Computational Prediction of the Mode of Binding of Antitumor Lankacidin C to Tubulin.
ABSTRACT: Lankacidin C, which is an antibiotic produced by the organism Streptomyces rochei, shows considerable antitumor activity. The mechanism of its antitumor activity remained elusive for decades until it was recently shown to overstabilize microtubules by binding at the taxol binding site of tubulin, causing mitotic arrest followed by apoptosis. However, the exact binding mode of lankacidin C inside the tubulin binding pocket remains unknown, an issue that impedes proper structure-based design, modification, and optimization of the drug. Here, we have used computational methods to predict the most likely binding mode of lankacidin C to tubulin. We employed ensemble-based docking in different software packages, supplemented with molecular dynamics simulation and subsequent binding-energy prediction. The molecular dynamics simulations performed on lankacidin C were collectively 1.1 ?s long. Also, a multiple-trajectory approach was performed to assess the stability of different potential binding modes. The identified binding mode could serve as an ideal starting point for structural modification and optimization of lankacidin C to enhance its affinity to the tubulin binding site and therefore improve its antitumor activity.
Project description:Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.
Project description:Our previous studies revealed that the srrX and srrA genes carried on the large linear plasmid pSLA2-L constitute a gamma-butyrolactone-receptor system in Streptomyces rochei. Extensive transcriptional analysis has now showed that the Streptomyces antibiotic regulatory protein gene srrY, which is also carried on pSLA2-L, is a target of the receptor/repressor SrrA and plays a central role in lankacidin and lankamycin production. The srrY gene was expressed in a growth-dependent manner, slightly preceding antibiotic production. The expression of srrY was undetectable in the srrX mutant but was restored in the srrX srrA double mutant. In addition, SrrA was bound specifically to the promoter region of srrY, and this binding was prevented by the addition of the S. rochei gamma-butyrolactone fraction, while the W119A mutant receptor SrrA was kept bound even in the presence of S. rochei gamma-butyrolactone. Furthermore, the introduction of an intact srrY gene under the control of a foreign promoter into the srrX or srrA(W119A) mutant restored antibiotic production. All of these results confirmed the signaling pathway from srrX through srrA to srrY, leading to lankacidin and lankamycin production.
Project description:Streptomyces rochei 7434AN4, a producer of lankacidin (LC) and lankamycin (LM), carries many regulatory genes including a biosynthesis gene for signaling molecules SRBs (srrX), an SRB receptor gene (srrA), and a SARP (Streptomyces antibiotic regulatory protein) family activator gene (srrY). Our previous study revealed that the main regulatory cascade goes from srrX through srrA to srrY, leading to LC production, whereas srrY further regulates a second SARP gene srrZ to synthesize LM. In this study we extensively investigated the function of srrB, a pseudo-receptor gene, by analyzing antibiotic production and transcription. Metabolite analysis showed that the srrB mutation increased both LC and LM production over four-folds. Transcription, gel shift, and DNase I footprinting experiments revealed that srrB and srrY are expressed under the SRB/SrrA regulatory system, and at the later stage, SrrB represses srrY expression by binding to the promoter region of srrY. These findings confirmed that SrrB acts as a negative regulator of the activator gene srrY to control LC and LM production at the later stage of fermentation in S. rochei.
Project description:The structures of the large ribosomal subunit of Deinococcus radiodurans (D50S) in complex with the antibiotic lankamycin (3.2 Å) and a double antibiotic complex of lankamycin and lankacidin C (3.45 Å) have been determined, in continuation of previous crystallographic studies on lankacidin-D50S complex. These two drugs have been previously reported to inhibit ribosomal function with mild synergistic effect. Lankamycin, a member of the macrolide family, binds in a similar manner to erythromycin. However, when in complex with lankacidin, lankamycin is located so that it can form interactions with lankacidin in the adjacent ribosomal binding site. When compared to the well-documented synergistic antibiotics, Streptogramins A and B, the pair of lankacidin and lankamycin bind in similar sites, the peptidyl transferase center and nascent peptide exit tunnel, respectively. Herein, we discuss the structural basis for antibiotic synergism and highlight the key factors involved in ribosomal inhibition.
Project description:Acquisition of new catalytic activity is a relatively rare evolutionary event. A striking example appears in the pathway to the antibiotic lankacidin, as a monoamine oxidase (MAO) family member, LkcE, catalyzes both an unusual amide oxidation, and a subsequent intramolecular Mannich reaction to form the polyketide macrocycle. We report evidence here for the molecular basis for this dual activity. The reaction sequence involves several essential active site residues and a conformational change likely comprising an interdomain hinge movement. These features, which have not previously been described in the MAO family, both depend on a unique dimerization mode relative to all structurally characterized members. Taken together, these data add weight to the idea that designing new multifunctional enzymes may require changes in both architecture and catalytic machinery. Encouragingly, however, our data also show LkcE to bind alternative substrates, supporting its potential utility as a general cyclization catalyst in synthetic biology.
Project description:Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics, lankacidin and lankamycin, and carries three linear plasmids, pSLA2-L (211?kb), -M (113?kb), and -S (18?kb), whose nucleotide sequences were previously reported. The complete nucleotide sequence of the S. rochei chromosome has now been determined using the long-read PacBio RS-II sequencing together with short-read Illumina Genome Analyzer IIx sequencing and Roche 454 pyrosequencing techniques. The assembled sequence revealed an 8,364,802-bp linear chromosome with a high G?+?C content of 71.7% and 7,568 protein-coding ORFs. Thus, the gross genome size of S. rochei 7434AN4 was confirmed to be 8,706,406?bp including the three linear plasmids. Consistent with our previous study, a tap-tpg gene pair, which is essential for the maintenance of a linear topology of Streptomyces genomes, was not found on the chromosome. Remarkably, the S. rochei chromosome contains seven ribosomal RNA (rrn) operons (16S-23S-5S), although Streptomyces species generally contain six rrn operons. Based on 2ndFind and antiSMASH platforms, the S. rochei chromosome harbors at least 35 secondary metabolite biosynthetic gene clusters, including those for the 28-membered polyene macrolide pentamycin and the azoxyalkene compound KA57-A.
