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

The inhibitory-kappaB kinases (IKKs) IKKα and IKKß 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 IKKß-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. Herein, we report the development of an aminoindazole-pyrrolo[2,3-b]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKß in vitro and explain the structure-activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds (SU1261 [IKKα Ki = 10 nM; IKKß Ki = 680 nM] and SU1349 [IKKα Ki = 16 nM; IKKß Ki = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKß in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents.

Mitokyne: A Ratiometric Raman Probe for Mitochondrial pH.

Mitochondrial pH (pHmito) is intimately related to mitochondrial function, and aberrant values for pHmito are linked to several disease states. We report the design, synthesis, and application of mitokyne 1-the first small molecule pHmito sensor for stimulated Raman scattering (SRS) microscopy. This ratiometric probe can determine subtle changes in pHmito in response to external stimuli and the inhibition of both the electron transport chain and ATP synthase with small molecule inhibitors. In addition, 1 was also used to monitor mitochondrial dynamics in a time-resolved manner with subcellular spatial resolution during mitophagy providing a powerful tool for dissecting the molecular and cell biology of this critical organelle.

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. METHODS: A cohort of 115 patients with hormone-naïve PC (HNPC) and CRPC specimens available were used to assess tumor cell expression of proteins within both the cytoplasm and the nucleus by immunohistochemistry. The expression levels were dichotomized (low vs high) to determine the associations between IKKα, AR, Ki67, and patients'Isurvival. In addition, an analysis was performed to assess potential IKKα associations with clinicopathological and inflammatory features, and potential IKKα correlations with other cancer pathways essential for CRPC growth. RESULTS: High levels of cytoplasmic IKKα were associated with a higher cancer-specific survival in HNPC patients with low AR expression (hazards ratio [HR], 0.33; 95% confidence interval [CI] log-rank, 0.11-0.98; P = .04). Furthermore, nuclear IKKα (HR, 2.60; 95% CI, 1.27-5.33; P = .01) and cytoplasmic p-IKKα S180 (HR, 2.10; 95% CI, 1.17-3.76; P = .01) were associated with a lower time to death from recurrence in patients with CRPC. In addition, high IKKα expression was associated with high levels of T-cells (CD3+ P = .01 and CD8+ P = .03) in HNPC; however, under castration conditions, high IKKα expression was associated with high levels of CD68+ macrophages (P = .04), higher Gleason score (P = .01) and more prostate-specific antigen concentration (P = .03). Finally, we identified crosstalk between IKKα and members of the canonical NF-κB pathway in the nucleus of HNPC. Otherwise, IKKα phosphorylated by noncanonical NF-κB and Akt pathways correlated with members of the canonical NF-κB pathway in CRPC. CONCLUSION: The present study reports that patients with CRPC expressing high levels of nuclear IKKα or cytoplasmic p-IKKα S180, which associated with a lower time to death from recurrence, may benefit from IKKα inhibitors.

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

Intracellular pH (pHi) homeostasis is intertwined with a myriad of normal cellular behaviors as well as pathological processes. As such, small molecule probes for the measurement of pHi are invaluable tools for chemical biology, facilitating the study of the role of pH in cellular function and disease. The field of small molecule pHi sensors has traditionally been dominated with probes based on fluorescent scaffolds. In this study, a series of low molecular weight (<260) oligoyne compounds have been developed which exhibit pH sensitive alkyne stretching frequencies (νalkyne) in Raman spectroscopy. The modular design of the compounds enabled tuneability of their pKa(H) through simple structural modification, such that continuous pH sensitivity is achieved over the range 2-10. Alkyne stretching bands reside in the 'cell-silent' region of the Raman spectrum (1800-2600 cm-1) and are readily detectable in a cellular environment with subcellular spatial resolution. This enabled the application of a pH sensitive oligoyne compound to the ratiometric sensing of pHi in prostate cancer (PC3) cells in response to drug treatment. We propose that probes based on Alkyne Tag Raman Imaging offer an entirely new platform for the sensing of pHi, complementary to fluorescence microscopy.

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. The structures and associated thermodynamic parameters of self-assembled ligand aggregates and their complexes with their respective DNA targets are considered in the context of cluster targeting of DNA by minor-groove complexes.

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. Moreover, resveratrol forms higher order oligomers that exhibit better selectivity and potency in modulating sphingolipid metabolism. This review evaluates the evidence supporting the modulation of sphingolipid metabolism and signaling as a mechanism of action underlying the therapeutic efficacy of resveratrol and oligomers in diseases, such as cancer.

