport behaviours of collagen hydrogels is therefore extremely important, albeit difficult due to their very high water content (typically >99.5%).

Identification and development of the 1,4-benzodiazepin-2-one and quinazoline-2,4-dione scaffolds as submicromolar inhibitors of HAT.

A library of 1,4-benzodiazepines has been synthesised and evaluated for activity against Trypanosoma brucei, a causative parasite of Human African Trypanosomiasis (HAT). The most potent of these derivatives has an MIC value of 0.97 μM.

Design and synthesis of EGFR dimerization inhibitors and evaluation of their potential in the treatment of psoriasis.

Hit compounds from in silico screening for inhibitors of the EGFR dimerization process were evaluated for their anti-proliferative (CCD-1106 keratinocytes) and anti-oxidant (TBA assay) activity and their effect on EGFR dimerization (BS(3) chemical crosslinking assay). 7-Benzyl-8-{N'-[1-(3-ethoxy-4-hydroxyphenyl)meth-(Z)-ylidene]hydrazino}-1,3-dimethylxanthine 2a (127 μM) leads to 37% inhibition of p-EGFR dimerization in the CCD-1106 cell line and also inhibits phosphorylation of proteins in the MAPK/ERK pathway, ERK 1/2 and p-38. Based on this initial data, 2a was selected for further study and was evaluated for its anti-proliferative activity in a range of keratinocyte (CCD-1106, HaCaT and NHEK) and monocyte (ThP1 and U937) cell lines.

Triphenylbutanamines: kinesin spindle protein inhibitors with in vivo antitumor activity.

The human mitotic kinesin Eg5 represents a novel mitotic spindle target for cancer chemotherapy. We previously identified S-trityl-l-cysteine (STLC) and related analogues as selective potent inhibitors of Eg5. We herein report on the development of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from the STLC scaffold.