Comparative characterization of the lactimidomycin and iso-migrastatin biosynthetic machineries revealing unusual features for acyltransferase-less type I polyketide synthases and providing an opportunity to engineer new analogues
Seo J.-W.,University of Wisconsin - Madison |
Seo J.-W.,Korea Research Institute of Bioscience and Biotechnology |
Ma M.,Scripps Research Institute |
Kwong T.,Scripps Research Institute |
And 14 more authors.
Biochemistry | Year: 2014
(Chemical Equation Presented) Lactimidomycin (LTM, 1) and iso-migrastatin (iso-MGS, 2) belong to the glutarimide-containing polyketide family of natural products. We previously cloned and characterized the mgs biosynthetic gene cluster from Streptomyces platensis NRRL 18993. The iso-MGS biosynthetic machinery featured an acyltransferase (AT)-less type I polyketide synthase (PKS) and three tailoring enzymes (MgsIJK). We now report cloning and characterization of the ltm biosynthetic gene cluster from Streptomyces amphibiosporus ATCC 53964, which consists of nine genes that encode an AT-less type I PKS (LtmBCDEFGHL) and one tailoring enzyme (LtmK). Inactivation of ltmE or ltmH afforded the mutant strain SB15001 or SB15002, respectively, that abolished the production of 1, as well as the three cometabolites 8,9-dihydro-LTM (14), 8,9-dihydro-8S-hydroxy-LTM (15), and 8,9-dihydro-9 R-hydroxy-LTM (13 ). Inactivation of ltmK yielded the mutant strain SB15003 that abolished the production of 1, 13, and 15 but led to the accumulation of 14. Complementation of the ΔltmK mutation in SB15003 by expressing ltmK in trans restored the production of 1, as well as that of 13 and 15. These results support the model for 1 biosynthesis, featuring an AT-less type I PKS that synthesizes 14 as the nascent polyketide intermediate and a cytochrome P450 desaturase that converts 14 to 1, with 13 and 15 as minor cometabolites. Comparative analysis of the LTM and iso-MGS AT-less type I PKSs revealed several unusual features that deviate from those of the collinear type I PKS model. Exploitation of the tailoring enzymes for 1 and 2 biosynthesis afforded two analogues, 8,9-dihydro-8R-hydroxy-LTM (16) and 8,9-dihydro-8 R-methoxy-LTM (17), that provided new insights into the structure - activity relationship of 1 and 2. While 12-membered macrolides, featuring a combination of a hydroxyl group at C-17 and a double bond at C-8 and C-9 as found in 1, exhibit the most potent activity, analogues with a single hydroxyl or methoxy group at C-8 or C-9 retain most of the activity whereas analogues with double substitutions at C-8 and C-9 lose significant activity. © 2014 American Chemical Society.
Mason W.P.,University of Toronto |
Belanger K.,Hopital Notre Dame |
Nicholas G.,Ottawa Hospital Regional Cancer Center |
Vallieres I.,Hotel Dieu de Quebec |
And 5 more authors.
Journal of Neuro-Oncology | Year: 2012
This phase II trial was undertaken to evaluate the efficacy of TLN-4601 in patients with glioblastoma (GBM) at first progression. TLN-4601 inhibits the RasMAPK signaling pathway, and in animal models crosses the blood-brain barrier and accumulates in implanted gliomas, possibly by binding specifically to the peripheral benzodiazepine receptor. A maximum of 40 patients with recurrent GBM were to be enrolled in this study. TLN4601 was administered at a dose of 480 mg/m2/day by continuous intravenous (CIV) administration. Each 21-day cycle consisted of a 14-day CIV administration and a 7-day recovery period. Samples were obtained from all patients for pharmacokinetic evaluations (PK) and for Raf1 and pERK biomarker assessment using immunohistochemistry and flow cytometry. Following enrollment of 20 patients, this study was terminated due to a lack of efficacy. Of 17 evaluable patients, 14 had MR scans performed after two cycles of TLN-4601. Of these 14 patients, three had stable disease and 11 had disease progression. Only three patients had MR scans performed after four cycles and all had evidence of radiographic progression. Serum PKs confirmed that patients were exposed to TLN-4601 at targeted drug levels. TLN-4601 was generally well tolerated although two patients discontinued treatment due to adverse events. Biomarker analysis did not show consistent changes. TLN-4601 infused via CIV at 480 mg/m2/day for 14 of 21 days is well tolerated by patients with progressive GBM. However, this agent is ineffective in progressive GBM when administered as monotherapy in this schedule. © Springer Science+Business Media, LLC. 2011.
Bitzan M.,McGill University |
Schaefer F.,University of Heidelberg |
Reymond D.,Thallion Pharmaceuticals Inc.