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. This was in contrast with the action of traditional JAK/STAT3 inhibitors and the polyphenol resveratrol, which effectively suppress SOCS3 gene expression. Both naringenin and flavone also effectively suppressed IL-6-stimulated phosphorylation of STAT3 (Tyr7°5) which led to suppression of IL-6-induction of the atherogenic STAT3 target gene MCP1 (monocyte chemotactic protein-1), suggesting that their ability to induce SOCS3 gene expression is STAT3-independent. Supporting this idea was the observation that the general kinase inhibitor compound C inhibits flavone- and cAMP-dependent, but not JAK-dependent, SOCS3 induction in VECs. Indeed, the ability of flavanoids to induce SOCS3 expression requires activation of the ERK (extracellular-signal-regulated kinase)-dependent transcription factor SP3, and not STAT3. In the present paper we therefore describe novel molecular actions of flavanoids, which control SOCS3 gene induction and suppression of STAT3 signalling in VECs. These mechanisms could potentially be exploited to develop novel anti-atherogenic therapies.

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.

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. Herein we report the design, synthesis and biological evaluation of the abovementioned compounds.

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. Xanthine 2a is pro-apoptotic in HaCaT keratinocytes, as shown by electron microscopy, caspase 3/7, and annexin V-FITC/PI flow cytometric assays. It is significantly less cytotoxic than the established antipsoriatic agent dithranol 14, as determined by MTT and LDH release assays, and thus has potential as a lead compound for the treatment of psoriasis.

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 (p = 0.01; r2 = 0.39). Co-culture on CD40L-expressing cells induced both canonical and non-canonical subunit expression in nuclear extracts, which promoted in vitro resistance against fludarabine and ABT-199 (venetoclax) but not CW15337. Furthermore, the combination of CW15337 with fludarabine or ABT-199 showed cytotoxic synergy. Mechanistically, CW15337 caused the selective inhibition of non-canonical NF-κB subunits and the transcriptional repression of BCL2L1, BCL2A1 and MCL1 gene transcription. Taken together, these data suggest that the NIK inhibitor, CW15337, exerts its effects via suppression of the non-canonical NF-κB signaling pathway, which reverses BCL2 family-mediated resistance in the context of CD40L stimulation.

Synthesis and biological evaluation of 1,4-benzodiazepin-2-ones with antitrypanosomal activity.

A library of 1,4-benzodiazepines has been synthesized and evaluated against Trypanosoma brucei, a causative parasite of Human African trypanosomiasis. Benzodiazepines possessing a P2- transporter motif were found to have MIC values as low as 0.78 µM.

Manganese dioxide mediated one-pot synthesis of methyl 9H-pyrido[3,4-b]indole-1-carboxylate: Concise synthesis of alangiobussinine.

The carboline ring system is an important pharmacophore found in a number of biologically important targets. Development of synthetic routes for the preparation of these compounds is important in order to prepare a range of analogues containing the carboline heterocyclic moiety. A manganese dioxide mediated one-pot method starting with an activated alcohol and consisting of alcohol oxidation, Pictet-Spengler cyclisation, and oxidative aromatisation, offers a convenient process that allows access to ß-carbolines. This one-pot process for the preparation of methyl 9H-pyrido[3,4-b]indole-1-carboxylate has subsequently been used as the key step in the synthesis of alangiobussinine and a closely related analogue.

Therapeutic strategies in the treatment of diabetic nephropathy - a translational medicine approach.

Type 2 Diabetes (T2D) is an important cause of renal dysfunction and the most common cause of end-stage renal disease (ESRD). Diabetic nephropathy is also associated with an increased risk of vascular disease and patient mortality. Aggressive management of hypertension to reduce microalbuminuria, together with tight glycaemic control are important therapeutic strategies for renal and vascular disease prevention in T2D. The main pathophysiological mechanisms associated with diabetic nephropathy result from activation of the renin-angiotensin-aldosterone system (RAAS), protein kinase C pathway, pro-inflammatory cytokines and various growth factors. Angiotensin II and transforming growth factor-beta (TGF-beta) are two important molecular mediators. The production of advanced glycation end-products (AGEs) and increased oxidative stress further exacerbates renal injury. These molecular changes within the renal tissue result in mesangial expansion, increased extracellular matrix deposition and an alteration in podocyte structure and function. Therapeutic targeting of these molecular pathways is an important area of translational research in diabetes. The elucidation of new genetic associations and proteomic biomarkers of diabetic kidney disease will also assist in the identification and treatment of high-risk patients. This review article will discuss both the molecular and clinical aspects of diabetic nephropathy, providing a bench-to-bedside research perspective to potential new therapeutic strategies.

A new synthesis of alkene-containing minor-groove binders and essential hydrogen bonding in binding to DNA and in antibacterial activity.

A practical synthesis of alkene-containing minor-groove binders for DNA, related to distamycin, with potential for wide structural diversity is described, based upon the Wittig chemistry of N-alkylpyrrole aldehydes. The compounds prepared have been evaluated for binding to DNA by physical methods (melting temperature and NMR) and for their antibacterial activity. Significantly, it was found that alkenes linking the aryl head group of the minor-groove binder promote strong binding to DNA and high antibacterial activity against Gram-positive bacteria. Conversely, a minor-groove binder containing an alkene located towards the alkylamino tail group has a low affinity for DNA and does not show antibacterial activity. These observations suggest an important role for specific hydrogen bonds in the binding of compounds of this type to DNA, and in their antibacterial activity.

