Two-dimensional combinatorial screening and the RNA Privileged Space Predictor program efficiently identify aminoglycoside–RNA hairpin loop interactions

Herein, we report the identification of RNA hairpin loops that bind derivatives of kanamycin A, tobramycin, neamine, and neomycin B via two-dimensional combinatorial screening, a method that screens chemical and RNA spaces simultaneously. An arrayed aminoglycoside library was probed for binding to a 6-nucleotide RNA hairpin loop library (4096 members). Members of the loop library that bound each aminoglycoside were excised from the array, amplified and sequenced. Sequences were analyzed with our newly developed RNA Privileged Space Predictor (RNA-PSP) program, which analyzes selected sequences to identify statistically significant trends. RNA-PSP identified the following unique trends: 5′UNNNC3′ loops for the kanamycin A derivative (where N is any nucleotide); 5′UNNC3′ loops for the tobramycin derivative; 5′UNC3′ loops for the neamine derivative; and 5′UNNG3′ loops for the neomycin B derivative. The affinities and selectivities of a subset of the ligand–hairpin loop interactions were determined. The selected interactions have K_d values ranging from 10 nM to 605 nM. Selectivities ranged from 0.4 to >200-fold. Interestingly, the results from RNA-PSP are able to qualitatively predict specificity based on overlap between the RNA sequences selected for the ligands. These studies expand the information available on small molecule–RNA motif interactions, which could be useful to design ligands targeting RNA.     

Autotaxin structure–activity relationships revealed through lysophosphatidylcholine analogs

Autotaxin (ATX) catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to form the bioactive lipid lysophosphatidic acid (LPA). LPA stimulates cell proliferation, cell survival, and cell migration and is involved in obesity, rheumatoid arthritis, neuropathic pain, atherosclerosis and various cancers, suggesting that ATX inhibitors have broad therapeutic potential. Product feedback inhibition of ATX by LPA has stimulated structure–activity studies focused on LPA analogs. However, LPA displays mixed mode inhibition, indicating that it can bind to both the enzyme and the enzyme–substrate complex. This suggests that LPA may not interact solely with the catalytic site. In this report we have prepared LPC analogs to help map out substrate structure–activity relationships. The structural variances include length and unsaturation of the fatty tail, choline and polar linker presence, acyl versus ether linkage of the hydrocarbon chain, and methylene and nitrogen replacement of the choline oxygen. All LPC analogs were assayed in competition with the synthetic substrate, FS-3, to show the preference ATX has for each alteration. Choline presence and methylene replacement of the choline oxygen were detrimental to ATX recognition. These findings provide insights into the structure of the enzyme in the vicinity of the catalytic site as well as suggesting that ATX produces rate enhancement, at least in part, by substrate destabilization.     

Larkspur poisoning: toxicology and alkaloid structure–activity relationships

Systematic approaches to taxonomic classifications of the tall larkspur spp. have been developed using traditional chemical methods to profile alkaloids, comparison of relative toxicity of individual alkaloids, plant morphology/taxonomy and molecular genetics. Using these methods (papers published in this series) toxicology of three distinct species of tall larkspurs including Delphinium glaucum, Delphinium barbeyi and Delphinium occidentale is described. Tall larkspurs (Delphinium spp.) continue to be the most serious cause of cattle losses on mountain rangelands in the western US. Over 40 norditerpenoid alkaloids have been reported in species of larkspurs and toxicology data on 25 of these have been reported by the authors. These alkaloids can be classified into three general types based on their structural characteristics and toxicity: the N-(methylsuccinyl) anthranoyl lycoctonine (MSAL)-type, having high toxicity; the lycoctonine-type, with moderate toxicity; and the 7,8-methylenedioxylycoctonine (MDL)-type, of low toxicity. The structural importance of the methylsuccinimido anthranilic acid ester group at the C18 position is evident in the high toxicity of MSAL alkaloids, particularly methyllycaconitine (MLA), Nudicauline (NUD) and 14-deacetylnudicauline (14-DAN). Other structural aspects of these alkaloids such as the C14 functionality are also important, as demonstrated by the reduced toxicity of barbinine. MLA is the alkaloid of most importance in toxicity of larkspurs on mountain rangelands because of its prevalence in most larkspurs and high toxicity. While NUD and 14-DAN also possess high toxicity, they are relatively minor components in few larkspur species (generally the plains and low larkspurs), but when present at concentrations approaching 1 mg/g dry weight they contribute significantly to overall toxicity. Deltaline (DLT) is often found in high concentrations in many larkspurs but because of low toxicity, its contribution to larkspur poisoning in the field is relatively minor and it will probably not cause toxicosis in the absence of the MSAL-type alkaloids.     