Seminars in Thrombosis and Hemostasis | Year: 2010
Typical enteropathic HUS (eHUS) is triggered by Shiga toxin (Stx)-producing bacteria (STPB), predominantly Stx-producing Escherichia coli O157. The cell biological aspects of Stx have been well defined, but host factors potentially predisposing to the development or severity of HUS remain elusive. Treatment of eHUS includes supportive measures and invasive extracorporeal therapies. Thirty to 60% of children with eHUS require dialysis. Peritoneal and hemodialysis appear equally effective. Patient age, center experience, and equipment availability determine the choice of the modality; circulatory instability may require continuous renal replacement therapies. At present, no evidence indicates that plasma infusion or exchange therapies improve outcome of Stx-induced HUS. However, the traditional separation between diarrhea-positive (D +) and negative (D -) HUS, implying two entirely different pathological pathways, requires a fresh look: Atypical HUS may follow nonspecific diarrhea, and, conversely, STPB and fecal Stx may not be detected anymore at the time of the diagnosis of HUS. Recently, Stx has been found to directly interfere with the alternative complement pathway regulator factor H in vitro, whereas some patients with Stx-HUS demonstrate evidence of complement activation. Among newer treatments for eHUS, development of Stx-neutralizing monoclonal antibodies is the most advanced. This review concludes with a discussion of the rationale, mode of action, and status of presently available therapeutic antibodies against Stx2 and Stx1. Copyright © 2010 by Thieme Medical Publishers, Inc.
Melton-Celsa A.R.,Uniformed Services University of the Health Sciences |
Carvalho H.M.,Uniformed Services University of the Health Sciences |
Thuning-Roberson C.,Thallion Pharmaceuticals Inc. |
O'Brien A.D.,Uniformed Services University of the Health Sciences
Clinical and Vaccine Immunology | Year: 2015
In the United States, Shiga toxin (Stx)-producing Escherichia coli (STEC) is the most frequent infectious cause of hemorrhagic colitis. Hemolytic uremic syndrome (HUS) is a serious sequela that may develop after STEC infection that can lead to renal failure and death in up to 10% of cases. STEC can produce one or more types of Stx, Stx1 and/or Stx2, and Stx1 and Stx2 are responsible for HUS-mediated kidney damage. We previously generated two monoclonal antibodies (MAbs) that neutralize the toxicity of Stx1 or Stx2. In this study, we evaluated the protective efficacy of human/mouse chimeric versions of those monoclonal antibodies, named cαStx1 and cαStx2. Mice given an otherwise lethal dose of Stx1 were protected from death when injected with cαStx1 either 1 h before or 1 h after toxin injection. Additionally, streptomycin-treated mice fed the mouse-lethal STEC strain B2F1 that produces the Stx2 variant Stx2d were protected when given a dose of 0.1 mg of cαStx2/kg of body weight administered up to 72 h post-oral bacterial challenge. Since many STEC strains produce both Stx1 and Stx2 and since either toxin may lead to the HUS, we also assessed the protective efficacy of the combined MAbs. We found that both antibodies were required to protect mice from the presence of both Stx1 and Stx2. Pharmacokinetic studies indicated that cαStx1 and cαStx2 had serum half-lives (t1/2) of about 50 and 145 h, respectively. We propose that cαStx1 and cαStx2, both of which have been tested for safety in humans, could be used therapeutically for prevention or treatment early in the development of HUS. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Bertomeu T.,Thallion Pharmaceuticals Inc. |
Zvereff V.,Thallion Pharmaceuticals Inc. |
Ibrahim A.,Thallion Pharmaceuticals Inc. |
Zehntner S.P.,Biospective Inc. |
And 6 more authors.
Biochemical Pharmacology | Year: 2010
TLN-4601 is a farnesylated dibenzodiazepinone isolated from Micromonospora sp. with an antiproliferative effect on several human cancer cell lines. Although the mechanism of action of TLN-4601 is unknown, our earlier work indicated that TLN-4601 binds the PBR (peripheral benzodiazepine receptor; more recently known as the translocator protein or TSPO), an 18. kDa protein associated with the mitochondrial permeability transition (mPT) pore. While the exact function of the PBR remains a matter of debate, it has been implicated in heme and steroid synthesis, cellular growth and differentiation, oxygen consumption and apoptosis. Using the Jurkat immortalized T-lymphocyte cell line, documented to have negligible PBR expression, and Jurkat cells stably transfected with a human PBR cDNA, the present study demonstrates that TLN-4601 induces apoptosis independently of PBR expression. As PBRs are overexpressed in brain tumors compared to normal brain, we examined if TLN-4601 would preferentially accumulate in tumors using an intra-cerebral tumor model. Our results demonstrate the ability of TLN-4601 to effectively bind the PBR in vivo as determined by competitive binding assay and receptor occupancy. Analysis of TLN-4601 tissue and plasma indicated that TLN-4601 preferentially accumulates in the tumor. Indeed, drug levels were 200-fold higher in the tumor compared to the normal brain. TLN-4601 accumulation in the tumor (176μg/g) was also significant compared to liver (24.8μg/g; 7-fold) and plasma (16.2μg/mL; 11-fold). Taken together our data indicate that while PBR binding does not mediate cell growth inhibition and apoptosis, PBR binding may allow for the specific accumulation of TLN-4601 in PBR positive tumors. © 2010 Elsevier Inc.
Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc and Thallion Pharmaceuticals Inc. | Date: 2012-03-06
The invention features methods, compositions, and kits for treating a subject having a Shiga toxin associated disease with chimeric anti-Shiga Toxin 1 (cStx1) and anti-Shiga Toxin 2 (cStx2) antibodies.
Thallion Pharmaceuticals Inc. and Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc | Date: 2013-01-16
The invention features methods, compositions, and kits for treating a subject having a Shiga toxin associated disease with chimeric anti-Shiga Toxin (cStx1) and anti-Shiga Toxin 2 (cStx2) antibodies
Thallion Pharmaceuticals Inc. | Date: 2010-04-21
The present invention provides a scalable process for producing a concentrate containing a mass of a farnesylated dibenzodiazepinone by fermenting in an aqueous culture medium a strain of a microorganism that is capable of producing the farnesylated dibenzodiazepinone, upon completion of fermentation harvesting the fermentation broth and extracting the fermentation broth to provide an extract, and thereafter treating the extract to form the concentrate. The concentrate so produced may be utilized in downstream processes for producing pharmaceutical compounds. A strain of a Micromonospora species capable of producing a farnesylated dibenzodiazepinone at a high yield rate is provided, together with culture media for culturing microorganisms, and fermentation conditions for production of the farnesylated dibenzodiazepinone of the concentrate.
Boufaied N.,Thallion Pharmaceuticals Inc. |
Wioland M.-A.,Thallion Pharmaceuticals Inc. |
Falardeau P.,Thallion Pharmaceuticals Inc. |
Gourdeau H.,Thallion Pharmaceuticals Inc.
Anti-Cancer Drugs | Year: 2010
TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through DECIPHER, Thallion's proprietary drug discovery platform. The compound was shown to have a broad cytotoxic activity (low μmol/l) when tested in the NCI 60 tumor cell line panel and has shown in-vivo antitumor activity in several xenograft models. Related to its farnesylated moiety, the effect of TLN-4601 on Ras mitogen-activated protein kinase signaling was assessed. Downstream Ras signaling events, Raf-1, MEK, and ERK1/2 phosphorylation in MCF7 cells were evaluated by western blot analysis. TLN-4601 prevented epidermal growth factor-induced phosphorylation of Raf-1, MEK, and ERK1/2. This effect was time-dependent and dose-dependent with complete inhibition of protein phosphorylation within 4-6 h at 10 μmol/l. The inhibition of Ras signaling was not mediated by the inhibition of protein prenylation, documented by the lack of effect TLN-4601 on the prenylation of HDJ2 (specific substrate of farnesyltransferase), RAP1A (specific substrate of geranylgeranyl transferase-1), or Ras. As TLN-4601 did not inhibit EGFR, Raf-1, MEK or ERK1/2 kinase activities, the inhibitory effect of TLN-4601 on Ras signaling is not mediated by direct kinase inhibition. Using an Elk-1 trans-activation reporter assay, we found that TLN-4601 inhibits the MEK/ERK pathway at the level of Raf-1. Interestingly, TLN-4601 induces Raf-1 proteasomal-dependent degradation. These data indicate that TLN-4601 may inhibit the Ras-mitogen-activated protein kinase-signaling pathway by depleting the Raf-1 protein. © 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.
PubMed | Thallion Pharmaceuticals Inc. and Uniformed Services University of the Health Sciences
Type: Journal Article | Journal: Clinical and vaccine immunology : CVI | Year: 2015
In the United States, Shiga toxin (Stx)-producing Escherichia coli (STEC) is the most frequent infectious cause of hemorrhagic colitis. Hemolytic uremic syndrome (HUS) is a serious sequela that may develop after STEC infection that can lead to renal failure and death in up to 10% of cases. STEC can produce one or more types of Stx, Stx1 and/or Stx2, and Stx1 and Stx2 are responsible for HUS-mediated kidney damage. We previously generated two monoclonal antibodies (MAbs) that neutralize the toxicity of Stx1 or Stx2. In this study, we evaluated the protective efficacy of human/mouse chimeric versions of those monoclonal antibodies, named cStx1 and cStx2. Mice given an otherwise lethal dose of Stx1 were protected from death when injected with cStx1 either 1 h before or 1 h after toxin injection. Additionally, streptomycin-treated mice fed the mouse-lethal STEC strain B2F1 that produces the Stx2 variant Stx2d were protected when given a dose of 0.1 mg of cStx2/kg of body weight administered up to 72 h post-oral bacterial challenge. Since many STEC strains produce both Stx1 and Stx2 and since either toxin may lead to the HUS, we also assessed the protective efficacy of the combined MAbs. We found that both antibodies were required to protect mice from the presence of both Stx1 and Stx2. Pharmacokinetic studies indicated that cStx1 and cStx2 had serum half-lives (t1/2) of about 50 and 145 h, respectively. We propose that cStx1 and cStx2, both of which have been tested for safety in humans, could be used therapeutically for prevention or treatment early in the development of HUS.