Development of novobiocin analogues that manifest anti-proliferative activity against several cancer cell lines

Recent studies have shown that the DNA gyrase inhibitor, novobiocin, binds to a previously unrecognized ATP-binding site located at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at approximately 700 microM. As a result of these studies, several analogues of the coumarin family of antibiotics have been reported and shown to exhibit increased Hsp90 inhibitory activity; however, the monomeric species lacked the ability to manifest anti-proliferative activity against cancer cell lines at concentrations tested. In an effort to develop more efficacious compounds that produce growth inhibitory activity against cancer cell lines, structure-activity relationships were investigated surrounding the prenylated benzamide side chain of the natural product. Results obtained from these studies have produced the first novobiocin analogues that manifest anti-proliferative activity against several cancer cell lines.     

The design, synthesis, and evaluation of coumarin ring derivatives of the novobiocin scaffold that exhibit antiproliferative activity

Novobiocin, a known DNA gyrase inhibitor, binds to a nucleotide-binding site located on the Hsp90 C-terminus and induces degradation of Hsp90-dependent client proteins at approximately 700 microM in breast cancer cells (SKBr3). Although many analogues of novobiocin have been synthesized, it was only recently demonstrated that monomeric species exhibit antiproliferative activity against various cancer cell lines. To further refine the essential elements of the coumarin core, a series of modified coumarin derivatives was synthesized and evaluated to elucidate structure-activity relationships for novobiocin as an anticancer agent. Results obtained from these studies have produced novobiocin analogues that manifest low micromolar activity against several cancer cell lines.     

A library of noviosylated coumarin analogues

The DNA gyrase inhibitor, novobiocin, was recently shown to inhibit Hsp90 via a previously unrecognized C-terminal ATP-binding site. Previous structure-activity relationship studies identified key moieties that appear important for Hsp90 inhibitory activity. In an effort to provide a more efficacious lead compound, a parallel library of noviosylated coumarin analogues was prepared.     

Synthesis and evaluation of coumermycin A1 analogues that inhibit the Hsp90 protein folding machinery

[structure: see text] The coumarin antibiotics are not only potent inhibitors of DNA gyrase but also represent the most effective C-terminal inhibitors of 90 kDa heat shock proteins (Hsp90) reported thus far. In contrast to the N-terminal ATP-binding site, little is known about the Hsp90 C-terminus. In addition, very limited structure-activity relationships exist between this class of natural products and Hsp90. In this letter, the syntheses of dimeric coumarin analogues are presented along with their inhibitory values in breast cancer cell lines.     

In vitro and in vivo production of new aminocoumarins by a combined biochemical, genetic, and synthetic approach

The aminocoumarin antibiotics clorobiocin, novobiocin, and coumermycin A(1) are inhibitors of bacterial gyrase. Their chemical structures contain amide bonds, formed between an aminocoumarin ring and an aromatic acyl component, which is 3-dimethylallyl-4-hydroxybenzoate in the case of novobiocin and clorobiocin. These amide bonds are formed under catalysis of the gene products of cloL, novL, and couL, respectively. We first examined the substrate specificity of the purified amide synthetases CloL, NovL, and CouL for the various analogs of the prenylated benzoate moiety. We then generated new aminocoumarin antibiotics by feeding synthetic analogs of the 3-dimethylallyl-4-hydroxybenzoate moiety to a mutant strain defective in the biosynthesis of the prenylated benzoate moiety. This resulted in the formation of 32 new aminocoumarin compounds. The structures of these compounds were elucidated using FAB-MS and (1)H-NMR spectroscopy.     

Amycolamicin: A Novel Broad‐Spectrum Antibiotic Inhibiting Bacterial Topoisomerase

The abuse of antibacterial drugs imposes a selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens. Our efforts to discover novel classes of antibiotics to combat these pathogens resulted in the discovery of amycolamicin (AMM). The absolute structure of AMM was determined by NMR spectroscopy, X‐ray analysis, chemical degradation, and modification of its functional groups. AMM consists of trans‐decalin, tetramic acid, two unusual sugars (amycolose and amykitanose), and dichloropyrrole carboxylic acid. The pyranose ring named as amykitanose undergoes anomerization in methanol. AMM is a potent and broad‐spectrum antibiotic against Gram‐positive pathogenic bacteria by inhibiting DNA gyrase and bacterial topoisomerase IV. The target of AMM has been proved to be the DNA gyrase B subunit and its binding mode to DNA gyrase is different from those of novobiocin and coumermycin, the known DNA gyrase inhibitors.     

