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We studied the role of the karyopherin a2 nuclear import carrier (also known as importin a2) in glucose signaling. In mhAT3F hepatoma cells, GFP-karyopherin a2 accumu- lated massively in the cytoplasm within minutes of glu- cose extracellular addition and returned to the nucleus after glucose removal. In contrast, GFP-karyopherin a1 distribution was unaffected regardless of glucose concentration. Glucose increased GFP-karyopherin a2 nuclear efflux by a factor 80 and its shuttling by a factor 4. These glucose-induced movements were not due to glycolytic ATP production. The mechanism involved was leptomycin B-insensitive, but phosphatase- and energy- dependent. HepG2 and COS-7 cells displayed no glucose- induced GFP-karyopherin a2 movements. In pancreatic MIN-6 cells, the glucose-induced movements of karyo- pherin a2 and the stimulation of glucose-induced gene transcription were simultaneously lost between pas- sages 28 and 33. Thus, extracellular glucose regulates a nuclear transport pathway by increasing the nuclear efflux and shuttling of karyopherin a2 in cells in which glucose can stimulate the transcription of sugar-responsive genes.

Parathyroid hormone-related protein is responsible for hypercalcemia induced by various tumors. The similarity of its N-terminus to that of parathyroid hormone enables parathyroid hormone-related protein to share parathyroid hormone's signaling properties, but the rest of the molecule possesses distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. We have previously shown that parathyroid hormone-related protein nuclear import is mediated by importin β1. Here we use fluorescence recovery after photobleaching for the first time to show that, in living cells, parathyroid hormone-related protein is exported from the nucleus in a leptomycin B-sensitive manner, implicating CRM1 as the parathyroid hormone-related protein nuclear export receptor. Leptomycin B treatment significantly reduced the rate of nuclear export 4 −10-fold, thereby increasing parathyroid hormone-related protein concentration in the nucleus/nucleolus about 2-fold. Intriguingly, this also led to a 2-fold reduced nuclear import rate. Inhibiting the nuclear export of a protein able to shuttle between nucleus and cytoplasm through distinct receptors thus can also affect nuclear import, indicating that the subcellular localization of a protein containing distinct nuclear import and export signals is the product of an integrated system. Although there have been several recent studies examining the dynamics of intranuclear transport using fluorescence recovery after photobleaching, this represents, to our knowledge, the first use of the technique to examine the kinetics of nucleocytoplasmic flux in living cells.

Trichostatin A (TSA), an inhibitor of the eukaryotic cell cycle and an inducer of morphological reversion of transformed cells, inhibits histone deacetylase (HDAC) at nanomolar concentrations. Recently, trapoxin, oxamflatin, and FR901228, antitumor agents structurally unrelated to TSA, were found to be potent HDAC inhibitors. These inhibitors activate expression of p21Waf1 and 16INK4A in a p53-independent manner. Changes in the expression of these cell cycle regulators by an increase in histone acetylation may be responsible for cell cycle arrest and antitumor activity by HDAC inhibitors. The target molecule of leptomycin B (LMB), a potent antitumor agent, was genetically and biochemically identified as CRM1, a protein reported as being required for chromosome structure control. We showed that CRM1 was a receptor for the nuclear export signal (NES) and that LMB inhibited nuclear export of proteins. Using LMB, we identified a novel NES in fission yeast transcription factor Pap1, the function of which is abolished by oxidative stress in a manner conserved in eukaryotes.

As one of the regulatory gene products in the HIV-1 genome, Rev protein must be translocated from the nucleus to the cytoplasm to exert its function. Therefore, inhibition of Rev protein translocation could be a useful target for HIV therapy. An extract from the Streptomyces strain A92-308902 with very potent inhibitory activity was found in the course of a high throughput screening with a Rev translocation assay (RTA). Bioassay-guided fractionation with gel filtration, normal-phase and reversed-phase chromatography yielded six RTA-active metabolites belonging to the leptomycin family, the known leptomycin A (1), leptomycin B (2), kazusamycin B (3), and kazusamycin A (4). and the hitherto unknown dilactonmycin (5) and delactonmycin (6), together with an inactive cyclic hexadepsipeptide L-156,620 (7). The structures were established mainly by spectroscopic methods (UV, FT-IR, FAB-MS, 1H-NMR, 13C-NMR(JMOD), DQ-COSY, ROESY, HSQC, and HMBC). The configuration of all CC bonds of 1–6 was unambiguously established by analysis of coupling constants and ROESY spectra. All isolated leptomycins 1–6 inhibit Rev translocation at nanomolar concentrations. Six derivatives (2a–c and 4a–c) of leptomycin B (2) and kazusamycin A (4) were also prepared and tested in the RTA for preliminary investigations on structure-activity relationships.

