Recombinant strains for the enhanced production of bioengineered rapalogs.

The rapK gene required for biosynthesis of the DHCHC starter acid that initiates rapamycin biosynthesis was deleted from strain BIOT-3410, a derivative of Streptomyces rapamycinicus which had been subjected to classical strain and process development and capable of robust rapamycin production at titres up to 250mg/L. The resulting strain BIOT-4010 could no longer produce rapamycin, but when supplied exogenously with DHCHC produced rapamycin at titres equivalent to its parent strain. This strain enabled mutasynthetic access to new rapalogs that could not readily be isolated from lower titre strains when fed DHCHC analogs. Mutasynthesis of some rapalogs resulted predominantly in compounds lacking late post polyketide synthase biosynthetic modifications. To enhance the relative production of fully elaborated rapalogs, genes encoding late-acting biosynthetic pathway enzymes which failed to act efficiently on the novel compounds were expressed ectopically to give strain BIOT-4110. Strains BIOT-4010 and BIOT-4110 represent valuable tools for natural product lead optimization using biosynthetic medicinal chemistry and for the production of rapalogs for pre-clinical and early stage clinical trials.

Discovery and assembly-line biosynthesis of the lymphostin pyrroloquinoline alkaloid family of mTOR inhibitors in Salinispora bacteria.

The pyrroloquinoline alkaloid family of natural products, which includes the immunosuppressant lymphostin, has long been postulated to arise from tryptophan. We now report the molecular basis of lymphostin biosynthesis in three marine Salinispora species that maintain conserved biosynthetic gene clusters harboring a hybrid nonribosomal peptide synthetase-polyketide synthase that is central to lymphostin assembly. Through a series of experiments involving gene mutations, stable isotope profiling, and natural product discovery, we report the assembly-line biosynthesis of lymphostin and nine new analogues that exhibit potent mTOR inhibitory activity.

Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities.

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the beta1-subunit of L-type voltage-dependent Ca(2+) channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca(2+) channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca(2+)-induced cell death by modulating Ca(2+) channels and promote neurite outgrowth via FKBP52 binding.

Depsides isolated from the Sri Lankan lichen Parmotrema sp. exhibit selective Plk1 inhibitory activity.

CONTEXT: Mitotic kinase enzymes regulate critical stages of mitosis and are amenable to pharmacological inhibition. Since natural products have been a rich source of antimitotic inhibitors, we postulated that natural products would also provide effective inhibitors of mitotic kinases. OBJECTIVE: To explore unique marine and terrestrial natural product sources for new anticancer drug leads, we screened our natural product extract library for polo-like kinase-1 (Plk1) kinase inhibitors. MATERIALS AND METHODS: Extracts of the lichen Parmotrema sp. (Parmeliaceae) exhibited in vitro inhibitory activity. Bioassay-guided fractionation of the Parmotrema sp. extract led to the isolation of depside inhibitors. RESULTS: A new depside 1 has been isolated from the Sri Lankan lichen Parmotrema sp. along with the known metabolites 2 (ß-collatolic acid) and 3 (ß-alectoronic acid). The structure of depside 1 was elucidated by spectroscopic analysis. The three depsides 1-3 exhibited moderate inhibition of purified recombinant Plk1 kinase with IC50 of 2.8, 0.7, and 1.7 µM, respectively, at 1 µM ATP. Inhibitory activity was also observed at high concentrations of ATP, suggesting the potential for activity in a cellular environment. The depsides were also tested against a panel of 23 other recombinant kinases and were found to possess up to 30-fold selectivity toward Plk1. DISCUSSION AND CONCLUSION: These data suggest that the depsides 1-3 may serve as core structures that can be further explored as potential inhibitors of Plk1 and other kinases.

Stereoselective synthesis of an active metabolite of the potent PI3 kinase inhibitor PKI-179.

The synthesis and stereochemical determination of 1-(4-(4-((1R,5R,6R)-6-hydroxy-3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-yl)phenyl)-3-(pyridin-4-yl)urea (2), an active metabolite of the potent PI3 kinase inhibitor PKI-179 (1), is described. Stereospecific hydroboration of the double bond of 2,5-dihydro-1H-pyrrole 8 gave the 2,3-trans alcohol 9 exclusively. The configuration of the 3-hydroxyl group in 9 was inverted by an oxidation and stereoselective reduction sequence to give the corresponding 2,3-cis isomer 23. Both exo (21) and endo (27) isomers of the metabolite 2 were prepared via a practical synthetic route from 9 and 23, respectively, and the stereochemistry of 2 was determined to be endo. The endo isomer (27) was separated into two enantiomers 28 and 29 by chiral HPLC. Compound 2 was found to be enantiomerically pure and identical to the enantiomer 28. The absolute stereochemistry of the enantiomer 28 was determined by Mosher's method, thus establishing the stereochemistry of the active metabolite 2.

