Search

Mitochondria are found in the extracellular space in rheumatoid arthritis (RA). However, whether mitochondria are a source of autoantigens in RA has not been carefully addressed. Thus, we investigated the presence and significance of anti-mitochondrial antibodies (AMA) in patients with RA.AMAs were measured in sera from three cross-sectional RA cohorts (n=95, n=192, and n=117) and healthy individuals (n=38, n=72, n=50) using a flow cytometry-based assay. Further, AMAs were detected using anti-mitofusin-1 (MFN1) IgG ELISA and Western blot. A longitudinal inception cohort, followed for a median of 8 years, was used to study disease progression.AMA levels were elevated in all three RA cohorts as compared to healthy individuals (p0.001, p0.05, and p0.01), with a range of 14-26% positivity. Levels of anti-MFN1 antibodies correlated with AMA levels (r=0.31, p=0.006) and were elevated in RA as compared to healthy individuals (p0.001). Presence of AMA was associated with erosive disease (p0.05) and interstitial lung disease (p0.01). Further, AMA levels could predict erosive disease (OR=4.59, p=0.006) and joint space narrowing (OR=3.08, p=0.02) independent of anti-citrullinated protein antibodies. Finally, anti-MFN1 antibodies identified seronegative patients developing erosive disease (OR=9.33, p=0.02).Our findings demonstrate the presence of novel autoantibodies targeting mitochondria in RA. AMAs stratified patients based on disease phenotype and predicted development of erosive disease, including in patients with seronegative disease. Our results highlight the essential role of mitochondria in RA pathogenesis and suggest a possible benefit of therapies targeting mitochondrial-mediated inflammation and clearance in these patients. This article is protected by copyright. All rights reserved.

Diffuse alveolar hemorrhage (DAH), although rare, is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans, although increasingly neutrophils, NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model, we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b+Ly6G+ neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress, namely Chop and Bip. Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model, with accompanying reduction in IFN-driven genes and pathology. Lastly, coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH.

Background/Aims To examine if serum fibrosis biomarkers could accurately identify the stage of liver disease amongst hepatitis C (HCV) and HIV co-infected patients. Methods One hundred and thirty seven HIV/HCV co-infected persons were randomly selected from the Johns Hopkins HIV Clinic cohort. Ninety five had complete testing for fibrosis markers in sera collected at the time of liver biopsy. Biopsies were scored according to Ishak modified histological activity index (F0 no fibrosis to F6 cirrhosis). Fibrosis was evaluated against alanine aminotransferase (ALT), aspartate aminotransferase (AST), AST to platelet ratio (APRI), albumin, total bilirubin, hyaluronic acid (HA) and YKL-40. Results Sixty nine (73%) had no or minimal portal fibrosis (F0-2) and were compared with remaining subjects (F3-6). Fibrosis scores ≥F3 were found 27 times more often in persons with HA levels >86ng/ml and 5.5 times more often in persons with HA levels 41–86ng/ml. Less substantial associations were detected with levels of albumin 60iu (OR 5.91). All 35 subjects who had favorable results of HA, albumin, and AST had minimal fibrosis (F0-2). Conclusions Amongst HIV/HCV co-infected patients, serum testing for HA, albumin, and AST (SHASTA Index) was able to accurately stage mild and advanced fibrosis.

