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

IKKß 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 IKKß, is less well understood. One major reason for this is the absence of chemical tools designed as selective inhibitors for IKKα over IKKß. Herein, we report for the first time a series of novel, potent, and selective inhibitors of IKKα. We demonstrate effective target engagement and selectivity with IKKα in U2OS cells through inhibition of IKKα-driven p100 phosphorylation in the noncanonical NF-kB pathway without affecting IKKß-dependent IKappa-Bα loss in the canonical pathway. These compounds represent the first chemical tools that can be used to further characterize the role of IKKα in cellular signaling, to dissect this from IKKß and to validate it in its own right as a target in inflammatory diseases.

Activation of a primed RING E3-E2-ubiquitin complex by non-covalent ubiquitin.

RING ubiquitin ligases (E3) recruit ubiquitin-conjugate enzymes (E2) charged with ubiquitin (Ub) to catalyze ubiquitination. Non-covalent Ub binding to the backside of certain E2s promotes processive polyUb formation, but the mechanism remains elusive. Here, we show that backside bound Ub (Ub(B)) enhances both RING-independent and RING-dependent UbcH5B-catalyzed donor Ub (Ub(D)) transfer, but with a more prominent effect in RING-dependent transfer. Ub(B) enhances RING E3s' affinities for UbcH5B-Ub, and RING E3-UbcH5B-Ub complex improves Ub(B)'s affinity for UbcH5B. A comparison of the crystal structures of a RING E3, RNF38, bound to UbcH5B-Ub in the absence and presence of Ub(B), together with molecular dynamics simulation and biochemical analyses, suggests Ub(B) restricts the flexibility of UbcH5B's α1 and α1ß1 loop. Ub(B) supports E3 function by stabilizing the RING E3-UbcH5B-Ub complex, thereby improving the catalytic efficiency of Ub transfer. Thus, Ub(B) serves as an allosteric activator of RING E3-mediated Ub transfer.

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.

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.

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. We provide a basis for the key residues targeted by our profiled series and provide further evidence for the ability to discriminate between the two isoforms using pharmacological intervention.

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. Surprisingly, the structures of wild-type and mutant Eg5 in complex with 1 display no apparent structural changes in the binding configuration of the drug candidate. Furthermore, ITC and modeling approaches reveal that resistance to 1 is not through conventional steric effects at the binding site but through reduced flexibility and changes in energy fluctuation pathways through the protein that influence its function. This is a phenomenon we have called "resistance by allostery".

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. Molecular self-association of AIK-18/51 is consistent with anti-parallel, face-to-face dimer formation, the building block on which the molecule aggregates. This underlying structure is closely allied to the form found in the ligand's DNA complex. NMR chemical shift and diffusion measurements yield a self-association constant Kass=(61±19)×10(3)M(-1) for AIK-18/51 that fits with a stepwise self-assembly model and is consistent with ITC data. The deconstructed energetics of this assembly process are reported with respect to a design strategy for ligand/DNA recognition.

Doing the methylene shuffle--further insights into the inhibition of mitotic kinesin Eg5 with S-trityl L-cysteine.

S-Trityl L-cysteine (STLC) is an inhibitor of the mitotic kinesin Eg5 with potential as an antimitotic chemotherapeutic agent. We previously reported the crystal structure of the ligand-protein complex, and now for the first time, have quantified the interactions using a molecular dynamics based approach. Based on these data, we have explored the SAR of the trityl head group using the methylene shuffle strategy to expand the occupation of one of the hydrophobic pockets. The most potent compounds exhibit strong (<100 nM) inhibition of Eg5 in the basal ATPase assay and inhibit growth in a variety of tumour-derived cell lines.

Solid state stabilisation of the orally delivered drugs atenolol, glibenclamide, memantine and paracetamol through their complexation with cucurbit[7]uril.