Fermentative production of self-toxic fungal secondary metabolites.

Fungi are well known for their vast diversity of secondary metabolites that include many life-saving drugs and highly toxic mycotoxins. In general, fungal cultures producing such metabolites are immune to their toxic effects. However, some are known to produce self-toxic compounds that can pose production optimization challenges if the metabolites are needed in large amounts for chemical modification. One such culture, LV-2841, was identified as the lead for one of our exploratory projects. This culture was found to be a slow grower that produced trace amounts of a known metabolite, cercosporamide, under the standard flask fermentation conditions, and extensive medium optimization studies failed to yield higher titers. Poor growth of the culture in liquid media was attributed to the self-toxicity of cercosporamide to the producing organism, and the minimum inhibitory concentration (MIC) of cercosporamide was estimated to be in the range of 8-16 microg/ml. Fermentations carried out in media containing Diaion HP20 resin afforded significantly higher titers of the desired compound. While several examples of resin-based fermentations of soil streptomyces have been published, this approach has rarely been used for fungal fermentations. Over a 100-fold increase in the production titer of cercosporamide, a self-toxic secondary metabolite, was achieved by supplementing the production medium with a commercially available neutral adsorbent resin.

Fibrosterol sulfates from the Philippine sponge Lissodendoryx (Acanthodoryx) fibrosa: sterol dimers that inhibit PKCzeta.

Three new sulfated sterol dimers, fibrosterol sulfates A-C (1-3), have been isolated from the sponge Lissodendoryx (Acanthodoryx) fibrosa, collected in the Philippines. The structures were assigned on the basis of extensive 1D and 2D NMR studies as well as analysis by HRESIMS. Compounds 1 and 2 inhibited PKCzeta with IC(50) values of 16.4 and 5.6 microM, respectively.

The lecanindoles, nonsteroidal progestins from the terrestrial fungus Verticillium lecanii 6144.

Four new indolosesquiterpenes, lecanindoles A-D (1-4), were isolated from fermentations of the terrestrial fungus Verticillium lecanii 6144. The structures of compounds 1-4 were elucidated from analysis of spectroscopic data. Compound 2 was reduced to give 4 and its isomer 5. Compound 4 was found to be a potent and selective progesterone receptor agonist with an EC50 of 1.1 +/- 0.4 nM in a cell-based luciferase reporter assay.

Discovery of lactoquinomycin and related pyranonaphthoquinones as potent and allosteric inhibitors of AKT/PKB: mechanistic involvement of AKT catalytic activation loop cysteines.

The serine/threonine kinase AKT/PKB plays a critical role in cancer and represents a rational target for therapy. Although efforts in targeting AKT pathway have accelerated in recent years, relatively few small molecule inhibitors of AKT have been reported. The development of selective AKT inhibitors is further challenged by the extensive conservation of the ATP-binding sites of the AGC kinase family. In this report, we have conducted a high-throughput screen for inhibitors of activated AKT1. We have identified lactoquinomycin as a potent inhibitor of AKT kinases (AKT1 IC(50), 0.149 +/- 0.045 micromol/L). Biochemical studies implicated a novel irreversible interaction of the inhibitor and AKT involving a critical cysteine residue(s). To examine the role of conserved cysteines in the activation loop (T-loop), we studied mutant AKT1 harboring C296A, C310A, and C296A/C310A. Whereas the ATP-pocket inhibitor, staurosporine, indiscriminately targeted the wild-type and all three mutant-enzymes, the inhibition by lactoquinomycin was drastically diminished in the single mutants C296A and C310A, and completely abolished in the double mutant C296A/C310A. These data strongly implicate the binding of lactoquinomycin to the T-loop cysteines as critical for abrogation of catalysis, and define an unprecedented mechanism of AKT inhibition by a small molecule. Lactoquinomycin inhibited cellular AKT substrate phosphorylation induced by growth factor, loss of PTEN, and myristoylated AKT. The inhibition was substantially attenuated by coexpression of C296A/C310A. Moreover, lactoquinomycin reduced cellular mammalian target of rapamycin signaling and cap-dependent mRNA translation initiation. Our results highlight T-loop targeting as a new strategy for the generation of selective AKT inhibitors.

Diversity oriented biosynthesis via accelerated evolution of modular gene clusters.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.

In support of the original medermycin/lactoquinomycin A structure.

[structure: see text] Herein we report a significant body of spectroscopic data that supports the originally proposed structure of medermycin/lactoquinomycin A. In addition, we demonstrate that these data are inconsistent with the revised structure reported recently in the literature.

Discovery of (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride (GAP-134)13, an orally active small molecule gap-junction modifier for the treatment of atrial fibrillation.