Cryptophycins-1 and 52 (epoxides) were discovered to have in-vitro and in-vivo antitumor activity in the early 1990s. The chlorohydrins of these, Cryptophycins-8 and 55 (also discovered in the early 1990s) were markedly more active, but could not be formulated as stable solutions. With no method to adequately stabilize the chlorohydrins at the time, Cryptophycin-52 (LY 355073) entered clinical trials, producing only marginal antitumor activity. Since that time, glycinate esters of the hydroxyl group of the chlorohydrins have been synthesized and found to provide stability. Three of the most active were compared herein. Cryptophycin-309 (C-309) is a glycinate ester of the chlorohydrin Cryptophycin-296. The glycinate derivative provided both chemical stability and improved aqueous solubility. After the examination of 81 different Cryptophycin analogs in tumor bearing animals, C-309 has emerged as superior to all others. The following %T/C and Log Kill (LK) values were obtained from a single course of IV treatment (Q2d x 5) against early staged SC transplantable tumors of mouse and human origin: Mam 17/Adr [a pgp (+) MDR tumor]: 0%T/C, 3.2 LK; Mam 16/C/Adr [a pgp (-) MDR tumor]: 0%T/C, 3.3 LK; Mam 16/C: 0%T/C, 3.8 LK; Colon 26: 0%T/C, 2.2 LK; Colon 51: 0%T/C, 2.4 LK; Pancreatic Ductal Adenocarcinoma 02 (Panc 02): 0%T/C, 2.4 LK; Human Colon HCT15 [a pgp (+) MDR tumor]: 0%T/C, 3.3 LK; Human Colon HCT116: 0%T/C, 4.1 LK. One additional analog, Cryptophycin-249 (C-249, the glycinate of Cryptophycin-8), also emerged with efficacy rivaling or superior to C-309. However, there was sufficient material for only a single C-249 trial in which a 4.0 LK was obtained against the multidrug resistant breast adenocarcinoma Mam-16/C/Adr. C-309 and C-249 are being considered as second-generation clinical candidates.

Two new cytotoxins have been isolated from a species of marine cyanobacterium belonging to the genus Symploca that was collected in Guam. These new compounds, micromide (1) and guamamide (2), were accompanied by the known lipopeptides apramides A (3), B (4), and G (5). The planar structures of both alkaloids were elucidated by standard 2D NMR techniques, and the configurations of the amino acid-derived units in 1 were determined by chiral HPLC. The stereochemistry of the beta-methoxyhexanoic acid in 1 was determined by derivatization with methyl d-mandelate, after acid hydrolysis, and comparison with synthetic standards.

Bioassay-guided fractionation of the extract of a species of Lyngbya from Palau has yielded Palau'amide (1), which had an IC(50) value of 13 nM against KB cells. The structure of 1 was elucidated by NMR analysis in a variety of solvents. In particular, a gradient-enhanced band-selective HMBC experiment allowed unambiguous assignment of HMBC correlations to carbon signals that were separated by 0.1 ppm. The relative stereochemistry and the absolute configurations of all but one (C-37) of the nine chiral centers were determined by NMR analysis of the alpha-methoxyphenylacetic acid (MPA) derivatives and chiral HPLC of the degradation products of 1. The assignment of S rather than R stereochemistry at C-37 is discussed in detail.

A new cytotoxic peptide, which displayed an IC(50) value of 0.8 microM against KB cells, has been isolated from the marine cyanobacterium Symploca sp. The planar structure of tasiamide B (1), deduced by 2D NMR experiments, contains the unusual amino acid-derived residue 4-amino-3-hydroxy-5-phenylpentanoic acid (Ahppa). The configuration of 1 was deduced by HPLC analysis of degradation products.

Cryptophycin 52 is a synthetic derivative of Cryptophycin 1, a potent antimicrotubule agent isolated from cyanobacteria. In an effort to increase the potency and water solubility of the molecule, a structure-activity relationship study (SAR) was initiated around the phenyl ring of fragment A. These Cryptophycin 52 analogues were accessed using a Wittig olefination reaction between various triphenylphosphonium salts and a key intermediate aldehyde prepared from Cryptophycin 53. Substitution on the phenyl ring of fragment A was well tolerated, and several of these analogues were equally or more potent than Cryptophycin 52 when evaluated in vitro in the CCRF-CEM leukemia cell line and in vivo against a murine pancreatic adenocarcinoma.

Ulongapeptin (1), a cyclic depsipeptide, was isolated from a Palauan marine cyanobacterium Lyngbya sp. The gross structure was elucidated through one-dimensional TOCSY experiments and other spectroscopic techniques. The absolute and relative stereochemistry of the beta-amino acid, 3-amino-2-methyl-7-octynoic acid (AMO), in 1 was determined by synthesis of the saturated alpha-alkyl-beta-amino acid and Marfey's analysis of the acid hydrolysate of tetrahydro-1. Ulongapeptin (1) was cytotoxic against KB cells at an IC(50) value of 0.63 microM.