Synthesis and structure–activity relationships of novel benzofuran farnesyltransferase inhibitors

A series of benzofuran-based farnesyltransferase inhibitors have been designed and synthesized as antitumor agents. Among them, 11f showed the most potent enzyme inhibitory activity (IC₅₀ = 1.1 nM) and antitumor activity in human cancer xenografts in mice.     

Computational identification of RNA functional determinants by three-dimensional quantitative structure–activity relationships

Anti-infection drugs target vital functions of infectious agents, including their ribosome and other essential non-coding RNAs. One of the reasons infectious agents become resistant to drugs is due to mutations that eliminate drug-binding affinity while maintaining vital elements. Identifying these elements is based on the determination of viable and lethal mutants and associated structures. However, determining the structure of enough mutants at high resolution is not always possible. Here, we introduce a new computational method, MC-3DQSAR, to determine the vital elements of target RNA structure from mutagenesis and available high-resolution data. We applied the method to further characterize the structural determinants of the bacterial 23S ribosomal RNA sarcin–ricin loop (SRL), as well as those of the lead-activated and hammerhead ribozymes. The method was accurate in confirming experimentally determined essential structural elements and predicting the viability of new SRL variants, which were either observed in bacteria or validated in bacterial growth assays. Our results indicate that MC-3DQSAR could be used systematically to evaluate the drug-target potentials of any RNA sites using current high-resolution structural data.     

Synthesis,characterization, antimicrobial activity and structure–activity relationships of new aryldisulfonamides

A series of aromatic disulfonamide (1-8) derivatives and 4-methylbenzenesulfonyl hydrazide (9) were synthesized and characterized. All compounds were evaluated in vitro for their antimicrobial activity against Staphylococcus aureus ATCC 25953, Bacillus cereus ATCC 6633, Bacillus magaterium RSKK 5117, Escherichia coli ATCC 11230, Salmonella enterititis ATCC 13076 by microdilution and disc diffusion methods. Antimicrobial activity of the aromatic disulfonamides decreased as the length of the carbon chain increased. An analysis of the structure- activity relationship (SAR) along with computational studies showed that the most active compound (9) possessed low lipophilicity (AlogP=0.59) and high solubility (logS = -1.33).     

New insights into viral structure and virus–cell interactions through proteomics

Although genomics techniques such as DNA microarrays have been widely used in virology, much more limited use has been made of proteomics. Although difficult, proteomics can greatly contribute to an understanding of virus–cell interactions, including the ternary structure of viral receptors at the cell surface, post-translational modifications and isoforms of critical viral and cellular proteins and even to the structure of viruses. Proteomics techniques also offer the potential for discovering markers for diagnostic and prognostic tests of viral infections in vivo. This review describes the use of several proteomic approaches for the analysis of HIV–cellular receptor interactions, the molecular mechanisms of transport of herpes simplex virus within neurons, and the structure of the tegument of herpes simplex virus.     

Synthesis and structure–activity relationships of cassiarin A as potential antimalarials with vasorelaxant activity

Cassiarin A 1, a tricyclic alkaloid, isolated from the leaves of Cassia siamea (Leguminosae), shows powerful antimalarial activity against Plasmodium falciparum in vitro as well as P. berghei in vivo, which may be valuable leads for novel antimalarials. Interactions of parasitized red blood cells (pRBCs) with endothelium in aorta are especially important in the processes contribute to the pathogenesis of severe malaria. Nitric oxide (NO) reduces endothelial expression of receptors/adhesion molecules used by pRBC to adhere to vascular endothelium, and reduces cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 showed vasorelaxation activity against rat aortic ring, which may be related with NO production. A series of a hydroxyl and a nitrogen-substituted derivatives and a dehydroxy derivative of 1 have been synthesized as having potent antimalarials against P. falciparum with vasodilator activity, which may reduce cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 exhibited a potent antimalarial activity and a high selectivity index in vitro, suggesting that the presence of a hydroxyl and a nitrogen atom without any substituents may be important to show antimalarial activity. Relative to cassiarin A, a methoxy derivative showed more potent vasorelaxant activity, although it did not show improvement for inhibition of P. falciparum in vitro. These cassiarin derivatives may be promising candidates as antimalarials with different mode of actions.     