New aminocoumarin antibiotics formed by a combined mutational and chemoenzymatic approach utilizing the carbamoyltransferase NovN

Five new aminocoumarin antibiotics were produced by a combined mutational and chemoenzymatic approach. For this purpose, the 3"-carbamoyltransferase NovN from the novobiocin producer Streptomyces spheroides was overexpressed in the heterologous host S. lividans as an N-terminal His(6) fusion protein and purified by nickel affinity chromatography. Five different 3"-unsubstituted aminocoumarin derivatives were isolated from mutants of the clorobiocin producer S. roseochromogenes, carrying single or multiple gene defects. All five compounds were readily accepted as substrates by NovN, and the 3"-carbamoylated products were isolated on a preparative scale. Their structures were elucidated by (1)H-NMR and mass spectroscopy, and their inhibitory activity on gyrase in vitro as well as their antibacterial activity was determined. The results give further insight into the structure-activity relationships of aminocoumarin antibiotics.     

Molecular design of anticancer drug leads based on three-dimensional quantitative structure-activity relationship

Heat shock protein 90 (Hsp90) takes part in the developments of several cancers. Novobiocin, a typically C-terminal inhibitor for Hsp90, will probably used as an important anticancer drug in the future. In this work, we explored the valuable information and designed new novobiocin derivatives based on a three-dimensional quantitative structure-activity relationship (3D QSAR). The comparative molecular field analysis and comparative molecular similarity indices analysis models with high predictive capability were established, and their reliabilities are supported by the statistical parameters. Based on the several important influence factors obtained from these models, six new novobiocin derivatives with higher inhibitory activities were designed and confirmed by the molecular simulation with our models, which provide the potential anticancer drug leads for further research.     

Hsp90 inhibitors identified from a library of novobiocin analogues

Novobiocin is a C-terminal inhibitor of the Hsp90 protein folding machinery, which is responsible for the conformational maturation of numerous proteins involved in cancer growth and survival. Due to novobiocin's poor inhibitory activity ( approximately 700 muM), very little attention has been paid toward the development of novobiocin analogues for Hsp90 inhibition. In this study, a parallel library of 20 novobiocin derivatives was prepared and the biological activity of each evaluated by Western blot analysis of Hsp90 client proteins. A4 was found to be a potent inhibitor of Hsp90 as determined by its ability to cause the degradation of several Hsp90 client proteins in both breast and prostate cancer cell lines. In the presence of 1 muM A4, several Hsp90 client proteins were degraded, including AKT, Her2, Hif-1alpha, and the androgen receptor.     

Coumarin formation in novobiocin biosynthesis: beta-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI

BACKGROUND: Coumarin group antibiotics, such as novobiocin, coumermycin A1 and clorobiocin, are potent inhibitors of DNA gyrase. These antibiotics have been isolated from various Streptomyces species and all possess a 3-amino-4-hydroxy-coumarin moiety as their structural core. Prior labeling experiments on novobiocin established that the coumarin moiety was derived from L-tyrosine, probably via a beta-hydroxy-tyrosine (beta-OH-Tyr) intermediate. Recently the novobiocin gene cluster from Streptomyces spheroides was cloned and sequenced and allows analysis of the biosynthesis of the coumarin at the biochemical level using overexpressed and purified proteins. RESULTS: Two open reading frames (ORFs), NovH and NovI, from the novobiocin producer S. spheroides have been overexpressed in Escherichia coli, purified and characterized for tyrosine activation and oxygenation which are the initial steps in coumarin formation. The 65 kDa NovH has two predicted domains, an adenylation (A) and a peptidyl carrier protein (PCP), reminiscent of non-ribosomal peptide synthetases. Purified NovH catalyzes L-tyrosyl-AMP formation by its A domain, can be posttranslationally phosphopantetheinylated on the PCP domain, and accumulates the covalent L-tyrosyl-S-enzyme intermediate on the holo PCP domain. The second enzyme in the pathway, NovI, is a 45 kDa heme protein that functions as a cytochrome P450-type monooxygenase with specificity for the tyrosyl-S-NovH acyl enzyme. The product beta-OH-tyrosyl-S-NovH was detected by alkaline release and high performance liquid chromatography analysis of radioactive [3H]beta-OH-Tyr and by mass spectrometry. Also detected was 4-OH-benzaldehyde, a retro aldol breakdown product of beta-OH-Tyr. The amino acid released was (3R,2S)-3-OH-Tyr by comparison with authentic standards. CONCLUSIONS: This work establishes that NovH and NovI are responsible for the formation of a beta-OH-Tyr intermediate that is covalently tethered to NovH in novobiocin biosynthesis. Comparable A-PCP/P450 pairs for amino acid beta-hydroxylation are found in various biosynthetic gene clusters, such as ORF19/ORF20 in the chloroeremomycin cluster for tyrosine, CumC/CumD in the coumermycin A1 cluster for tyrosine, and NikP1/NikQ in the nikkomycin cluster for histidine. This phenomenon of covalent docking of the amino acid in a kinetically stable thioester linkage prior to chemical modification by downstream tailoring enzymes, could represent a common strategy for controlling the partitioning of the amino acid for incorporation into secondary metabolites.     