Re-collections of the cyanobacterium Lyngbya sp. have yielded two more members of the lyngbyapeptin and lyngbyabellin families. The gross structures of 15-norlyngbyapeptin A (1) and lyngbyabellin D (3) were deduced through standard 2D NMR techniques, with the absolute configuration of both elucidated through degradation and comparison with commercially available and synthetic standards. Degradation to the alpha-amino acid and NOE correlations determined the absolute and relative configuration of the 4-amino-3-hydroxy-5-methylhexanoic acid unit in 3. Lyngbyabellin D (3) displayed an IC(50) value of 0.1 microM against the KB cell line.

Symplostatin 1, an analog of dolastatin 10, was recently isolated from cyanobacteria of the genus Symploca. Symplostatin 1 is a potent inhibitor of cell proliferation with IC(50) values in the low nanomolar range and it exhibits efficacy against a variety of cancer cell types. Symplostatin 1 caused the formation of abnormal mitotic spindles and accumulation of cells in metaphase at concentrations that had only minor effects on interphase microtubules. At higher concentrations, symplostatin 1 caused the loss of interphase microtubules. Cell cycle analysis revealed that symplostatin 1 caused G(2)/M arrest, consistent with its effects on mitotic spindles. Symplostatin 1 initiated the phosphorylation of Bcl-2, formation of micronuclei and activation of caspase 3, indicating induction of apoptosis. The cellular effects of symplostatin 1 are consistent with other antimitotic tubulin-targeting drugs. Tubulin polymerization experiments indicated that symplostatin 1 potently inhibits the assembly of purified tubulin, suggesting that tubulin may be its intracellular target. Some microtubule-targeting agents are reported to have antiangiogenic activity and therefore the effects of symplostatin 1 on endothelial cell proliferation and invasion were evaluated. Symplostatin 1 was found to be a potent inhibitor of both endothelial cell proliferation and invasion. Because of its potent and broad activity in vitro, symplostatin 1 was evaluated in vivo. Symplostatin 1 was active against murine colon 38 and murine mammary 16/C; however, it was poorly tolerated and the mice were slow to recover from the toxicity. The data indicate that symplostatin 1 has a mechanism of action similar to dolastatin 10.

The biosynthesis of the unusual amino acid 4-methylproline in the Nostoc genus of cyanobacteria was investigated on the genetic and enzymatic level. Two genes involved in the biosynthesis were cloned and the corresponding enzymes, a zinc-dependent long-chain dehydrogenase and a Delta(1)-pyrroline-5-carboxylic acid (P5C) reductase homologue, were overexpressed in Escherichia coli and biochemically characterized. Putative substrates were synthesized to test enzyme substrate specificities, and deuterium labeling studies were carried out to reveal the stereospecificities of the enzymatic reactions with respect to the substrates as well as to the coenzymes.

The synthesis of a lyngbyastatin 1-Ibu-epilyngbyastatin 1 mixture combined with NMR and molecular modeling studies proved that natural lyngbyastatin 1 was only one Ibu epimer rather than a mixture of both and that the configuration of this epimer in the Ibu unit was R. The substance isolated with lyngbyastatin 1 was Ibu-epidolastatin 12. The extreme broadness in the proton NMR spectra of lyngbyastatin 1 and Ibu-epidolastatin 12 was exchange broadening due to rotation about the Ibu-Ala amide bond. It was a consequence of (1) a small energy difference between the cis and trans forms of this bond, (2) a substantial difference in conformation between these forms, and (3) a lowered barrier between them compared to most amide bonds (due to steric hindrance). The synthetic lyngbyastatin 1-Ibu-epilyngbyastatin 1 mixture had significant activities against cancer cells and in stimulating actin polymerization, but was less active than dolastatin 11 in all assays.