The inclusion of the cardiovascular beta-blocker drug atenolol, the antidiabetic drug glibenclamide, the Alzheimer's NMDA glutamate receptor drug memantine and the analgesic/antipyretic drug paracetamol by cucurbit[7]uril (CB[7]) has been studied by (1)H nuclear magnetic resonance spectroscopy, electrospray ionisation mass spectrometry, molecular modelling, fluorescence displacement assays and differential scanning calorimetry. All four drugs form 1 : 1 host-guest complexes with CB[7], but the exchange kinetics and location of the binding is different for each drug. Atenolol is bound over the central phenyl ring with a binding constant of 4.2 x 10(4) M(-1), whereas glibenclamide is bound over the terminal cyclohexyl group with a binding constant of 1.7 x 10(5) M(-1), and memantine is totally bound within the CB[7] cavity. Paracetamol is bound in two locations, over the central phenyl ring and over the methyl group, with the CB[7] molecule shuttling quickly between the two sites. Inclusion by CB[7] was shown by differential scanning calorimetry to physically stabilise all four drugs, which has applications preventing drug degradation and improving drug processing and formulation.

Ranking ligand affinity for the DNA minor groove by experiment and simulation.

The structural and thermodynamic basis for the strength and selectivity of the interactions of minor groove binders (MGBs) with DNA is not fully understood. In 2003, we reported the first example of a thiazole-containing MGB that bound in a phase-shifted pattern that spanned six base pairs rather than the usual four (for tricyclic distamycin-like compounds). Since then, using DNA footprinting, NMR spectroscopy, isothermal titration calorimetry, and molecular dynamics, we have established that the flanking bases around the central four being read by the ligand have subtle effects on recognition. We have investigated the effect of these flanking sequences on binding and the reasons for the differences and established a computational method to rank ligand affinity against varying DNA sequences.

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.

Identification of 2-aminothiazole-4-carboxylate derivatives active against Mycobacterium tuberculosis H37Rv and the beta-ketoacyl-ACP synthase mtFabH.

BACKGROUND: Tuberculosis (TB) is a disease which kills two million people every year and infects approximately over one-third of the world's population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration. METHODOLOGY/PRINCIPAL FINDINGS: Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM) to the more tractable 2-aminothiazole-4-carboxylate scaffold to generate compounds that mimic TLM's novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H(37)R(v) and, dissociatively, against the beta-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H(37)R(v) with an MIC of 0.06 microg/ml (240 nM), but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido)-5-(3-chlorophenyl)thiazole-4-carboxylate inhibited mtFabH with an IC(50) of 0.95+/-0.05 microg/ml (2.43+/-0.13 microM) but was not active against the whole cell organism. CONCLUSIONS/SIGNIFICANCE: These findings clearly identify the 2-aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of anti-tubercular agents.

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.

3D-QSAR studies on chromone derivatives as HIV-1 protease inhibitors: application of molecular field analysis.

Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed for chromone derivatives against HIV-1 protease using molecular field analysis (MFA) with genetic partial least square algorithms (G/PLS). Three different alignment methods: field fit, pharmacophore-based, and receptor-based were used to derive three MFA models. All models produced good predictive ability with high cross-validated r(2) (r(2) (cv)), conventional r(2), and predictive r(2)(r(2)(pred)) values. The receptor-based MFA showed the best statistical results with r(2) (cv) = 0.789, r(2)= 0.886, and r(2)(pred) = 0.995. The result obtained from the receptor-based model was compared with the docking simulation of the most active compound 21 in this chromone series to the binding pocket of HIV-1 protease (PDB entry 1AJX). It was shown that the MFA model related well with the binding structure of the complex and can provide guidelines to design more potent HIV-1 protease inhibitors.

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.

Exploring DNA topoisomerase I inhibition by the benzo[c]phenanthridines fagaronine and ethoxidine using steered molecular dynamics.

The benzo[c]phenanthridines (BCPs) are a group of compounds that are believed to express their antitumor activity through the inhibition of topoisomerase I. The enzyme is crucial to cell cycle division and progression, and regulates the equilibrium between relaxed and supercoiled DNA that occurs during DNA replication. Over the years, we have prepared a number of BCPs and employed a number of biophysical techniques to explore their mechanism of action and improve their activity against this particular enzyme. The naturally occurring alkaloid fagaronine 1 and the synthetic compound ethoxidine 3 are two of the most active compounds, although their inhibitory mechanisms are different, being a poison and suppressor, respectively. We have modified the approach of steered molecular dynamics to create a torque on the intercalator to comprehensively sample the DNA binding site, and using topoisomerase I crystal structures, have proposed a model to explain the different mechanisms of action for these two BCP compounds.