Synthesis and Biological Evaluation of Novobiocin Core Analogues as Hsp90 Inhibitors

Development of heat shock protein 90 (Hsp90) C‐terminal inhibitors has emerged as an exciting strategy for the treatment of cancer. Previous efforts have focused on modifications to the natural products novobiocin and coumermycin. Moreover, variations in both the sugar and amide moieties have been extensively studied, whereas replacements for the coumarin core have received less attention. Herein, 24 cores were synthesized with varying distances and angles between the sugar and amide moieties. Compounds that exhibited good anti‐proliferative activity against multiple cancer cell lines and Hsp90 inhibitory activity, were those that placed the sugar and amide moieties between 7.7 and 12.1 Å apart along with angles of 180°.     

Synthesis of a versatile metacyclophane macrolactam

As Hsp90 has emerged as a promising target for the development of cancer chemotherapeutics, so has the need for systematic evaluation of structure-activity relationships between natural product inhibitors and this molecular chaperone. Utilizing our chimera approach, which encompasses the quinone moiety of geldanamycin and the resorcinol moiety of radicicol, molecules have been produced that are highly effective inhibitors of the Hsp90 protein folding machinery. These chimeric inhibitors contain metacyclophane macrolactams that are difficult to cyclize and modify for incorporation of functional diversity. To circumvent this problem, a highly diversifiable α-bromo-α,β-unsaturated ester has been prepared, which allows for the introduction of various functionalities that enable elucidation of structure-activity relationships between chimeric compounds and Hsp90. The rationale, synthesis, and optimization for such a molecule is reported herein.     

Synthesis and Characterization of PEG‐Based Drug‐Responsive Biohybrid Hydrogels

Interactive materials being responsive to a biocompatible stimulus represent a promising approach for future therapeutic applications. In this study, we present a novel biohybrid material synthesized from biocompatible components being stimulus‐responsive to the pharmaceutically approved small‐molecule novobiocin. The hydrogel design is based on the gyrase B (GyrB) protein, which is covalently grafted to multi‐arm polyethylene glycol (PEG) using a Michael‐type addition reaction. Upon addition of the GyrB‐dimerizing substance coumermycin, stable hydrogels form which can be dissolved in a dose‐adjustable manner by the antibiotic novobiocin. The switchable properties of this PEG‐based hydrogel are favorable for future applications in tissue engineering and as externally controlled drug depot.     

Covalent CouN7 enzyme intermediate for acyl group shuttling in aminocoumarin biosynthesis

The last stages of assembly of the aminocoumarin antibiotics, clorobiocin and coumermycin A(1), which target the GyrB subunits of bacterial DNA gyrase, involve enzymatic transfer of the pyrrolyl-2-carbonyl acyl group from a carrier protein (CloN1/CouN1) to the 3'-OH of the noviosyl moiety of the antibiotic scaffold. The enzyme, CouN7, will catalyze both the forward and back reaction on both arms of the coumermycin scaffold. This occurs via an O-acyl-Ser(101)-CouN7 intermediate, as shown by transient labeling of the enzyme with [(14)C]acetyl-S-CouN1 as donor and by inactivating mutation of the active site, Ser(101), to Ala. The intermediacy of the pyrrolyl-2-carbonyl-O-CouN7 allows net pyrrole transfer between distinct aminocoumarin scaffolds, for example, between the descarbamoylnovobiocin scaffold and coumermycin A(1) and vice versa. CouN7 also allows shuttling of surrogate acyl groups between noviosyl-aminocoumarin scaffolds to generate new antibiotic variants.     

Indeno[1,2-b]pyridin-5-one derivatives containing azo groups and their hydrazonal precursors: Synthesis,antimicrobial profile,DNA gyrase binding affinity,and molecular docking

The prevalence of germs that are resistant to many antibiotics is rising rapidly the world over. There is a large group of researchers actively looking for better medicines. Here, we designed two series of hydrazonal and indeno[1,2-b]pyridin-5-one bearing hydrazone and azo-groups to test their antimicrobial activity. Molecular structures of all derivatives were assured based on their spectral data and elemental analyses. Results of the antimicrobial activity of the tested hydrazone and azo compounds showed promising potential for several derivatives. The minimum inhibitory concentrations (MICs) of hydrazones 4a - h and 6a - g displayed good antibacterial reactivities with a range of 3.91–250 μg/mL and moderate antifungal activity with a range of 15.6–500 μg/mL. The most promising hydrazone 4f and azo- 6a compounds demonstrated MIC values against Streptococcus faecalis and Escherichia coli equal to 3.91 and 7.81 μg/mL, respectively. Moreover, azo compound 6a showed MIC value equal to 3.91 μg/mL against Enterobacter cloacae species. Additionally, derivative 4f exhibited a significant inhibitory profile against the E. coli gyrase A enzyme (IC₅₀ = 5.53 μg/mL). On the other hand, compound 6a (IC₅₀ 14.05 μg/mL) exhibited the lowest DNA gyrase inhibitory activity as compared to compounds 4f and reference standard drug novobiocin, IC₅₀ 5.53 and 1.88 μg/mL, respectively. Pharmacokinetic and pharmacodynamic profiles and molecular docking studies for the two most promising molecules 4f and 6a were computed and revealed that both compounds have good ADME profiles and high binding affinity to DNA gyrase binding site.     