Project description:Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics lankacidin and lankamycin, and their biosynthesis is tightly controlled by butenolide-type signaling molecules SRB1 and SRB2. SRBs are synthesized by SRB synthase SrrX, and induce lankacidin and lankamycin production at 40 nM concentration. We here investigated the role of a P450 monooxygenase gene srrO (orf84), which is located adjacent to srrX (orf85), in SRB biosynthesis. An srrO mutant KA54 accumulated lankacidin and lankamycin at a normal level when compared with the parent strain. To elucidate the chemical structures of the signaling molecules accumulated in KA54 (termed as KA54-SRBs), this mutant was cultured (30 L) and the active components were purified. Two active components (KA54-SRB1 and KA54-SRB2) were detected in ESI-MS and chiral HPLC analysis. The molecular formulae for KA54-SRB1 and KA54-SRB2 are C15H26O4 and C16H28O4, whose values are one oxygen smaller and two hydrogen larger when compared with those for SRB1 and SRB2, respectively. Based on extensive NMR analysis, the signaling molecules in KA54 were determined to be 6'-deoxo-SRB1 and 6'-deoxo-SRB2. Gel shift analysis indicated that a ligand affinity of 6'-deoxo-SRB1 to the specific receptor SrrA was 100-fold less than that of SRB1. We performed bioconversion of the synthetic 6'-deoxo-SRB1 in the Streptomyces lividans recombinant carrying SrrO-expression plasmid. Substrate 6'-deoxo-SRB1 was converted through 6'-deoxo-6'-hydroxy-SRB1 to SRB1 in a time-dependent manner. Thus, these results clearly indicated that SrrO catalyzes the C-6' oxidation at a final step in SRB biosynthesis.
Project description:New target compounds were designed as inhibitors of tubulin polymerization relying on using two types of ring B models (cyclohexenone and indazole) to replace the central ring in colchicine. Different functional groups (R1) were attached to manipulate their physicochemical properties and/or their biological activity. The designed compounds were assessed for their antitumor activity on HCT-116 and MCF-7 cancer cell lines. Compounds 4b, 5e and 5f exhibited comparable or higher potency than colchicine against colon HCT-116 and MCF-7 tumor cells. The mechanism of the antitumor activity was investigated through evaluating the tubulin inhibition potential of the active compounds. Compounds 4b, 5e and 5f showed percentage inhibition of tubulin in both cell line homogenates ranging from 79.72% to 89.31%. Cell cycle analysis of compounds 4b, 5e and 5f revealed cell cycle arrest at G2/M phase. Molecular docking revealed the binding mode of these new compounds into the colchicine binding site of tubulin.[Formula: see text].
Project description:Aiming at development of potent antitubulin agents targeting colchicine-binding site, a series of novel 5-indolyl-7-arylimidazo[1,2-a]pyridine-8-carbonitrilederivatives (5a-5v and 7a-7h) were designed based on bioisosterism and hybridization strategies. All these compounds were concisely synthesized via a three-step process and examined against five human cancer cell lines (HT-29, A549, MKN-45, MDA-MB-231 and SMMC-7721) along with a normal human cell (L02) in vitro. A structure-activity relationships (SARs) study was carried out and optimization towards this series of compounds in cellular assay resulted in the discovery of 5k, which displayed similar or better antitumor potency against the tested cancer cells with IC50 value ranging from 0.02 to 1.22??M superior to CA-4 and Crolibulin. Significantly, a cell cycle study disclosed the ability of 5k to arrest cell cycle at the G2/M phase, and immunofluorescence assay as well as a colchicine competition assay revealed that tubulin polymerization was disturbed by 5k by binding to the colchicine site. Moreover, the molecular modeling mode showed the posture of 5k and Crolibulin was similar in the colchcine-binding pocket of tubulin as identified with the SARs and pharmacological results. Together, all these results rationalized 5k might serve as a promising lead for a novel class of antitubulin agents for cancer treatments.
Project description:Microtubules are unique cytoskeletal structures that have structural subunits of αβ tubulin. Taxol is a typical microtubule stabilizing drug. The epothilones are other natural products with similar mechanism of action totaxol. Despite the highly conserved nature of β-tubulin, some organism like Saccharomyces cerevesia (S.cerevesia) is resistance to taxol, but sensitive to epothilones. In order to find differences in sensitivity of yeast tubulin to these molecules, we investigated binding mode of the taxol and epothilone A to yeast tubulin using molecular modeling. The multiple sequence alignment of β-tubulin of different species was performed using ClustalW2. Protein structure of yeast β-tubulin was constructed with Swiss Model 8.05 by using 1TVK. Modeled tubulin was superimposed with PyMol on1JFF for comparison of three-dimensional structure of two proteins. Our results showed that one of the most interesting differences in binding mode of these molecules is residue 227. The His227 in bovine makes a hydrogen bond by means of its δ-nitrogen with epothilone A and by means of its ɛ-nitrogen with taxol. The Asn227 of yeast can play role of the δ-nitrogen of imidazole ring of H227, but not of ɛ-nitrogen of it. So yeast tubulin in contrast to taxol can interact with epothilone A. Due to conservation of essential residues for binding (T274, R282 and Q292), epothilone A in comparison with taxol can tolerate the interchange in the binding pocket (R276I). Our findings may be of a great aid in the rational design of antitumor agents that bind to the taxol binding region of tubulin.