Engineered Peptide Macrocycles Can Inhibit Matrix Metalloproteinases with High Selectivity.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases at the intersection of health and disease due to their involvement in processes such as tissue repair and immunity as well as cancer and inflammation. Because of the high structural conservation in the catalytic domains and shallow substrate binding sites, selective, small-molecule inhibitors of MMPs have remained elusive. In a tour-de-force peptide engineering approach combining phage-display selections, rational design of enhanced zinc chelation, and d-amino acid screening, we succeeded in developing a first synthetic MMP-2 inhibitor that combines high potency (Ki =1.9±0.5 nm), high target selectivity, and proteolytic stability, and thus fulfills all the required qualities for in cell culture and in vivo application. Our work suggests that selective MMP inhibition is achievable with peptide macrocycles and paves the way for developing specific inhibitors for application as chemical probes and potentially therapeutics.

Influence of S-Oxidation on Cytotoxic Activity of Oxathiole-Fused Chalcones.

Synthesis, in vitro cytotoxic activity, and interaction with tubulin of oxidized, isomeric 1-(5-alkoxybenzo[d][1,3]oxathiol-6-yl)-3-phenylprop-2-en-1-ones and 1-(6-alkoxybenzo[d][1,3]oxathiol-5-yl)-3-phenylprop-2-en-1-ones are described. Most of the compounds demonstrated cytotoxic activity at submicromolar concentrations. It was found that oxidation of sulfur atom of the oxathiole-fused chalcones strongly influenced activity of the parent compounds, and that depending on relative position of the sulfur atom in the molecule, the activity was either increased or diminished. For isomers with sulfur atom para to the chalcone carbonyl group, oxidation led to increase in activity, while for isomers with sulfur atom meta to the carbonyl the activity dropped down. It was demonstrated that the compounds interact with tubulin at the colchicine binding site, and the interaction was evaluated using molecular modeling. It was concluded that the observed profound influence of oxidation of the sulfur atom on cytotoxic activity cannot be solely related to interaction of the compounds with tubulin.

Structural factors affecting affinity of cytotoxic oxathiole-fused chalcones toward tubulin.

Synthesis, in vitro cytotoxic activity, and interaction with tubulin of (E)-1-(6-alkoxybenzo[d][1,3]oxathiol-5-yl)-3-phenylprop-2-en-1-one derivatives (2) are described. Some of the compounds demonstrated cytotoxic activity at submicromolar concentrations, and the activity could be related to interaction with tubulin at the colchicine binding site. Interaction of selected derivatives with tubulin was evaluated using molecular modeling, and two different modes of the interaction were identified. The proposed models demonstrate how particular structural fragments participate in binding to the tubulin and explain the importance of the fragments for cytotoxic activity. It was demonstrated that concerning binding to tubulin, the 6-alkoxybenzoxathiole ring can be considered as structural equivalent of trimethoxyphenyl motif of colchicine, podophyllotoxin or combretastatin A4. The observation opened new ways of rational modifications of several groups of tubulin binders.

Identification of target-binding peptide motifs by high-throughput sequencing of phage-selected peptides.

High-throughput sequencing was previously applied to phage-selected peptides in order to gain insight into the abundance and diversity of isolated peptides. Herein we developed a procedure to efficiently compare the sequences of large numbers of phage-selected peptides for the purpose of identifying target-binding peptide motifs. We applied the procedure to analyze bicyclic peptides isolated against five different protein targets: sortase A, urokinase-type plasminogen activator, coagulation factor XII, plasma kallikrein and streptavidin. We optimized sequence data filters to reduce biases originating from the sequencing method and developed sequence correction algorithms to prevent identification of false consensus motifs. With our strategy, we were able to identify rare target-binding peptide motifs, as well as to define more precisely consensus sequences and sub-groups of consensus sequences. This information is valuable to choose peptide leads for drug development and it facilitates identification of epitopes. We furthermore show that binding motifs can be identified after a single round of phage selection. Such a selection regimen reduces propagation-related bias and may facilitate application of phage display in non-specialized laboratories, as procedures such as bacterial infection, phage propagation and purification are not required.

Structural factors affecting cytotoxic activity of (E)-1-(Benzo[d ][1,3]oxathiol-6-yl)-3-phenylprop-2-en-1-one derivatives.

Derivatives of (E)-1-(5-alkoxybenzo[d][1,3]oxathiol-6-yl)-3-phenylprop-2-en-1-one demonstrated exceptionally high in vitro cytotoxic activity, with IC50 values of the most active derivatives in the nanomolar range. To identify structural fragments necessary for the activity, several analogs deprived of selected fragments were prepared, and their cytotoxic activity was tested. It was found that the activity depends on combined effects of (i) the heterocyclic ring, (ii) the alkoxy group at position 5 of the benzoxathiole ring, and (iii) the substituents in the phenyl ring B. Replacement of the sulfur atom by oxygen does not influence the activity. None of the listed structural fragments alone assured high cytotoxic activity.

