An approach to identifying novel substrates of bacterial arylamine N-acetyltransferases

Arylamine N-acetyltransferases (NATs) catalyse the acetylation of arylamine, arylhydrazine and arylhydroxylamine substrates by acetyl Coenzyme A. NAT has been discovered in a wide range of eukaryotic and prokaryotic species. Although prokaryotic NATs have been implicated in xenobiotic metabolism, to date no endogenous role has been identified for the arylamine N-acetyl transfer reaction in prokaryotes. Investigating the substrate specificity of these enzymes is one approach to determining a possible endogenous role for prokaryotic NATs. We describe an accurate and efficient assay for NAT activity that is suitable for high-throughput screening of potential NAT ligands. This assay has been utilised to identify novel substrates for pure NAT from Salmonella typhimurium and Mycobacterium smegmatis which show a relationship between the lipophilicity of the arylamine and its activity as a substrate. The lipophilic structure/activity relationship observed is proposed to depend on the topology of the active site using docking studies of the crystal structures of these NAT isoenzymes. The evidence suggests an endogenous role of NAT in the protection of bacteria from aromatic and lipophilic toxins.     

Synthesis and structure activity relationships of novel small molecule cathepsin D inhibitors.

Cathepsin D, a lysosomal aspartyl protease, has been implicated in the pathology of Alzheimer's disease as well as breast and ovarian cancer. A weakly active cathepsin D inhibitor was identified by high throughput screening. Subsequent optimization led to the discovery of a new class of small molecule inhibitors of this enzyme, culminating with the sulfonamide 13 (IC50 = 250 nM).     

Design, synthesis and in vitro evaluation of potent, novel, small molecule inhibitors of plasminogen activator inhibitor-1

We have synthesized and evaluated a series of tetramic acid-based and hydroxyquinolinone-based inhibitors of plasminogen activator inhibitor-1 (PAI-1). These studies resulted in the identification of several compounds which showed excellent potency against PAI-1. The design, synthesis and SAR of these compounds are described.     

Disruption of Kv1.1 N-type inactivation by novel small molecule inhibitors (disinactivators)

Kv1.1 channels are expressed in many regions of the brain and spinal cord [Monaghan, M. M.; Trimmer, J. S.; Rhodes, K. J. J. Neurosci.2001, 21, 5973; Rasband, M. N.; Trimmer, J. S. J. Comp. Neurol.2001, 429, 166; Trimmer, J. S.; Rhodes, K. J. Ann. Rev. Physiol.2004, 66, 477]. When expressed alone, they produce a delayed rectifier slowly inactivating type current that contributes to hyperpolarizing the neuron following depolarization. In the hippocampus Kv1.1 is co-expressed with Kvbeta1 (and other beta subunits), which converts Kv1.1 into a transient, fast inactivating current, reducing its ability to hyperpolarize the cell and thus increasing neuronal excitability. To reduce neuronal excitability, screening for compounds that prevent inactivation of Kv1.1 channels by Kvbeta1 was performed using a yeast two-hybrid screen. A variety of compounds were discovered in this assay and subsequently determined to disrupt inactivation of the ionic currents, and hence were termed 'disinactivators'. Several of these disinactivators also inhibited pentylenetetrazole-induced seizures (PTZ) in mice. Compounds were found to act by several mechanisms to prevent Kvbeta1 inactivation of Kv1.1 channels, including enhancement of Ca(2+) release/influx and by direct mechanisms. Two structural classes were identified that act on a Kvbeta1N70-Kv1.1 chimera where the N-terminal 70 amino acids of Kvbeta1 were attached to the N-terminus of Kv1.1. It is likely that these disinactivators act directly on the Kvbeta1 N-terminus or its receptor site on Kv1.1, thus preventing it from blocking Kv1.1 channels. Compounds acting by this mechanism may be useful for reducing neuronal hyperexcitability in diseases such as epilepsy and neuropathic pain.     

Chromosome mapping of the genes for murine arylamine N-acetyltransferases (NATs), enzymes involved in the metabolism of carcinogens: identification of a novel upstream noncoding exon for murine Nat2

Arylamine N-acetyltransferases (NATs) catalyse acetylation reactions which can result in either detoxification or activation of arylamine carcinogens. The human NAT loci (NAT1, NAT2, and a pseudogene, NATP) have been mapped to human chromosome 8p22, a region frequently deleted in tumours. There are three functional genes in mice (Nat1, Nat2, and Nat3) encoding for three NAT isoenzymes. Different alleles at the Nat2 locus are responsible for the acetylation polymorphism identified in different mouse strains. We show that Nat3 is close to Nat1 and Nat2, by screening of a P1 artificial chromosome (PAC) library and provide cytogenetic evidence for co-localisation of the three genes in chromosome region 8 B3.1-B3.3. The Nat region of mouse and human is homologous. We also provide sequence information and a restriction map in the vicinity of Nat1 and Nat2 and describe a noncoding exon located 6 kb upstream of the Nat2 coding region.     