Many proteins are transported from the nucleus to the cytoplasm by the exportin CRM1, which recognizes cargo proteins through a leucine rich nuclear export signal (NES). This nuclear export process can be inhibited by several small molecules, both natural products and fully synthetic compounds. The structural basis for the inhibition of nuclear export by leptomycin (LMB) based on disruption of the protein/protein interaction between CRM1 and cargo proteins is discussed. The chemistry and inhibition of nucleocytoplasmic transport of leptomycin, anguinomycin and derivatives, goniothalamin, JBIR-02, valtrate, dihydrovaltrate, ACA, peumusolide A and several synthetic compounds is presented. Consequences for the design of nuclear export inhibitors are discussed, and the potential of these compounds as anticancer agents is evaluated.

This account describes several examples from the literature, as well as from our own work, of efforts to reduce the structural complexity of natural products through truncation by organic synthesis. This process, sometimes called diverted total synthesis or function oriented synthesis, also aims to deliver improved compounds for clinical evaluation. The discussed examples from the literature of this type of chemical editing are the truncation of halichondrin B to E7389/eribulin, bryostatin 1 to the bryologs, migrastatin to the migrastatin core, macfarlandin E, and somatostatin (SRIF14) to L-363,301 and octreotide. From our work, efforts on the truncation of anguinomycin/leptomycin, farinosone C to tyrosinol propionamide, and anachelin to the anachelin chromophore are discussed. A hypothesis as to why nature retains apparently nonfunctional appendages on biologically active core structures (related to the pharmacophore) in such ‘less than perfect' natural products is presented. It is proposed that such appendages are generated to increase chemotype plasticity, and that such plasticity might eventually lead to secondary benefits, such as improved physicochemical properties or additional bioactivity.

As one of the regulatory gene products in the HIV-1 genome, Rev protein must be translocated from the nucleus to the cytoplasm to exert its function. Therefore, inhibition of Rev protein translocation could be a useful target for HIV therapy. An extract from the Streptomyces strain A92-308902 with very potent inhibitory activity was found in the course of a high throughput screening with a Rev translocation assay (RTA). Bioassay-guided fractionation with gel filtration, normal-phase and reversed-phase chromatography yielded six RTA-active metabolites belonging to the leptomycin family, the known leptomycin A (1), leptomycin B (2), kazusamycin B (3), and kazusamycin A (4). and the hitherto unknown dilactonmycin (5) and delactonmycin (6), together with an inactive cyclic hexadepsipeptide L-156,620 (7). The structures were established mainly by spectroscopic methods (UV, FT-IR, FAB-MS, 1H-NMR, 13C-NMR(JMOD), DQ-COSY, ROESY, HSQC, and HMBC). The configuration of all CC bonds of 1–6 was unambiguously established by analysis of coupling constants and ROESY spectra. All isolated leptomycins 1–6 inhibit Rev translocation at nanomolar concentrations. Six derivatives (2a–c and 4a–c) of leptomycin B (2) and kazusamycin A (4) were also prepared and tested in the RTA for preliminary investigations on structure-activity relationships.

An effective total synthesis of (−)-callystatin A (1), member of the leptomycin family of antibiotics, has been achieved. The synthesis features Evans extended aldol methodology to construct the northern polypropionate subunit and two separate Julia olefinations to assemble the conjugated dienes. The total synthesis proceeded in 2.3% overall yield with the longest linear sequence of 15 steps.

Natural products play a dominant role in providing ligands that interfere with biopolymers such as receptors or enzymes. In many cases the exact mode of action or the targets are not known and synthesis of analogs provides a more detailed insight into the mode of action and helps to identify the protein target. We discuss the synthetic endeavors that led to the synthesis of members of the leptomycin family and will provide a picture of the mode of action.