Rotigaptide (3) is an antiarrhythmic peptide that improves cardiac conduction by modifying gap-junction communication. Small molecule gap-junction modifiers with improved physical properties were identified from a Zealand Pharma peptide library using pharmaceutical profiling, established SAR around 3, and a putative pharmacophore model for rotigaptide. Activity of the compounds was confirmed in a mouse cardiac conduction block model of arrhythmia. Dipeptide 9f (GAP-134) was identified as a potent, orally active gap-junction modifier for clinical development.

Pyranonaphthoquinone lactones: a new class of AKT selective kinase inhibitors alkylate a regulatory loop cysteine.

The naturally occurring pyranonaphthoquinone (PNQ) antibiotic lactoquinomycin and related aglycones were found to be selective inhibitors of the serine-threonine kinase AKT. A set of synthetic PNQs were prepared and a minimum active feature set and preliminary SAR were determined. PNQ lactones inhibit the proliferation of human tumor cell lines containing constitutively activated AKT and show expected effects on cellular biomarkers. Biochemical data are presented supporting a proposed bioreductive alkylation mechanism of action.

Enantiomeric separation and determination of absolute stereochemistry of asymmetric molecules in drug discovery: building chiral technology toolboxes.

The application of Chiral Technology, or the (extensive) use of techniques or tools for the determination of absolute stereochemistry and the enantiomeric or chiral separation of racemic small molecule potential lead compounds, has been critical to successfully discovering and developing chiral drugs in the pharmaceutical industry. This has been due to the rapid increase over the past 10-15 years in potential drug candidates containing one or more asymmetric centers. Based on the experiences of one pharmaceutical company, a summary of the establishment of a Chiral Technology toolbox, including the implementation of known tools as well as the design, development, and implementation of new Chiral Technology tools, is provided.

07H239-A, a new cytotoxic eremophilane sesquiterpene from the marine-derived Xylariaceous fungus LL-07H239.

07H239-A (1), a new eremophilane sesquiterpene from a marine-derived xylariaceous fungus, was isolated, characterized, and shown to be cytotoxic toward a variety of cancer cell lines, with some selectivity for a CCRFCEM leukemia line (IC(50) = 0.9 microg/mL).

FTMS structure elucidation of natural products: application to muraymycin antibiotics using ESI multi-CHEF SORI-CID FTMS(n), the top-down/bottom-up approach, and HPLC ESI capillary-skimmer CID FTMS.

The molecular formulas for the structures and substructures of muraymycin antibiotics A1 (C52H90N14O19, MW 1214) and B1 (C49H83N11O18, MW 1113) were determined using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The muraymycin A1 and B1 structures were elucidated by utilizing capillary-skimmer fragmentation with up to five stages of mass spectrometry (MS5). Multi-CHEF, a multiple ion isolation method, was used at each stage of MS(n) to isolate a parent ion and up to four reference ions, for exact-mass calibration. The parent ions were fragmented by SORI-CID and the product ions internally calibrated with average absolute mass errors less than 1 ppm at each stage in the fragmentation processes. Using the top-down/bottom-up approach, the molecular formulas for the antibiotics were determined by summing the elemental formulas of the neutral losses, obtained by measuring the mass differences (<500 Da) between the genetically related sequential parent ion masses in the MS(n) spectra, with the unique elemental formula of the lowest parent ion mass (<500 Da). The structures of 12 additional compounds in the muraymycin complex were elucidated using HPLC ESI capillary-skimmer CID FTMS by correlating their fragmentation patterns with those of muraymycins A1 and B1. Sequential neutral losses of an aminosugar, a valine, a uridine, and an ester fatty acid from the muraymycin parent ions provided diagnostic fragments for characterization.

Structures of the muraymycins, novel peptidoglycan biosynthesis inhibitors.

The muraymycins, a family of nucleoside-lipopeptide antibiotics, were purified from the extract of Streptomyces sp. LL-AA896. The antibiotics were purified by chromatographic methods and characterized by NMR spectroscopy, degradation studies, and mass spectrometry. The structures of 19 compounds were established. The muraymycins constitute a new antibiotic family whose core structure contains a glycosylated uronic acid derivative joined by an aminopropane group to a hexahydro-2-imino-4-pyrimidylglycyl residue (epicapreomycidine) containing dipeptide that is further extended by a urea-valine moiety. Members of this family show broad-spectrum in vitro antimicrobial activity against a variety of clinical isolates (MIC 2 to >64 mug/mL). The muraymycins inhibited peptidoglycan biosynthesis. The fatty acid substituent and the presence or absence of the amino sugar play important roles in biological activity. One of the most active compounds, muraymycin A1, demonstrated protection in vivo against Staphylococcus aureus infection in mice (ED50 1.1 mg/kg).

Muraymycins, novel peptidoglycan biosynthesis inhibitors: semisynthesis and SAR of their derivatives.

Sixteen muraymycin derivatives 2-17 were synthesized based on selective reactions of the primary and secondary amino groups of muraymycin C1 (1) with isocyanates and aldehydes. Disubstituted derivatives 3-9 demonstrated no activity against either MraY or MurG at