Jason C. Kwan

Morphological and Pathological Evolution of the Brain Microcirculation in Aging and Alzheimer’s Disease

Key pathological hallmarks of Alzheimer’s disease (AD), including amyloid plaques, cerebral amyloid angiopathy (CAA) and neurofibrillary tangles do not completely account for cognitive impairment, therefore other factors such as cardiovascular and cerebrovascular pathologies, may contribute to AD. In order to elucidate the microvascular changes that contribute to aging and disease, direct neuropathological staining and immunohistochemistry, were used to quantify the structural integrity of the microvasculature and its innervation in three oldest-old cohorts: 1) nonagenarians with AD and a high amyloid plaque load; 2) nonagenarians with no dementia and a high amyloid plaque load; 3) nonagenarians without dementia or amyloid plaques. In addition, a non-demented (ND) group (average age 71 years) with no amyloid plaques was included for comparison. While gray matter thickness and overall brain mass were reduced in AD compared to ND control groups, overall capillary density was not different. However, degenerated string capillaries were elevated in AD, potentially suggesting greater microvascular “dysfunction” compared to ND groups. Intriguingly, apolipoprotein ε4 carriers had significantly higher string vessel counts relative to non-ε4 carriers. Taken together, these data suggest a concomitant loss of functional capillaries and brain volume in AD subjects. We also demonstrated a trend of decreasing vesicular acetylcholine transporter staining, a marker of cortical cholinergic afferents that contribute to arteriolar vasoregulation, in AD compared to ND control groups, suggesting impaired control of vasodilation in AD subjects. In addition, tyrosine hydroxylase, a marker of noradrenergic vascular innervation, was reduced which may also contribute to a loss of control of vasoconstriction. The data highlight the importance of the brain microcirculation in the pathogenesis and evolution of AD.

Genome streamlining and chemical defense in a coral reef symbiosis

Secondary metabolites are ubiquitous in bacteria, but by definition, they are thought to be nonessential. Highly toxic secondary metabolites such as patellazoles have been isolated from marine tunicates, where their exceptional potency and abundance implies a role in chemical defense, but their biological source is unknown. Here, we describe the association of the tunicate Lissoclinum patella with a symbiotic α-proteobacterium, Candidatus Endolissoclinum faulkneri, and present chemical and biological evidence that the bacterium synthesizes patellazoles. We sequenced and assembled the complete Ca. E. faulkneri genome, directly from metagenomic DNA obtained from the tunicate, where it accounted for 0.6% of sequence data. We show that the large patellazoles biosynthetic pathway is maintained, whereas the remainder of the genome is undergoing extensive streamlining to eliminate unneeded genes. The preservation of this pathway in streamlined bacteria demonstrates that secondary metabolism is an essential component of the symbiotic interaction.

Isolation and biological evaluation of 8-epi-malyngamide C from the Floridian marine cyanobacterium Lyngbya majuscula.

A new stereoisomer of malyngamide C, 8-epi-malyngamide C (1), and the known compound lyngbic acid [(4E,7S)-7-methoxytetradec-4-enoic acid] were isolated from a sample of Lyngbya majuscula collected near Bush Key, Dry Tortugas, Florida. The structure of 1 was determined by NMR and MS experiments. The absolute configuration of 1 was determined by selective Mitsunobu inversion of C-8 to give malyngamide C, as determined by NMR, MS, and comparison of specific rotation. Both 1 and malyngamide C were found to be cytotoxic to HT29 colon cancer cells (IC(50) 15.4 and 5.2 microM, respectively) and to inhibit bacterial quorum sensing in a reporter gene assay.

Anticolon Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor

Histone deacetylases (HDACs) are validated targets for anticancer therapy as attested by the approval of suberoylanilide hydroxamic acid (SAHA) and romidepsin (FK228) for treating cutaneous T cell lymphoma. We recently described the bioassay-guided isolation, structure determination, synthesis, and target identification of largazole, a marine-derived antiproliferative natural product that is a prodrug that releases a potent HDAC inhibitor, largazole thiol. Here, we characterize the anticancer activity of largazole by using in vitro and in vivo cancer models. Screening against the National Cancer Institutes 60 cell lines revealed that largazole is particularly active against several colon cancer cell types. Consequently, we tested largazole, along with several synthetic analogs, for HDAC inhibition in human HCT116 colon cancer cells. Enzyme inhibition strongly correlated with the growth inhibitory effects, and differential activity of largazole analogs was rationalized by molecular docking to an HDAC1 homology model. Comparative genomewide transcript profiling revealed a close overlap of genes that are regulated by largazole, FK228, and SAHA. Several of these genes can be related to largazoles ability to induce cell cycle arrest and apoptosis. Stability studies suggested reasonable bioavailability of the active species, largazole thiol. We established that largazole inhibits HDACs in tumor tissue in vivo by using a human HCT116 xenograft mouse model. Largazole strongly stimulated histone hyperacetylation in the tumor, showed efficacy in inhibiting tumor growth, and induced apoptosis in the tumor. This effect probably is mediated by the modulation of levels of cell cycle regulators, antagonism of the AKT pathway through insulin receptor substrate 1 down-regulation, and reduction of epidermal growth factor receptor levels.

