Novel and Specific Inhibitors of a Poxvirus Type I Topoisomerase

Abstract

Vaccinia DNA topoisomerase (vTopo) is a prototypic pox virus family topoisomerase that shares extensive structural and mechanistic properties with the human type IB enzyme (hTopo) and is important for viral replication. Despite their far-reaching similarities, vTopo and hTopo have surprisingly distinct pharmacological properties. To further exploit these differences, we have developed recently the first high-throughput screen for vTopo, which has allowed rapid screening of a 1990-member small-molecule library for inhibitors. Using this approach, 21 compounds were identified with IC 90 values less than 10 M, and 19 of these were also found to inhibit DNA supercoil relaxation by vTopo. Four of the most potent compounds were completely characterized and are structurally novel topo I inhibitors with efficacies at nanomolar concentrations. These inhibitors were highly specific for vTopo, showing no inhibition of the human enzyme even at 500-to 2000-fold greater concentrations. We describe a battery of efficient experiments to characterize the unique mechanisms of these vTopo inhibitors and discuss the surprising promiscuity of this enzyme to inhibition by structurally diverse small molecules.

Identification of Novel Keratinocyte Differentiation Modulating Compounds by High-Throughput Screening

“…Two of the compounds we identified, CCT001643 and CCT070795, were selective inhibitors of differentiation by the additional criteria and will be of interest in exploring the link between cell cycle withdrawal and initiation of terminal differentiation. 5,14 Of note was the compound CCT002080 (which has shown a number of other biological activities [32][33][34][35] ) that we found promoted keratinocyte differentiation. This compound is particularly interesting because it had the same effect, and at lower doses, on SCC cells, although not affecting a fibroblastic cell line, J2-3T3.…”

“…The chemical structure of the compound is shown in Figure 5B. In addition to its use as an antiseptic, 32 CCT002080 has been reported to inhibit anthrax lethal factor metalloprotease, 33 vaccinia DNA topoisomerase, 34 and Kaposi's sarcoma-associated herpesvirus DNA synthesis. 35 The induction of differentiation by 20 µM CCT002080 was confirmed by flow cytometry of cells stained with anti-involucrin (SY5) or antibodies to a 2nd terminal differentiation marker, transglutaminase 1 (BC1; Fig.…”

New Peptide Inhibitors of Type IB Topoisomerases: Similarities and Differences Vis-a-vis Inhibitors of Tyrosine Recombinases

“…To relate this to their affinity for HJ, we titrated these substituted peptides on HJ using gel-shift assays. Each alanine-substituted peptides bound with 2 to 3-fold less affinity than WYCRCK as judged by the amount of free HJ DNA in lanes with 150 nM compared to 50 nM WYCRCK (Figure 6 [18][19], showing a direct correlation between the number of cysteine residues present in each hexapeptide and the peptide-HJ complexes formed. Peptide WYARAK did not shift HJ substrates at all (data not shown).…”

“…The amount of remaining supercoiled substrate was compared among the treated reactions ( Fig. 1(b), lanes [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] and to untreated control reactions (Fig 1(b), lanes 1 and 2). In the example shown, the mixtures containing amino acids C, F, L, M, W and Y at the second position inhibit vTopo activity most.…”

Multicomponent Reactions in Ionic Liquids

“…Pyrimidoquinolinedione derivatives are known to possess antitumor, anticancer, antihypertensive, antibacterial activity and are Kaposi's sarcoma-associated herpesvirus and topoisomerase inhibitors. [89], [90] …”