Various collections of the apratoxin-producing marine cyanobacterium Lyngbya sp. from Palau have afforded several new nitrogenous metabolites. Lyngbyabellin C is a dichlorinated, thiazole hydroxy acid-containing, cytotoxic macrolide that is related to other lyngbyabellins, to dolabellin, and to hectochlorin. Its selective decomposition tendency to form homohydroxydolabellin suggests that the sea hare isolate dolabellin may be an isolation artifact. In addition to the known lyngbyapeptin A, two new related modified tetrapeptides, lyngbyapeptins B and C, have been isolated. Furthermore, a new cytotoxic N-acylpyrrolinone, termed palau'imide, was found. Structure elucidation of these metabolites involved extensive application of NMR spectroscopy.

The acyclic peptide tasiamide (1) has been isolated from the marine cyanobacterium Symploca sp. The planar structure and absolute stereochemistry of the peptide were deduced by 2D NMR techniques and chiral HPLC. Tasiamide (1) was cytotoxic against KB and LoVo cells with IC(50) values of 0.48 and 3.47 microg/mL, respectively.

In vitro growth inhibition assays were performed using human cancer cell lines at various concentrations with experimental anticancer drugs such as the cryptophycins and other cytotoxins. The effect of variations in assay parameters on the observed growth inhibition of these anticancer therapeutic agents was determined. The results demonstrated that the observed inhibitory activity of these compounds varied inversely with the cell concentrations used. The observed differences in activity between different cytotoxins were not necessarily proportionate. Thus, the relative activities of two toxins also varied with cell concentration. Furthermore, the sensitivity of these cell lines to the cytostatic purine analog, 6-mercaptopurine (used as a control), varied with cell concentration as well. The activity of this compound was dependent on the medium used for cell growth, yielding good activity in Eagle's minimum essential medium, but not in Ham's F-12 (Kaigin) medium. Moreover, growth inhibition by cryptophycin as well as 6-mercaptopurine was also dependent on the serum concentration in the medium. Finally, the sensitivity of the cancer cell lines to various organic solvents commonly used as drug vehicles for in vitro testing, such as ethanol, dimethylformamide, and dimethylsulfoxide, was likewise found to vary inversely with cell concentration.

The potent antitumor agent dolastatin 10 (1) was originally isolated from the sea hare Dolabella auricularia, and we now report its isolation from the marine cyanobacterium Symploca sp. VP642 from Palau. The chemically related analogue symplostatin 1 (2) has been reisolated from Guamanian and Hawaiian varieties of S. hydnoides and its total stereochemistry completed by determining the N,N-dimethylisoleucine unit to be L. Symplostatin 1 (2), like dolastatin 10 (1), is a potent microtubule inhibitor. The antitumor activity of 2 was assessed in vivo against several murine tumors. Symplostatin 1 (2) was effective against a drug-insensitive mammary tumor and a drug-insensitive colon tumor; however, it was only slightly effective against two MDR tumors.

The isolation and total structure determination of nostocyclopeptides A1 (1) and A2 (2) are described. These cyclic heptapeptides, which possess a unique imino linkage in the macrocyclic ring, are characteristic constituents of the cryptophycin-producing cyanobacterium Nostoc sp. ATCC53789. 1D TOCSY experiments proved to be very useful in identifying the seven amino acid residues in each compound, and HMBC and NOESY correlations made it possible to sequence the seven units into a total gross structure. The absolute stereochemistry was determined by directly comparing the amino acids in the acid hydrolyzate of each natural product and its peroxide oxidation and borohydride reduction products with authentic standards. Studies were carried out on the biosynthesis and initiated on the biological activity of these cyclic peptides.

The isolation and total structure determinations of nostopeptolides A1 (1), A2 (2) and A3 (3) are described. These cyclic depsipeptides, which are devoid of cytotoxic, antifungal and inhibition of protease activities, are characteristic constituents of the cryptophycin-producing cyanobacterium Nostoc sp. GSV224. Structure elucidation by NMR analysis was made more challenging by the existence of each nostopeptolide in three conformations. One-dimensional TOCSY experiments proved to be very useful in isolating and identifying the nine amino acid residues and the butyryl group in each compound. The absolute stereochemistries of 1–3 were determined by comparing the amino acids in the acid hydrolyzates directly with authentic standards.