Peptide deformylase inhibitors with retro-amide scaffold: Synthesis and structure–activity relationships

Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins. Thus inhibition of PDF activity is considered to be one of the most effective antibiotic strategies. Reported herein are the synthesis and structure–activity relationship studies of retro-amide inhibitors based on actinonin, a naturally occurring PDF inhibitor. Analysis of the structure–activity relationships led to the discovery of 7a, which exhibits potent enzyme inhibition and antibacterial activity against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis.     

Synthesis and structure–activity relationships of novel indazolyl glucocorticoid receptor partial agonists

SAR was used to further develop an indazole class of non-steroidal glucocorticoid receptor agonists aided by a GR LBD (ligand-binding domain)-agonist co-crystal structure described in the accompanying paper. Progress towards discovering a dissociated GR agonist guided by human in vitro assays biased the optimization of this compound series towards partial agonists that possessed excellent selectivity against other nuclear hormone receptors.     

Synthesis and structure–activity relationships of phenyl-substituted coumarins with anti-tubercular activity that target FadD32

In an effort to develop new and potent agents for therapy against tuberculosis, a high-throughput screen was performed against Mycobacterium tuberculosis strain H37Rv. Two 6-aryl-5,7-dimethyl-4-phenylcoumarin compounds 1a and 1b were found with modest activity. A series of coumarin derivatives were synthesized to improve potency and to investigate the structure–activity relationship of the series. Among them, compounds 1o and 2d showed improved activity with IC₉₀ of 2 μM and 0.5 μM, respectively. Further optimization provided compound 3b with better physiochemical properties with IC₉₀ 0.4 μM which had activity in a mouse model of infection. The role of the conformation of the 4- and 6-aryl substituents is also described.     

Design,synthesis and structure–activity relationships of novel biarylamine-based Met kinase inhibitors

Biarylamine-based inhibitors of Met kinase have been identified. Lead compounds demonstrate nanomolar potency in Met kinase biochemical assays and significant activity in the Met-driven GTL-16 human gastric carcinoma cell line. X-ray crystallography revealed that these compounds adopt a bioactive conformation, in the kinase domain, consistent with that previously seen with 2-pyridone-based Met kinase inhibitors. Compound 9b demonstrated potent in vivo antitumor activity in the GTL-16 human tumor xenograft model.     

Discovery and structure–activity relationships of a novel class of quinazoline glucokinase activators

We describe design, syntheses and structure–activity relationships of a novel class of 4,6-disubstituted quinazoline glucokinase activators. Prototype quinazoline leads (4 and 5) were designed based on the X-ray analyses of the previous 2-aminobenzamide lead classes. Modifications of the quinazoline leads led to the identification of a potent GK activator (21d).     

Synthesis and structure–activity relationships evaluation of benzothiazinone derivatives as potential anti-tubercular agents

N-Alkyl and heterocycle substituted 1,3-benzothiazin-4-one (BTZ) derivatives were synthesized. The anti-mycobacterial activities of these compounds were evaluated by determination of minimal inhibitory concentration (MIC) for Mycobacterium tuberculosis H37Ra and M. tuberculosis H37Rv. It was found that an extended or branched alkyl chain analog could enhance the potency, and activities of N-alkyl substituted BTZs were not affected by either nitro or trifluoromethyl at 6-position. Trifluoromethyl plays an important role in maintaining anti-tubercular activity in the piperazine or piperidine analogs. Compound 8o, which contains an azaspirodithiolane group, showed a MIC of 0.0001 μM against M. tuberculosis H37Rv, 20-fold more potent than BTZ043 racemate. These results suggested that the volume and lipophilicity of the substituents were important in maintaining activity. In addition, compound 8o was nontoxic to Vero cells and orally bioavailable in a preliminary pharmacokinetics study.     

