Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have ...low toxicity. We report the potent anti-HPV activity of two
N-methylpyrrole–imidazole polyamides of the hairpin type, polyamide 1 (PA
1) and polyamide 25 (PA
25). Both polyamides have potent anti-HPV activity against three different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4–18 cells), and HPV31 (in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication (
ori), four polyamides were identified that possessed apparent IC
50s
⩽
150
nM with no evidence of cytotoxicity. We report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA
1 and PA
25 are currently in preclinical development as antiviral compounds for treatment of HPV-related disease, including cervical dysplasia. PA
1, PA
25, and related polyamides offer promise as antiviral agents and as tools to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to our polyamides, is accompanied by significant cellular toxicity.
A small-molecule inhibitor of hepatitis C virus (HCV) designated AP89652 was identified by screening a compound library with an HCV genotype 1b subgenomic replicon assay. AP89652 contains two chiral ...centers, and testing of two syn enantiomers revealed that activity in the replicon assay resided with only one, AP80978, whose 50% effective concentration (EC50) (the concentration at which a 50% reduction in Renilla luciferase levels was observed relative to an untreated control) was 630 nM. AP80978 was inhibitory against HCV genotypes 1a and 1b but not genotype 2a. In a replicon clearance assay, the potency and clearance rate of AP80978 were similar to those of telaprevir (VX950) and cyclosporine (CsA). AP80978 was nontoxic when tested against a panel of human cell lines, and inhibitory activity was HCV specific in that there was limited activity against negative-strand viruses, an alphavirus, and flaviviruses. By selection of resistant replicons and assessment of activity in genotype 1b/2a intergenotypic replicons, the viral protein target of this compound was identified as NS4B. NS4B F98V/L substitutions were confirmed by site-directed mutagenesis as AP80978 resistance-associated mutations. When tested against HCV produced in cell culture, the compound was significantly more potent than other HCV inhibitors, including VX950, CsA, and 2'-C-methyladenosine (2'C-meA). In addition, AP80977, the enantiomer that was inactive in the replicon assay, had activity against the virus, although it was lower than the activity of AP80978. These results suggest that AP80978 has the potential to be optimized into an effective antiviral drug and is a useful tool to further study the role of NS4B in HCV replication.
New Mn(II) macrocyclic pentaamine complexes derived from the biscyclohexyl-pyridine complex, M40403 ...(manganese(II)dichloro{(4R,9R,14R,19R)-3,10,13,20,26-pentaazatetracyclo20.3.1.0.4,9014,19hexacosa-1(26),-22(23),24-triene}), are described here. The complex M40403 was previously shown to be a superoxide dismutase (SOD) catalyst with rates for the catalytic dismutation of superoxide to oxygen and hydrogen peroxide at pH = 7.4 of 1.2 × 10+7 M-1 s-1. The use of the computer-aided design paradigm reported previously for this class of Mn(II) complexes2,3 led to the prediction that the 2S,21S-dimethyl derivative of M40403 should possess superior catalytic SOD activity. The synthesis of this new macrocyclic Mn(II) complex, manganese(II)dichloro{2S, 21S-dimethyl-(4R,9R,14R,19R)-3,10,13,20,26-pentaazatetracyclo20.3.1.0.4,9014,19hexacosa-1(26),22(23),24-triene}, 5, was accomplished via a high yield template condensation utilizing the linear tetraamine, N,N‘-Bis{(1R,2R)-2-(amino)cyclohexyl}-1,2-diaminoethane, 1, 2,6-diacetylpyridine, and MnCl2 to form the macrocyclic diimine complex, 2, which then is reduced. The two other possible dimethyl diastereomers of 5 (2R,21R-dimethyl, 3, and 2R,21S-dimethyl, 6) were also prepared via reduction of the diimine complex 2. Two of these complexes, 3 and 5, were characterized by X-ray structure determination confirming their absolute stereochemistry as 2R,21R-dimethyl and 2S,21S-dimethyl, respectively. The results of the MM calculations which predict that the 2S,21S-dimethyl complex, 5, should be a high activity catalyst and that the 2R,21R-dimethyl complex, 3, should have little or no catalytic activity are presented. The catalytic SOD rates for these complexes are reported for each of these complexes and a correlation with the modeling predictions is established showing that 2R,21R-complex, 3, has no measurable catalytic rate, while the 2R,21S complex, 6, is identical to M40403, and the 2S,21S- complex, 5, possesses a very fast rate at pH = 7.4 of 1.6 × 10+9 M-1 s-1 exceeding that of the native mitochondrial MnSOD enzymes.
A discovery approach based on an intramolecular inhibitory mechanism was applied to a prototype calmodulin (CaM)-regulated protein kinase in order to demonstrate a proof-of-principle for the ...development of selective inhibitors. The overall approach used functional genomics analysis of myosin light chain kinase (MLCK) to identify short autoinhibitory sequences that lack CaM recognition activity, followed by recursive combinatorial peptide library production and comparative activity screens. Peptide 18 (Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2), one of several selective inhibitors discovered, has an IC50 = 50 nM for MLCK, inhibits CaM kinase II only at 4000-fold higher concentrations, and does not inhibit cyclic AMP-dependent protein kinase. Analogues of peptide 18 containing conformationally constrained cis-4-aminocyclohexanecarboxylic acid retained affinity and selectivity. The inhibitors add to the armamentarium available for the deconvolution of complex signal transduction pathways and their relationship to homeostasis and disease, and the approach is potentially applicable to enzymes in which the catalytic and regulatory domains are found within the same open reading frame of a cDNA.
A solid-phase approach for the total synthesis of methyl carboxymycobactins
1a–
d, with an on-resin cyclization leading to azopine
5 as the key step, was developed. The iron-affinity of these ...compounds was assessed by a competitive sulfoxine-binding assay, and antimycobacterial activity was tested against the growth of
Mycobacterium avium.
The solid-phase syntheses of methyl carboxymycobactin T 7 (
1a) and its analogues are reported.
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