Six new metabolites have been isolated from a lyngbyastatin 2-producing strain of the marine cyanobacterium Lyngbya majuscula collected at Apra Harbor, Guam, and their structures elucidated. These linear lipopeptides have been assigned the trivial names apramides A-F (1-6). From a more recent collection of this cyanobacterium, a structurally related compound, apramide G (7), has been found instead of apramides A-F (1-6). Structure elucidation of the lipopeptides 1-7 is based on spectroscopic techniques and chiral chromatography of hydrolysis products. The apramides appear as NMR-spectroscopically distinguishable conformers in solution, and this has been ascribed to the presence of a thiazole-containing modified amino acid unit.

A total synthesis of cryptophycin 337 (1-aza-cryptophycin 1, 2 ), an analogue of the potent antitumor antibiotic cryptophycin 1 ( 1 ), is described. Cryptophycin 337 was unstable and underwent an unexpected skeletal rearrangement. ( R )-Mandelic acid was used as the sole source of asymmetry for the synthesis of unit A.

An analogue of dolastatin 13 (2) has been isolated from a marine cyanobacterium, Symploca hydnoides, collected near Guam. This new cyclic depsipeptide contains a L-methionine sulfoxide residue; however, the sulfoxide exists as both R- and S-forms, resulting in the doubling of several signals in the 1H and 13C NMR spectra. Structure elucidation required extensive application of 2-D NMR techniques such as COSY, HMQC, HMBC, and ROESY. The trivial name symplostatin 2 (1) has been assigned to the new metabolite and its isolation from S. hydnoides further supports the proposal that many compounds originally isolated from the sea hare Dolabella auricularia are most probably of cyanobacterial origin.

Lyngbyastatin 1 (1a), a new cytotoxic analogue of dolastatins 12 (2a) and 11 (4), was isolated as an inseparable mixture with its C-15 epimer (1b) from extracts of a Lyngbya majuscula/Schizothrix calcicola assemblage and a L. majuscula strain collected near Guam. Dolastatin 12 (2a) was also encountered as an inseparable mixture with its C-15 epimer (2b) in L. majuscula/S. calcicola assemblages. At least one of the compounds in each mixture appeared to exist in solution as a mixture of slowly interconverting conformers resulting in broadened signals in 1H NMR spectra. Structure elucidation therefore relied principally on mass spectroscopy and chemical degradation studies. Both 1ab and 2ab proved toxic with only marginal or no antitumor activity when tested against colon adenocarcinoma #38 or mammary adenocarcinoma #16/C. Both 1ab and 2ab were shown to be potent disrupters of cellular microfilament networks.

Both the PC-3 and the TSU-PR1 prostate tumor models were found to be satisfactory for chemotherapeutic investigations in ICR-SCID mice. The 30 to 60 mg fragments implanted took in all mice (as judged by 100% takes in the controls of all experiments as well as the passage mice). The tumor volume doubling time was 4.0 days for PC-3 and 2.5 days for TSU-Pr1. Nine agents were evaluated IV against early stage subcutaneous PC-3 tumors, with Nano-piposulfan being the only agent highly active (4.9 log kill). Three other agents were moderately active: Taxol (1.5 log kill), Cryptophycin-8 (1.6 log kill), Vinblastine (1.0 log kill). Five agents were inactive: VP-16, Adriamycin, CisDDPt, 5-FUra, and Cyclophosphamide. Ten agents were evaluated IV against early stage subcutaneous TSU-Pr1 tumors. Three agent were highly active, producing > 6 log kill and cures: Taxol (5/5 cures), Cryptophycin-8 (5/5 cures), Vinblastine (2/4 cures). Two other agents were moderately active: Nano-piposulfan (1.2 log kill), and Cyclophosphamide (1.1 log kill). Five agents were inactive: VP-16, Adriamycin, CisDDPt, 5-FUra, and BCNU. In part, activity was determined by the ability of the SCID mice to tolerate meaningful dosages of the agents. Agents producing granulocyte toxicity (e.g., Adriamycin) were poorly tolerated and appeared less active than expected. Vinblastine, producing little or no granulocyte toxicity was very well tolerated and appeared to be more active than expected.