In silico footprinting of ligands binding to the minor groove of DNA.

The sequence selectivity of small molecules binding to the minor groove of DNA can be predicted by "in silico footprinting". Any potential ligand can be docked in the minor groove and then moved along it using simple simulation techniques. By applying a simple scoring function to the trajectory after energy minimization, the preferred binding site can be identified. We show application to all known noncovalent binding modes, namely 1:1 ligand:DNA binding (including hairpin ligands) and 2:1 side-by-side binding, with various DNA base pair sequences and show excellent agreement with experimental results from X-ray crystallography, NMR, and gel-based footprinting.

Synthesis and antimicrobial activity of some netropsin analogues.

Nine novel lexitropsins were synthesized by linking two netropsin-like moieties through three different dicarboxylic acids; 9,10-dihydro-2,7-phenanthrenedicarboxylic acid; [(3-[[(carboxymethyl)amino]carbonyl]benzoyl)amino]acetic acid and indole-2,5-dicarboxylic acid. The netropsin residues were modified by the use of N-isopentylpyrrole, 5-methylthiophene or 5-isopropylthiazole heterocyclic building blocks in place of the usual N-methylpyrrole. The compounds were tested against five gram-positive bacteria: Staphylococcus aureus, Streptomyces faecalis, methicillin resistant Staphylococcus aureus, Enterobacter cloacae, Mycobacterium fortuitum, three gram-negative bacteria: Klebsiella aerogenes, Proteus vulgaris, Escherichia coli and three fungi: Aspergillus niger, Candida albicans and Aspergillus nidulans. Some of the compounds showed significant inhibitory effects on the growth of the microorganisms.

Short lexitropsin that recognizes the DNA minor groove at 5'-ACTAGT-3': understanding the role of isopropyl-thiazole.

Isopropyl-thiazole ((iPr)Th) represents a new addition to the building blocks of nucleic acid minor groove-binding molecules. The DNA decamer duplex d(CGACTAGTCG)(2) is bound by a short lexitropsin of sequence formyl-PyPy(iPr)Th-Dp (where Py represents N-methyl pyrrole, (iPr)Th represents thiazole with an isopropyl group attached, and Dp represents dimethylaminopropyl). NMR data indicate ligand binding in the minor groove of DNA to the sequence 5'-ACT(5)AG(7)T-3' at a 2:1 ratio of ligand to DNA duplex. Ligand binding, assisted by the enhanced hydrophobicity of the (iPr)Th group, occurs in a head-to-tail fashion, the formyl headgroups being located toward the 5'-ends of the DNA sequence. Sequence reading is augmented through hydrogen bond formation between the exocyclic amine protons of G(7) and the (iPr)Th nitrogen, which lies on the minor groove floor. The B(I)/B(II) DNA backbone equilibrium is altered at the T(5) 3'-phosphate position to accommodate a B(II) configuration. The ligands bind in a staggered mode with respect to one another creating a six base pair DNA reading frame. The introduction of a new DNA sequence-reading element into the recognition jigsaw, combined with an extended reading frame for a small lexitropsin with enhanced hydrophobicity, holds great promise in the development of new, potentially commercially viable drug lead candidates for gene targeting.

DNA binding of a short lexitropsin.

Footprinting, capillary electrophoresis, molecular modelling and NMR studies have been used to examine the binding of a short polyamide to DNA. This molecule, which contains an isopropyl-substituted thiazole in place of one of the N-methylpyrroles, is selective for the sequence 5'-ACTAGT-3' to which it binds with high affinity. Two molecules bind side-by-side in the minor groove, but their binding is staggered so that the molecule reads six base pairs, unlike the related natural products, which tend to bind to four-base-pair sequences. The result suggests that high affinity and selectivity may be gained without resort to very large molecules, which may be difficult to deliver to the site of action.