A detailed binding free energy study of 2:1 ligand-DNA complex formation by experiment and simulation.

In 2004, we used NMR to solve the structure of the minor groove binder thiazotropsin A bound in a 2:1 complex to the DNA duplex, d(CGACTAGTCG)2. In this current work, we have combined theory and experiment to confirm the binding thermodynamics of this system. Molecular dynamics simulations that use polarizable or non-polarizable force fields with single and separate trajectory approaches have been used to explore complexation at the molecular level. We have shown that the binding process invokes large conformational changes in both the receptor and ligand, which is reflected by large adaptation energies. This is compensated for by the net binding free energy, which is enthalpy driven and entropically opposed. Such a conformational change upon binding directly impacts on how the process must be simulated in order to yield accurate results. Our MM-PBSA binding calculations from snapshots obtained from MD simulations of the polarizable force field using separate trajectories yield an absolute binding free energy (-15.4 kcal mol(-1)) very close to that determined by isothermal titration calorimetry (-10.2 kcal mol(-1)). Analysis of the major energy components reveals that favorable non-bonded van der Waals and electrostatic interactions contribute predominantly to the enthalpy term, whilst the unfavorable entropy appears to be driven by stabilization of the complex and the associated loss of conformational freedom. Our results have led to a deeper understanding of the nature of side-by-side minor groove ligand binding, which has significant implications for structure-based ligand development.

Metabolism of two new benzodiazepine-type anti-leishmanial agents in rat hepatocytes and hepatic microsomes and their interaction with glutathione in macrophages.

OBJECTIVES: To measure the metabolism and toxicity of 7-chloro-4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-1) and 4-cyclohexylmethyl-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-2), two new benzodiazepine analogues found to be effective against Leishmania amastigotes in vitro. METHODS: The metabolism of BNZ-1 and -2 was investigated in isolated rat hepatocytes and rat liver microsomes. The toxicity of the compounds was assessed in a murine macrophage cell line by determining cell viability and reduced glutathione (GSH) content. The metabolism and toxicity of flurazepam was assessed for comparison. KEY FINDINGS: BNZ-1 and BNZ-2 underwent similar metabolic transformations by the liver systems, forming N-demethylated and hydroxylated metabolites, with subsequent O-glucuronidation. Flurazepam and both analogue compounds depleted macrophage GSH levels without affecting cell viability at the concentrations used (up to 100 microM), but only flurazepam inhibited glutathione reductase activity, indicating that it is acting by a different mechanism. CONCLUSIONS: The exact mechanism responsible for GSH depletion is unknown at present. Further experiments are needed to fully understand the effects of BNZs on the parasite GSH analogue, trypanothione, which may be a direct or indirect target for these agents. Pharmacokinetic evaluation of these compounds is required to further progress their development as potential new treatments for leishmaniasis.

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. When Arry-520 was assessed in two cell lines selected for the expression of either Eg5(D130A) or Eg5(L214A) STLC-resistant alleles, mutations previously shown to convey resistance to this class of inhibitors, it was inactive in both. Surprisingly, when the cells were challenged with ispinesib, another Eg5 inhibitor, the Eg5(D130A) cells were resistant, but those expressing Eg5(L214A) were strikingly sensitive. Molecular dynamics simulations suggest that subtle differences in ligand binding and flexibility in both compound and protein may alter allosteric transmission from the loop L5 site that do not necessarily result in reduced inhibitory activity in mutated Eg5 structures. Although we predict that cells challenged with Arry-520 in the clinical setting are likely to acquire resistance through point mutations in the Eg5-binding site, the data for ispinesib suggest that this resistance mechanism is not scaffold independent as previously thought, and new inhibitors can be designed that retain inhibitory activity in these resistant cells.

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. Here we show how it is possible, even with relatively conservative changes in compound structure, to systematically tune the activity profile of a ligand from ca. 100-fold SK1-selective inhibition, through equipotent SK1/SK2 inhibition, to reversed 100-fold SK2 selectivity, with retention of nanomolar potency.

Antimicrobial lexitropsins containing amide, amidine, and alkene linking groups.

The synthesis and properties of 80 short minor groove binders related to distamycin and the thiazotropsins are described. The design of the compounds was principally predicated upon increased affinity arising from hydrophobic interactions between minor groove binders and DNA. The introduction of hydrophobic aromatic head groups, including quinolyl and benzoyl derivatives, and of alkenes as linkers led to several strongly active antibacterial compounds with MIC for Staphylococcus aureus, both methicillin-sensitive and -resistant strains, in the range of 0.1-5 microg mL-1, which is comparable to many established antibacterial agents. Antifungal activity was also found in the range of 20-50 microg mL-1 MIC against Aspergillus niger and Candida albicans, again comparable with established antifungal drugs. A quinoline derivative was found to protect mice against S. aureus infection for a period of up to six days after a single intraperitoneal dose of 40 mg kg-1.