Abstract: Six to ten percent of the extracts of laboratory-cultured blue-green algae are significantly cytotoxic against human tumor cell lines. Structurally-unique cytotoxins have been isolated from most of the active extracts. Of the extracts of >2000 cyanophytes examined to date, approximately 0.8% showed solid tumor selective cytotoxicity in the Corbett assay. The lipophilic extract of Nostoc sp. GSV 224 was by far the most cytotoxic and one of the few extracts that were both solid tumor and tumor selective. Cyclic depsipeptides called cryptophycins were found to be the solid tumor and tumor selective agents. In vivo studies on naturally-occurring cryptophycin-1 and semi-synthetic cryptophycin-8 indicate that these compounds are very active against a broad spectrrum of tumors implanted in mice, including multidrug-resistant tumors, and that a cryptophycin analog may eventually be evaluated in the clinic. Several cytotoxins from terrestrial cyanophytes have•structures that resemble those of some antineoplastic agents from marine invertebrates, suggesting that a more extensive cultivation and screening effort on marine cyanophytes could lead to more discoveries of new antitumor drugs.

A large-scale screening program was initiated to evaluate laboratory-cultured blue-green algae (cyanobacteria) as a source of novel compounds with inhibitory activity against certain serine/threonine protein phosphatases. Over 1600 extracts, representing 816 cyanophyte strains with broad habitat and taxonomic diversity, were screened. Inhibitors were identified in extracts produced from all orders tested except Chamaesiphonales, and the family Stigonemataceae was found to contain proportionally more inhibitors as compared to the total screen.

The convergent total synthesis of cryptophycins C and D is described. It has been shown that in both natural products the absolute configuration of the a-amino acid corresponds to the D-series. The structural assignment for cryptophycin C has been corrected to reflect this fact. Since the structure of cryptophycin A has been correlated to cryptophycin C, the chloro-0-methyltyrosine unit in cryptophycin A has the D-configuration. Cryptophycins are potent tumor-selective cytotoxins associated with the terrestrial blue-green algae Nostoc sp. GSV 224' and Nostoc sp. ATCC 53789.2 The major cytotoxin in each alga, cryptophycin A, shows excellent activity against solid tumors implanted in mice, including a drug-resistant tumor. Over 20 related cytotoxins are present in the GSV 224 strain as minor constituent^,'^^ and some of these compounds, e.g., cryptophycins B and C, have been isolated in sufficient amounts for in vivo e~aluation.~ In order to acquire adequate quantities of selected naturally-occurring cryptophycins and synthetic analogs for structure-activity relationship (SAR) studies, preclinical evaluation, and human clinical trials, we have designed a general synthesis. Cryptophycins C and D, as described in the original paper, were chosen to be the initial targets as they represented examples from both of the alleged Land D-tyrosine series. We report here the total syntheses of cryptophycins C and D which (1) revise the structures of cryptophycins A and C to reflect the D-configuration for the a-amino acid unit as depicted in the structural drawings in this paper and (2) confirm the structures of cryptophycins B and D. Retrosynthetic analysis of the cryptophycins was straightforward: the structure is composed of four units (A-D, Figure 1); consequently several convergent approaches could be envisioned. The combination of two pairs of units (e.g., A-B and C-D) appeared to be optimally convergent. Since the success of the synthesis depended on the formation of a 16membered depsipeptide from an acyclic precursor, a macrolactamization involving the amino group of unit C and the carboxylate of unit B appeared to be the best choice. The acyclic precursor to cryptophycin D would therefore be 1. This, in turn, suggested a disconnection into two fragments, one represented by 2 and composed of (S)-( -)-2-hydroxy-4-methylvaleric (L-leucic) acid (D) and (R)-3-amino-2-methylpropanoic acid (C) units, and the other by 3 and composed of O-methylD-tyrosine (B) and (2E,7E,5S,6R)-5-hydroxy-6-methyl-8-phenyloctadienoic acid (A) units. In the direction of the synthesis, @ Abstract published in Advance ACS Absfracts, February 15, 1995. (1) Trimurtulu, G.; Ohtani, I.; Patterson, G. M. L.; Moore, R. E.; Corbett, T. H.; Valeriote, F. A.; Demchik, L. J. Am. Chem. SOC. 1994, 116, 47294731. (2) Schwartz, R. E.; Hirsch, C. F.; Sesin, D. F.; Flor, J. E.; Chartrain, M.; Fromtling, R. E.; Harris, G. H.; Salvatore, M. J.; Liesch, J. M.; Yudin, K. J. Ind. Microbiol. 1990, 5, 113-24. (3) Trimurtulu, G.; Ogino, J.; Heltzel, C. E.; Patterson, G. M. L.; Moore, R. E. Manuscript in preparation. An explanation of the structural misassignment for cryptophycins A and C is presented. (4) Heltzel, C. E.; Ogino, J.; Trimurtulu, G.; Mooberry, S. L.; Patterson, G. M. L.; Moore, R. E.; Corbett, T. H.; Valeriote, F. A,; Demchik, L. Manuscript in preparation. A Figure 1. Numbering system for each of the units of cryptophycins C and D. This numbering system is used for the NMR data.

