Search

Supplementary Figure 1 from Identification of Nuclear Export Inhibitors with Potent Anticancer Activity In vivo

Supplementary Figure 3 from Identification of Nuclear Export Inhibitors with Potent Anticancer Activity In vivo

Supplementary Methods and Materials from Identification of Nuclear Export Inhibitors with Potent Anticancer Activity In vivo

Supplementary Figure 2 from Identification of Nuclear Export Inhibitors with Potent Anticancer Activity In vivo

Cholera remains a major cause of infectious diarrhea globally. Despite the increased availability of cholera vaccines, there is still an urgent need for other effective interventions to reduce morbidity and mortality. Furthermore, increased prevalence of antibiotic-resistant Vibrio cholerae threatens the use of many drugs commonly used to treat cholera. We developed iOWH032, a synthetic small molecule inhibitor of the cystic fibrosis transmembrane conductance regulator chloride channel, as an antisecretory, host-directed therapeutic for cholera. In the study reported here, we tested iOWH032 in a Phase 2a cholera controlled human infection model. Forty-seven subjects were experimentally infected with V. cholerae El Tor Inaba strain N16961 in an inpatient setting and randomized to receive 500 mg iOWH032 or placebo by mouth every 8 hours for 3 days to determine the safety and efficacy of the compound as a potential treatment for cholera. We found that iOWH032 was generally safe and achieved a mean (± standard deviation) plasma level of 4,270 ng/mL (±2,170) after 3 days of oral dosing. However, the median (95% confidence interval) diarrheal stool output rate for the iOWH032 group was 25.4 mL/hour (8.9, 58.3), compared to 32.6 mL/hour (15.8, 48.2) for the placebo group, a reduction of 23%, which was not statistically significant. There was also no significant decrease in diarrhea severity and number or frequency of stools associated with iOWH032 treatment. We conclude that iOWH032 does not merit future development for treatment of cholera and offer lessons learned for others developing antisecretory therapeutic candidates that seek to demonstrate proof of principle in a cholera controlled human infection model study. Trial registration: This study is registered with ClinicalTrials.gov as NCT04150250., Author summary Cholera, a disease caused by infection with the bacterium Vibrio cholerae, remains a major cause of diarrheal illness and death, particularly in settings with poor sanitation and hygiene. We developed a synthetic chemical, named “iOWH032,” as a potential treatment for cholera, which is administered as oral tablets. The chemical acts by blocking secretions from cells in the intestine, and thereby was expected to prevent fluid loss and dehydration caused by cholera illness. We tested iOWH032 in a clinical study using a cholera human challenge model. Study volunteers were intentionally infected with V. cholerae in an inpatient clinic setting to better study the effects of iOWH032 on infected individuals. This challenge model had been used previously to test cholera vaccine candidates, but this study represents the first test of a potential cholera treatment using the model. We found that treatment of individuals with iOWH032 was safe, but did not result in a significant reduction of cholera illness, based on several different measurements of diarrheal symptoms and severity. This study demonstrates how human challenge models incorporating a relatively small number of subjects can help support decision-making about potential new therapeutics and other interventions for infectious diseases.

The export protein CRM1 is required for the nuclear export of a wide variety of cancer-related “cargo” proteins including p53, c-Abl, and FOXO-3A. Leptomycin B (LMB) is a highly specific inhibitor of CRM1 with significant in vitro potency but limited in vivo efficacy due to toxicity. We now report a series of semisynthetic LMB derivatives showing substantially improved therapeutic windows. Exposure of cancer cells to these compounds leads to a rapid and prolonged block of nuclear export and apoptosis. In contrast to what is observed in cancer cells, these agents induce cell cycle arrest, but not apoptosis, in normal lung fibroblasts. These new nuclear export inhibitors (NEI) maintain the high potency of LMB, are up to 16-fold better tolerated than LMB in vivo, and show significant efficacy in multiple mouse xenograft models. These NEIs show the potential of CRM1 inhibitors as novel and potent anticancer agents. [Cancer Res 2009;69(2):510–7]

Combinations of the five polyketide synthase (PKS) genes for biosynthesis of tylosin in Streptomyces fradiae (tylG), spiramycin in Streptomyces ambofaciens (srmG), or chalcomycin in Streptomyces bikiniensis (chmG) were expressed in engineered hosts derived from a tylosin-producing strain of S. fradiae. Surprisingly efficient synthesis of compounds predicted from the expressed hybrid PKS was obtained. The post-PKS tailoring enzymes of tylosin biosynthesis acted efficiently on the hybrid intermediates with the exception of TylH-catalyzed hydroxylation of the methyl group at C14, which was efficient if C4 bore a methyl group, but inefficient if a methoxyl was present. Moreover, for some compounds, oxidation of the C6 ethyl side chain to an unprecedented carboxylic acid was observed. By also expressing chmH, a homolog of tylH from the chalcomycin gene cluster, efficient hydroxylation of the 14-methyl group was restored.

A variety of regioisomeric bifunctionalized β-cyclodextrins derivatized with an imidazole and a metal ligand tris(2-aminoethyl)amine (tren) group were prepared. As their Zn2+ complexes, these compounds show bifunctional catalysis of phosphate hydrolysis with a regioisomeric preference.

Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15- R -6-dEB analogues were produced. The simpler “monoketide” acyl-thioester substrates required for this route of 15- R -6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1 o for the production of 15- R -6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N -acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both E. coli and S. coelicolor .

Analogs of the glycopeptide antibiotics vancomycin and teicoplanin with alterations in one or both sugar moieties of the disaccharide have been prepared by tandem action of the vancomycin pathway glycosyltransferases GtfE and GtfD. All four regioisomers (2-, 3-, 4-, 6-) of TDP-deoxyglucoses and UDP/TDP-aminoglucoses were prepared, predominantly by action of D-glucopyranosyl-1-phosphate thymidylyltransferase, E p . GtfE transferred the deoxyglucoses or aminoglucoses onto the 4-OH of 4-hydroxyphenylglycine of both the vancomycin and teicoplanin aglycone scaffolds. Kinetic analysis indicated the 2-, 3-, 4-, and 6-amino-glucoses were transferred by GtfE with only a 4- to 30-fold drop in k cat and no effect on K m compared to the native substrate, UDP/TDP-glucose, suggesting preparative utility. The next enzyme, GtfD, could utilize the variant glucosyl-peptides as substrates for transfer of L-4- epi -vancosamine. The aminosugar moieties in these variant glycopeptides introduce sites for acylation or reductive alkylation.

Two isomeric compounds have been synthesized carrying a pyridoxamine on C-6 of β-cyclodextrin and an imidazole unit on C-6 of the neighboring glucose residue. Each one stereoselectively transaminates phenylpyruvic acid to produce phenylalanine, and with opposite stereochemical preferences. Structure determinations by X-ray crystallography and NMR spectroscopy indicate that the imidazole units serve to block proton addition from their side, rather than acting to protonate the transamination intermediates. Related cyclodextrin–pyridoxamine compounds had been reported carrying ethylenediamine units instead of imidazoles, and high enantioselectivities in transamination were claimed. Our work indicates that these claims are incorrect, and that only poor selectivities are seen that are often unrelated to the position of the ethylenediamine units. Neither of these transaminating systems yet approaches the enantioselectivity seen with a pyridoxamine carrying a chirally mounted internal base group.