N-aryl-benzimidazolones as novel small molecule HSP90 inhibitors

We describe the development of a novel series of N-aryl-benzimidazolone HSP90 inhibitors (9) targeting the N-terminal ATP-ase site. SAR development was influenced by structure-based design based around X-ray structures of ligand bound HSP90 complexes. Lead compounds exhibited high binding affinities, ATP-ase inhibition and cellular client protein degradation.     

Synthesis of small molecule CDC25 phosphatases inhibitors

A targeted library of small molecules has been prepared to optimize the biological activity of BN82002, our initial lead compound, recently described as an original inhibitor of CDC25 phosphatases. Some of these compounds inhibit CDC25 in the micromolar range and therefore reinforce the interest of CDC25 as an anticancer target.     

Design,synthesis and in vitro evaluation studies of sulfonyl-amino-acetamides as small molecule BACE-1 inhibitors

The identification of a series of sulfonyl-amino-acetamides as BACE-1 (β-secretase) inhibitors for the treatment of Alzheimer’s disease is reported. The derivatives were designed based on the docking simulation study, synthesized and assessed for BACE-1 inhibition in vitro. The designed ligands revealed desired binding interactions with the catalytic aspartate dyad and occupance of S1 and S2′ active site regions. These in silico results correlated well with in vitro activity. Out of 33 compounds synthesized, 12 compounds showed significant inhibition at 10 μM concentration. The most active compound 2.17S had IC₅₀ of 7.90 μM against BACE-1, which was concomitant with results of in silico docking study.     

Synthesis and evaluation of a novel series of indoloisoquinolines as small molecule anti-malarial leads

A group of novel synthetic indoloisoquinolines was prepared and its potential as a novel series of small-molecule anti-malarial leads was assessed. The structure-activity relationship on variation of three distinct regions of chemical space was investigated. A lead compound was generated with an activity close to that observed for a known anti-malarial natural product, dihydrousambarensine, that shares the indoloisoquinoline template structure.     

Synthesis and in vitro evaluation of novel rhodanine derivatives as potential cholinesterase inhibitors

Based on a broad spectrum of biological activities of rhodanines, we synthesized aromatic amides and esters of 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid (rhodanine-3-acetic acid) via carbodiimide- or PCl₃-mediated coupling. Both esters and amides were investigated for their in vitro inhibitory potency and selectivity against acetylcholinesterase (AChE) from electric eel and butyrylcholinesterase (BChE) from equine serum using Ellman’s spectrophotometric method. The derivatives exhibited mostly a moderate activity against both cholinesterases. IC₅₀ values for AChE were in a closer concentration range of 24.05–86.85 μM when compared to BChE inhibition (7.92–227.19 μM). The esters caused the more efficient inhibition of AChE than amides and parent acid. The esterification and amidation of the rhodanine-3-acetic acid increased inhibition of BChE, even up to 26 times. Derivatives of 4-nitroaniline/phenol showed the activity superior to other substituents (H, Cl, CH₃, OCH₃, CF₃). Rhodanines produced a balanced inhibition of both cholinesterases. Seven derivatives produced the more potent inhibition of AChE than rivastigmine, a clinically used drug; additional three compounds were comparable. Two amides exceeded inhibitory potency of rivastigmine towards BChE. Importantly, this is the first evidence that rhodanine-based compounds are able to inhibit BChE.     

Synthesis and in vitro evaluation of novel anti-varicella-zoster virus (VZV) nucleosides

A series of alkyl-aryl, -phenoxy, and -thiophenoxy bicyclic furo pyrimidine nucleosides have been successfully synthesised by Pd-coupling of 5-iodo-2'-deoxyuridine (IDU) with terminal alkynes, followed by in situ copper-cyclisation. Synthesised compounds (4a-i) showed an anti-VZV activity at low microM concentration, comparable to that of current treatment acyclovir.     

Structure based discovery of small molecule suppressors targeting bacterial lysozyme inhibitors

The production of lysozyme inhibitors, competitively binding to the lysozyme active site, is a bacterial strategy to prevent the lytic activity of host lysozymes. Therefore, suppression of the lysozyme–inhibitor interaction is an interesting new approach for drug development since restoration of the bacterial lysozyme sensitivity will support bacterial clearance from the infected sites. Using molecular modelling techniques the interaction of the Salmonella PliC inhibitor with c-type lysozyme was studied and a protein–protein interaction based pharmacophore model was created. This model was used as a query to identify molecules, with potential affinity for the target, and subsequently, these molecules were filtered using molecular docking. The retained molecules were validated as suppressors of lysozyme inhibitory proteins using in vitro experiments revealing four active molecules.     

Potent small molecule inhibitors of spleen tyrosine kinase (Syk)

A series of oxindoles demonstrating inhibition of the phosphorylation of biotinylated substrates of Syk and IgE/Fc epsilon RI triggered basophil cell degranulation has been identified. A study of the SAR around sulfonamide 31 (IC(50)=5 nM, EC(50)=1400 nM) is discussed. The modest cellular activity representative of the sulfonamide series was overcome when the Polar Surface Area was lowered to <110 A(2), leading to the identification of amide 32 (IC(50)=145 nM, EC(50)=100 nM).     

