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-pentaazatetracyclo[20.3.1.0.(4,9)0(14,19)]hexacosa-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 x 10(+7) M(-1) s(-1).(1) The use of the computer-aided design paradigm reported previously for this class of Mn(II) complexes(2,3) 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-pentaazatetracyclo[20.3.1.0.(4,9)0(14,19)]hexacosa-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 MnCl(2) 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 x 10(+9) M(-1) s(-1) exceeding that of the native mitochondrial MnSOD enzymes.

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 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against 3 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 (tested 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), 4 polyamides were identified that possessed apparent IC50s ≤ 150 nM with no evidence of cytotoxicity and 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. PA1 and PA25 are currently in preclinical development as an antiviral compound for treatment of HPV-related disease, including cervical dysplasia. PA1 and related polyamides offer promise as antiviral agents and the ability 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 PA1, is accompanied by significant cellular toxicity.

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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.

This article describes the solid-phase combinatorial methods developed for the synthesis of polyhydroxamate-based siderophores. This strategy was applied to generate several libraries of structural DFO (1a) analogues that include DFO variants, non-amide analogues, C-terminal modified analogues, reverse-amide analogues, and hybrid analogues. To assess the relative iron-binding affinities of these compounds, a high-throughput spectrophotometric screening method based on competition with 8-hydroxyquinoline-5-sulfonic acid was developed. Some of the promising candidates containing various terminal functional groups were identified and prepared on large scale to enable future studies in animal models for iron-overload diseases.

Novel, highly constrained, 7,5-bicyclic dipeptides (1 -aza-6-oxa-2-oxobicyclo[5.3.0]decane ring skeletons, 3Sa and 7Sa) have been synthesized on a 40 mmol scale in -50% yield by a one-step electrochemical cyclization from the dipeptides Boc-L-homoserine-L-proline-OMe (Boc-Hse-Pro-OMe) and Boc-Hse-D-Pro-OMe. The reaction involved a selective anodic amide oxidation which was highly diastereoselective, generating a new chiral center having an S configuration from both precursors. In terms of conformation, the bicyclic system restricts two (q2 and 43) of the four torsion angles that characterize a reverse turn. Conformational analysis of these molecules and analogs having an R configuration at the ring fusion revealed some families of minimum energy conformations with torsion angles close to those of classical ,!?-turns, a secondary structural feature found in many bioactive peptides. This new ring skeleton was stable to trifluoroacetic acid, dilute base, and anhydrous hydrofluoric acid, making it compatible with standard solid phase peptide synthesis methodologies.

West Nile virus (WNV) has spread throughout the United States and Canada and now annually causes a clinical spectrum of human disease ranging from a self-limiting acute febrile illness to acute flaccid paralysis and lethal encephalitis. No therapy or vaccine is currently approved for use in humans. Using high-throughput screening assays that included a luciferase expressing WNV subgenomic replicon and an NS1 capture enzyme-linked immunosorbent assay, we evaluated a chemical library of over 80,000 compounds for their capacity to inhibit WNV replication. We identified 10 compounds with strong inhibitory activity against genetically diverse WNV and Kunjin virus isolates. Many of the inhibitory compounds belonged to a chemical family of secondary sulfonamides and have not been described previously to inhibit WNV or other related or unrelated viruses. Several of these compounds inhibited WNV infection in the submicromolar range, had selectivity indices of greater than 10, and inhibited replication of other flaviviruses, including dengue and yellow fever viruses. One of the most promising compounds, AP30451, specifically blocked translation of a yellow fever virus replicon but not a Sindbis virus replicon or an internal ribosome entry site containing mRNA. Overall, these compounds comprise a novel class of promising inhibitors for therapy against WNV and other flavivirus infections in humans.

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