Each independently arising tumor is a separate and unique biologic entity with its own unique histologic appearance, biologic behavior, and drug response profile. Thus, in drug discovery, no single tumor has been a perfect predictor for any other tumor. For this reason, new agents are evaluated in a variety of tumor models which is known as breadth of activity testing. In recent years, human tumors implanted in athymic nude mice and SCID mice have also become available for breadth of activity testing. In studies carried out in these laboratories, it was found that 10 human tumors metastasized in the SCID mice, but failed to metastasize in nude mice. In addition, tumor growth and tumor takes were superior in the SCID mice. The strengths and weaknesses of xenograft model systems are discussed. For example, most human tumor xenograft models are excessively sensitive to alkylating agents as well as to a new class of DNA binders (XE840 and XP315). Using human tumor models that are the least sensitive t...

Cellular in vitro assays have been both developed and employed by us to search for new, solid tumor specific anticancer agents. Parallel in vivo models are then used to assess the therapeutic efficacy of candidates selected by the in vitro assays. During the past 4 years, extracts as well as pure compounds from natural products have been obtained from collaborators at the University of Hawaii and Lilly Research Laboratories. The samples have been analyzed, the structures identified and a number of lead compounds proposed

Borophycin (1) is the potent cytotoxin in the lipophilic extract of a marine strain of the blue-green alga (cyanobacterium) Nostoc linckia (Roth) Bornet ex Bornet & Flahault (UH isolate GA-5-23). The gross structure of this boron-containing compound was determined by spectral methods and ita relative stereochemistry established by X-ray crystallography. Borophycin is made up of two identical halves with an overall structure reminiscent of the ionophoric antibiotics boromycin (2) and aplasmomycin (3). The biosynthesis of 1 differs from the biosynthesis of 2 and 3. All three compounds are acetate-derived polyketides that utilize a C3 precursor for the starter unit and methionine for the methyl branches on the polyketide chain. Whereas phosphoglycerate or phosphoenolpyruvate has been suggested to be the C3 starter unit in the biosynthesis of 2 and 3, the C3 starter unit for the biosynthesis of 1 is derived from acetate and methionine, but not propionate. In evaluating lipophilic and hydrophilic extracts of laboratory-cultured blue-green algae (cyanobacteria) for in uitro antitumor activity,' the lipophilic extract of a marine strain of Nostoc Zinckia (Roth) Bornet ex Bomet & Flahault (UH isolate GA-5-23)2 was found to display appreciable cytotoxicity against LoVo (MIC 0.066 pg/mL) and KB (MIC 3.3 pg/mL), two human tumor cell lines used routinely in our laboratory for screening.3 In the more meaningful Corbett and Valeriote assays,4 however, the extract was neither solid tumor selective nor tumor selective, suggesting that further investigation of this extract might not lead to a drug that would offer any distinct advantage over drugs already in clinical use for the treatment of human cancer. Nevertheless, this algal extract was among the most LoVo-cytotoxic found in screening extracts of 665 blue-green algae,l and this

Since 1981 we have cultured and prepared lipophilic and hydrophilic extracts from more than 1500 strains representing some 400 species of blue-green algae. Screening for a wide variety of potentially useful bioactivities, including cytotoxic, multi-drug-resistance reversal, antifungal, and antiviral effects, has led to the discovery and identification of numerous novel bioactive metabolites including peptides, macrolides and glycosides.