Synthesis and screening of small molecule inhibitors of anthrax edema factor

The synthesis and development of a novel class of molecules that inhibit anthrax edema factor, an adenylyl cyclase, is reported. These molecules are derived from the initial discovery that histidine and imidazole adducts of the prostaglandin PGE₂ reduce the net secretory response of cholera toxin-challenged mice and act directly on the action of anthrax edema factor, a calmodulin-dependent adenylyl cyclase. The simple enones examined in this letter were prepared by palladium-catalyzed Suzuki reaction.     

Synthesis and in vitro evaluation of novel highly potent coumarin inhibitors of gyrase B.

The design, synthesis, and in vitro biological activity of a series of novel coumarin inhibitors of gyrase B is presented. Replacement of the 3-acylamino residue (3-NHCOR) of coumarin drugs with reversed isosteres C(=O)R, C(=N-OR)R', COOR, CONHR and CONHOR leads to highly potent analogues which displayed excellent inhibition of the negative supercoiling of the relaxed DNA and antibacterial activity.     

Selective small molecule inhibitors of the potential breast cancer marker,human arylamine N-acetyltransferase 1, and its murine homologue,mouse arylamine N-acetyltransferase 2

The identification, synthesis and evaluation of a series of rhodanine and thiazolidin-2,4-dione derivatives as selective inhibitors of human arylamine N-acetyltransferase 1 and mouse arylamine N-acetyltransferase 2 is described. The most potent inhibitors identified have submicromolar activity and inhibit both the recombinant proteins and human NAT1 in ZR-75 cell lysates in a competitive manner. ¹H NMR studies on purified mouse Nat2 demonstrate that the inhibitors bind within the putative active site of the enzyme.     

Synthesis and evaluation of novel heterocyclic MMP inhibitors

A variety of novel heterocyclic compounds were synthesized and evaluated for MMP inhibition. Broad spectrum inhibition of MMPs 1, 2, 9, and 12 was found with pyridinone-based compounds while N-heterocyclic triazoles and tetrazoles were largely ineffective. A highly selective tetrazole inhibitor for MMP-2 was discovered.     

Synthesis and in vitro evaluation of aspartate transcarbamoylase inhibitors

The design, synthesis, and evaluation of a series of novel inhibitors of aspartate transcarbamoylase (ATCase) are reported. Several submicromolar phosphorus-containing inhibitors are described, but all-carboxylate compounds are inactive. Compounds were synthesized to probe the postulated cyclic transition-state of the enzyme-catalyzed reaction. In addition, the associated role of the protonation state at the phosphorus acid moiety was evaluated using phosphinic and carboxylic acids. Although none of the synthesized inhibitors is more potent than N-phosphonacetyl-l-aspartate (PALA), the compounds provide useful mechanistic information, as well as the basis for the design of future inhibitors and/or prodrugs.     

Identification of novel small molecule inhibitors of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase of Gram-negative bacteria

The biosyntheses of isoprenoids is essential for the survival in all living organisms, and requires one of the two biochemical pathways: (a) Mevalonate (MVA) Pathway or (b) Methylerythritol Phosphate (MEP) Pathway. The latter pathway, which is used by all Gram-negative bacteria, some Gram-positive bacteria and a few apicomplexan protozoa, provides an attractive target for the development of new antimicrobials because of its absence in humans. In this report, we describe two different approaches that we used to identify novel small molecule inhibitors of Escherichia coli and Yersinia pestis 4-diphosphocytidyl-2-C-methyl D-erythritol (CDP-ME) kinases, key enzymes of the MEP pathway encoded by the E. coli ispE and Y. pestisipk genes, respectively. In the first approach, we explored existing inhibitors of the GHMP kinases while in the second approach; we performed computational high-throughput screening of compound libraries by targeting the CDP-ME binding site of the two bacterial enzymes. From the first approach, we identified two compounds with 6-(benzylthio)-2-(2-hydroxyphenyl)-4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile and (Z)-3-methyl-4-((5-phenylfuran-2-yl)methylene)isoxazol-5(4H)-one scaffolds which inhibited E. coli CDP-ME kinase in vitro. We then performed substructure search and docking experiments based on these two scaffolds and identified twenty three analogs for structure-activity relationship (SAR) studies. Three new compounds from the isoxazol-5(4H)-one series have shown inhibitory activities against E. coli and Y. pestis CDP-ME kinases with the IC(50) values ranging from 7 to 13 μM. The second approach by computational high-throughput screening (HTS) of two million drug-like compounds yielded two compounds with benzenesulfonamide and acetamide moieties which, at a concentration of 20 μM, inhibited 80% and 65%, respectively, of control CDP-ME kinase activity.     

Synthesis and evaluation of novel oxazoline MMP inhibitors

MMP inhibitors with novel oxazoline zinc binding groups have been synthesized and evaluated. Selectivity for the inhibition of MMP-9 over MMP-1, MMP-2, and MMP-12 has been achieved in several cases.