Grassystatins A–C from Marine Cyanobacteria, Potent Cathepsin E Inhibitors that Reduce Antigen Presentation

In our efforts to explore marine cyanobacteria as a source of novel bioactive compounds, we discovered a statine unit-containing linear decadepsipeptide, grassystatin A (1), which we screened against a diverse set of 59 proteases. We describe the structure determination of 1 and two natural analogues, grassystatins B (2) and C (3), using NMR, MS, and chiral HPLC techniques. Compound 1 selectively inhibited cathepsins D and E with IC(50)s of 26.5 nM and 886 pM, respectively. Compound 2 showed similar potency and selectivity against cathepsins D and E (IC(50)s of 7.27 nM and 354 pM, respectively), whereas the truncated peptide analogue grassystatin C (3), which consists of two fewer residues than 1 and 2, was less potent against both but still selective for cathepsin E. The selectivity of compounds 1-3 for cathepsin E over D (20-38-fold) suggests that these natural products may be useful tools to probe cathepsin E function. We investigated the structural basis of this selectivity using molecular docking. We also show that 1 can reduce antigen presentation by dendritic cells, a process thought to rely on cathepsin E.

Structure and biosynthesis of the antibiotic bottromycin D.

Drug resistant infectious diseases are quickly becoming a global health crisis. While Streptomyces spp. have been a major source of antibiotics over the past 50 years, efficient methods are needed to identify new antibiotics and greatly improve the rate of discovery. LCMS-based metabolomics were applied to analyze extracts of 50 Streptomyes spp. Using this methodology, we discovered bottromycin D and used whole genome sequencing to determine its biosynthesis by a ribosomal pathway.

Lyngbyastatins 8-10, elastase inhibitors with cyclic depsipeptide scaffolds isolated from the marine cyanobacterium Lyngbya semiplena.

Investigation of an extract from the marine cyanobacterium Lyngbya semiplena, collected in Tumon Bay, Guam, led to the identification of three new cyclodepsipeptides, lyngbyastatins 8–10 (1–3). The structures of 1–3 were determined by NMR, MS, ESIMS fragmentation and chemical degradation. Compounds 1–3 are closely related to lyngbyastatins 4–7. Like the latter compounds, we found 1–3 to inhibit porcine pancreatic elastase, with IC50 values of 123 nM, 210 nM and 120 nM, respectively.

Total structure determination of grassypeptolide, a new marine cyanobacterial cytotoxin

A collection of the cyanobacterium Lyngbya confervoides off Grassy Key in Florida yielded grassypeptolide (1), a 31-membered macrocyclic depsipeptide with unusually high D-amino acid content, two thiazolines, and one beta-amino acid. We report the rigorous 3D structure determination and conformational analysis in solution and solid state by NMR, MS, X-ray crystallography, chemical degradation, and molecular modeling involving distance geometry and restrained molecular dynamics. Grassypeptolide (1) inhibited cancer cell growth with IC50 values from 1.0 to 4.2 microM.

A Bacterial Source for Mollusk Pyrone Polyketides

In the oceans, secondary metabolites often protect otherwise poorly defended invertebrates, such as shell-less mollusks, from predation. The origins of these metabolites are largely unknown, but many of them are thought to be made by symbiotic bacteria. In contrast, mollusks with thick shells and toxic venoms are thought to lack these secondary metabolites because of reduced defensive needs. Here, we show that heavily defended cone snails also occasionally contain abundant secondary metabolites, γ-pyrones known as nocapyrones, which are synthesized by symbiotic bacteria. The bacteria, Nocardiopsis alba CR167, are related to widespread actinomycetes that we propose to be casual symbionts of invertebrates on land and in the sea. The natural roles of nocapyrones are unknown, but they are active in neurological assays, revealing that mollusks with external shells are an overlooked source of secondary metabolite diversity.

Molassamide, a depsipeptide serine protease inhibitor from the marine cyanobacterium Dichothrix utahensis.

A new dolastatin 13 analogue, molassamide (1), was isolated from cyanobacterial assemblages of Dichothrix utahensis collected from the Molasses Reef, Key Largo, Florida, and from Brewers Bay, St. Thomas, U.S. Virgin Islands. This is the first peptide reported from the cyanobacterial genus Dichothrix and the first natural product isolated from marine Dichothrix spp. Its planar structure was determined by NMR spectroscopic techniques, and the configurations of the asymmetric centers were assigned after chiral HPLC analysis of the hydrolysis products. The depsipeptide 1 exhibited protease-inhibitory activity, with IC(50) values of 0.032 and 0.234 muM against elastase and chymotrypsin, respectively. There was no apparent inhibition of trypsin at 10 microM, the highest concentration tested.