Synthesis of the novel isotactic nonamethoy-1-pentacosene (3) isolated from the terrestrial blue-green alga Scytonema ocellatum was accomplished in a stereocontrolled manner and the absolute configuration was established.

Revised structures are presented for tantazoles A ( 3 ) and B ( 4 ) and preliminary studies on their biosyntheses in the terrestrial blue-green alga Scytonema mirabile BY-8-1 are reported. Marfey analysis of the acid hydrolyzates shows that the absolute configuration of C4 in thiazoline ring A of the two alkaloids is R . Feeding experiments with [1,2- 13 C]acetate, [1,2- 13 C]glycine, [2- 13 C, 15 N]glycine and [ methyl - 13 C]methionine indicate that 3 and 4 are modified pentapeptides assembled from acetate, four cysteines and a threonine and that the methyl groups on the four thiazoline rings, the amide nitrogen, and the methine attached to ring A originate from the tetrahydrofolate C 1 pool via methionine.

Preliminary studies on the biosynthesis of tolytoxin (1) in the terrestrial blue-green alga Scytonema mirabile BY-8-1 are reported. Incorporation experiments with [1,2-13C] acetate, [1-13C,18O]acetate, [1,2-13C]glycine, [2-13C,15N]glycine and [methyl- 13C]methionine indicate that the carbon chain of tolytoxin is a polyketide assembled from a glycine starter unit and 15 acetate units. The one-carbon branches on the polyketide chain originate from the tetrahydrofolate C1 pool.

The biosyntheses of nostocyclophane D ( 1 ) and cylindrocyclophane D ( 2 ) have been investigated by the administration of sodium [1,2- 13 C 2 ]acetate, sodium [1- 13 C, 2 H 3 ]acetate, sodium [2- 13 C, 2 H 3 ]acetate, sodium [ 2 H 3 ]acetate, sodium [1- 13 C, 18 O 2 ]acetate, sodium [2- 13 C, 18 O 2 ]acetate, l -[methyl- 13 C]methionine, and l -[methyl- 13 C, 2 H 3 ]methionine to cultures of the cyanobacteria Nostoc linckia and Cylindrospermum licheniforme , respectively. The isotopically labeled compounds produced by these feeding experiments indicate that the structural skeletons of these [7.7]paracyclophanes are polyketides that have been constructed by dimerization of acetate-derived nonaketides. Acetate has also been identified as the source of the branched methyl groups in cylindrocyclophane D.

Lipophilic and hydrophilic extracts from approximately 600 strains of cultured mcyanophytes, representing some 300 species, were examined for antiviral activity against three pathogenic viruses. Approximately 10% of the cultures produced substances that caused significant reduction in cytopathic effect normally associated with viral infection. The screening program identified the order Chroococcales as commonly producing antiviral agnets.

Mirabimide A, the major imide from the terrestrial blue-green alga Scytonema mirabile (Dillwyn) Bornet (strain BY-8-1), is N-[2-(N,N-dimethylleucyl)oxy-3-methylpentanoyl-N-methylvalyl-prolyl]-4-methoxy-5-isopropyl-3-pyrrolin-2-one. Mirabimides B–D are the valyl, 2-oxy-3-methylbutanoyl, and N-acetyl-N-methylleucyl analogs, respectively. The absolute configurations of the five asymmetric carbons in the mirabimides are proposed to be all S , but the assignments for the dimethylleucine units in A–C and the hydroxyacid units in B–D are tentative.

Puwainaphycins A–E, one of which (C) is a potent cardioactive agent in isolated mouse atria, are cyclic decapeptides that have beeen isolated from a terrestrial blue-green alga Annabaena sp. BQ-16-1. The structures of puwainaphycins A–E, including most of the stereochemical features, have been elucidated by a combination of spectral and chemical methods.