Synthesis and structure–activity relationships of C-glycosylated oxadiazoles as inhibitors of glycogen phosphorylase

A series of per-O-benzoylated 5-β-d-glucopyranosyl-2-substituted-1,3,4-oxadiazoles was prepared by acylation of the corresponding 5-(β-d-glucopyranosyl)tetrazole. As an alternative, oxidation of 2,6-anhydro-aldose benzoylhydrazones by iodobenzene I,I-diacetate afforded the same oxadiazoles. 1,3-Dipolar cycloaddition of nitrile oxides to per-O-benzoylated β-d-glucopyranosyl cyanide gave the corresponding 5-β-d-glucopyranosyl-3-substituted-1,2,4-oxadiazoles. The O-benzoyl protecting groups were removed by base-catalyzed transesterification. The 1,3,4-oxadiazoles were practically inefficient as inhibitors of rabbit muscle glycogen phosphorylase b while the 1,2,4-oxadiazoles displayed inhibitory activities in the micromolar range. The best inhibitors were the 5-β-d-glucopyranosyl-3-(4-methylphenyl- and -2-naphthyl)-1,2,4-oxadiazoles (K_i = 8.8 and 11.6 μM, respectively). A detailed analysis of the structure–activity relationships is presented.     

Assessing the structure–activity relationships of fluorotelomer unsaturated acids and aldehydes with glutathione

Fluorotelomer alcohols (FTOHs) have been shown to degrade via abiotic and biotic mechanisms to perfluorocarboxylates (PFCAs) which are environmentally persistent and bioaccumulate in humans and wildlife depending on their chain length. Fluorotelomer unsaturated aldehydes (FTUALs) and acids (FTUCAs) are intermediate metabolites that form from the degradation of FTOHs. Their potential for toxicity is not yet defined and may be more significant compared to PFCAs. Past studies have shown that these intermediates form adducts with glutathione (GSH). The purpose of this study was to further assess the reactivity of these intermediate compounds. In vitro experiments were carried out in an aqueous buffer system (pH 7.4) where FTUCAs and FTUALs of varying chain lengths were reacted with GSH. To quantify the reactivity of FTUCAs and FTUALs, unreacted free GSH was derivatized with 5,5′-dithiobis(2-nitrobenzoic acid), its absorbance measured at 412 nm, and the percentage of unconjugated free GSH evaluated over time. EC₅₀ values were obtained for the reactions of GSH with acrolein and methyl methacrylate to assess the accuracy of the method, as well as for acrylic acid, FTUCAs, and FTUALs. The results of this study indicated that α,β-unsaturated aldehydes are comparatively the most reactive and reaction with GSH may be influenced by the length of the fluorinated tail. This is the first study to examine the relationship of FTUCAs and FTUALs with biological nucleophiles by quantifying their intrinsic reactivity.     

Thermozymes: biotechnology and structure–function relationships

Recent findings on the biochemical and molecular features of the following thermozymes are presented, based on their biotechnological use: α-amylase and amylopullulanase, used in starch processing; glucose isomerase, used in sweetener production; alcohol dehydrogenase, used in chemical synthesis; and alkaline phosphatase, used in diagnostics. The corresponding genes and recombinant proteins have been characterized in terms of sequence similarities, specific activities, thermophilicity, and unfolding kinetics. Site-directed and nested deletion mutagenesis were used to understand structure–function relationships. All these thermozymes display higher stability and activity than their counterparts currently used in the biotechnology industry. Received: January 22, 1998 / Accepted: February 16, 1998     

Ruthenium complexes endowed with potent anti-Trypanosoma cruzi activity: Synthesis,biological characterization and structure–activity relationships

Although effective against epimastigotes (proliferative form) and of low cytotoxicity in mammals, the aryl-4-oxothiazolylhydrazones (ATZ) display only limited activity against trypomastigotes (bloodstream form) of Trypanosoma cruzi. Considering the metal complexation approach with bioactive ligands as one possible strategy for improving the biological efficacy of ATZ, a set of eight new ruthenium–ATZ complexes (RuCl₂ATZCOD, COD is 1,5-cyclooctadiene) were prepared, chemically and biologically characterized, including in vitro assays against epimastigotes and trypomastigote forms of the parasite and also assessment of cytotoxicity in mammals. Two of these complexes presented antitrypanosomal activity at non-cytotoxic concentrations on mammalian cells and of higher potency than its metal–free ligands, while the metallic precursor [RuCl₂COD(MeCN)₂] showed only moderate antitrypanosomal activity. Comparative analysis between the ruthenium complexes and metal–free ligands demonstrated the usefulness of this approach, with the establishment of new SAR data. Additional pharmacological tests, including a DNA bond assay, gave rise to the proposal of a single preliminary explanation for the molecular origin of the bioactivity.