Enantiospecific synthesis of (−)-d-noviose from (−)-pantolactone

Noviose, the rare sugar moiety present in novobiocin, is an important antimicrobial and antitumor agent that has been shown to inhibit DNA gyrase and HSP90. An enantiospecific synthesis of d-(−)-noviose starting from commercially available (−)-pantolactone is reported.     

Novel 1,2,4-oxadiazole/pyrrolidine hybrids as DNA gyrase and topoisomerase IV inhibitors with potential antibacterial activity

DNA gyrase is a promising target for antibacterial agents. Several classes of small-molecule inhibitors have been discovered in recent decades, but none of these have reached the market. We have designed a small library of 1,2,4-oxadiazole/pyrrolidine hybrids with mid nanomolar inhibitory and potent antibacterial activities against DNA gyrase and topoisomerase IV. Compounds 9, 15, 16, 19, and 21 inhibited Escherichia coli DNA gyrase to a similar extent as the reference compound, novobiocin, with inhibitory values ranging from 120 nM to 270 nM. Compound 16 was one of the most potent compounds in the series, with an IC₅₀ value of 120 nM against E. coli gyrase, which is lower than the IC₅₀ value of novobiocin (170 nM). Compound 16 had the highest inhibitory activity, with minimum inhibitory concentrations (MIC) of 24 and 62 ng/mL against Staphylococcus aureus and E. coli, respectively, which compared favorably with ciprofloxacin (30 and 60 ng/mL, respectively). Compounds 9, 15, 19, and 21 were similar to novobiocin in terms of their activity against E. coli and S. aureus topoisomerase IV, while compound 16 was more potent than novobiocin.     

Di-isatin heteronuclear compounds and their antibacterial activity

Twenty propylene and butylene tethered di-isatin heteronuclear compounds 5a-t were synthesized, and their antibacterial activities were evaluated. Most of the synthesized di-isatin heteronuclear derivatives were active against both Gram-positive and Gram-negative strains, and some of them exhibited considerable activities against drug-resistant organisms. In particular, di-isatin 5a (MIC: 32-512 μg/mL) was more active than the reference vancomycin against Gram-negative pathogens, demonstrating the antibacterial potential of di-isatin heteronuclear compounds. The inhibitory activity of di-isatin 5a was higher than mono-isatin against Escherichia coli DNA gyrase, suggesting the dimers could improve the inhibitory activity against DNA gyrase when compared with the isatin. The structure-activity relationship was discussed to provide an insight for rational designs of more efficient antibacterial candidates.     

具有全新机理的DNA旋转酶抑制剂的筛选及抑菌活性

利用具有新机制的抗耐药菌DNA旋转酶抑制剂GSK299423与DNA旋转酶的晶体复合物(PDB code:2XCS)构建基于配体-受体复合物的药效团模型, 诱骗集(Decoy set)验证结果表明该药效团模型具有较强的活性识别能力. 将药效团模型与分子对接相结合用于筛选化合物库, 通过抑菌活性测定, 获得了具有抗多药耐药菌活性的DNA旋转酶抑制剂LTH02.     

Biotinylated geldanamycin

Inhibition of the 90 kDa heat shock proteins (Hsp90) represents a promising new chemotherapeutic approach for the treatment of several cancers. Hsp90 is essential to the survival of cancer cells and is inhibited by members of the ansamycin family of antibiotics. In particular, the quinone-containing antibiotics geldanamycin (GDA) and herbimycin A inhibit Hsp90 function in vitro at low micromolar concentrations via interaction with an ATP binding domain. Many proteins bind ATP, and the discovery of selective Hsp90 inhibitors requires the identification of other proteins that bind GDA and may cause undesired effects. Biotinylated analogues of GDA with varying tether lengths have been synthesized to elucidate other proteins that competitively bind GDA. Analogues containing a photolabile tether have also been prepared as a complementary method for the removal of GDA-bound proteins from neutravidin-containing resin. Preliminary studies indicate several proteins other than Hsp90 are isolated with biotinylated GDA.