Chemical reactivity and antimicrobial activity of N-substituted maleimides.

Several N-substituted maleimides containing substituents of varying bulkiness and polarity were synthesised and tested for antimicrobial and cytostatic activity. Neutral maleimides displayed relatively strong antifungal effect minimum inhibitory concentrations (MICs in the 0.5-4 µg ml(-1) range); their antibacterial activity was structure dependent and all were highly cytostatic, with IC(50) values below 0.1 µg ml(-1). Low antimicrobial but high cytostatic activity was noted for basic maleimides containing tertiary aminoalkyl substituents. Chemical reactivity and lipophilicity influenced antibacterial activity of neutral maleimides but had little if any effect on their antifungal and cytostatic action. N-substituted maleimides affected biosynthesis of chitin and ß(1,3)glucan, components of the fungal cell wall. The membrane enzyme, ß(1,3)glucan synthase has been proposed as a putative primary target of N-ethylmaleimide and some of its analogues in Candida albicans cells.

Synthesis, structure, and tuberculostatic activity of dimethyl benzoylcarbonohydrazonodithioates.

ABSTRACT: New dimethyl benzoylcarbonohydrazonodithioates were obtained by CS2 addition to arylcarboxylic acid hydrazides and methylation of the formed adduct. The new derivatives were tested for their activity against Mycobacterium tuberculosis. Some compounds exhibited high activity toward sensitive and resistant strains.

Increased cytotoxicity of an unusual DNA topoisomerase II inhibitor compound C-1305 toward HeLa cells with downregulated PARP-1 activity results from re-activation of the p53 pathway and modulation of mitotic checkpoints.

Our previous studies have shown that murine fibroblast cells, in which PARP-1 gene was inactivated by gene disruption, are extremely sensitive to triazoloacridone compound C-1305, an inhibitor of DNA topoisomerase II with unusual properties. Here, we show that pharmacological inhibition of PARP-1 activity by its inhibitor compound NU1025, sensitizes human cervical carcinoma HeLa cells to compound C-1305 compared to treatment with drug alone. Cytotoxic effect of drug/NU1025 of other topoisomerase II inhibitors varied depending on the dose of PARP-1 inhibitor. Increased cytotoxicity of topoisomerase II inhibitor/NU1025 combinations was attributable to the re-activation of the p53 pathway in drug-treated HeLa cells. This lead to a more stringent cell cycle checkpoint control during G2 and M and enhanced cell death by mitotic catastrophe induced by drug/NU1025 combinations. Interestingly, treatment of HeLa cells with NU1025 alone also increased p53 expression. This effect is, at least in part, related to the inhibition of proteasome activity by drug treatments. Together, our results show that concomitant inhibition of topoisomerase II and PARP-1 leads to the synergistic cytotoxic effect toward tumor cells that may be important for combination therapies with NU1025 and topoisomerase II inhibitors. We also confirmed our earlier work and show the important role of PARP-1 activity in the maintenance of the G2 arrest induced by DNA damaging drugs. Finally, based on our studies we propose that NU1025 and possibly other inhibitors of PARP-1 may be used as non-genotoxic agents to activate p53 in tumor cells with non-functional p53 pathways.

Cancer stem cells and escape from drug-induced premature senescence in human lung tumor cells: implications for drug resistance and in vitro drug screening models.

In this study, using an in vitro human tumor model, we show that non-small lung adenocarcinoma A549 cells after treatment with DNA damaging antitumor drugs become permanently growth-arrested as a result of so-called drug-induced premature senescence (pseudo-senescence). However, a small fraction of drug-treated cells escapes pseudo-senescence that leads to re-growth of tumor cell population after drug treatment. We show that this re-growth is associated with the presence of cancer stem cells (CSCs) in lung tumor cell population. We also document that re-growth of CSCs can be greatly delayed if lung tumor cells are treated with drug/caffeine combination that leads to the inhibition of the ATM/ATR pathway and decreased phosphorylation of PKB/Akt at Ser473. We show that in non-treated A549 cells caffeine by itself induces a reversible growth arrest that is associated with increased fraction of so-called side population cells, containing CSCs. These results point to the existence of an unknown, caffeine-sensitive mechanism that controls the number of CSCs in lung tumor cell population. Full characterization of this mechanism may lead to the development of innovative cancer therapies, which are based on small molecular weight inhibitors of CSC differentiation and self-renewal, which mimic caffeine action. Our results have also important implications for drug screening tumor models in vitro.

Structural determinants of imidazoacridinones facilitating antitumor activity are crucial for substrate recognition by ABCG2.

Symadex is the lead acridine compound of a novel class of imidazoacridinones (IAs) currently undergoing phase II clinical trials for the treatment of various cancers. Recently, we have shown that Symadex is extruded by ABCG2-overexpressing lung cancer A549/K1.5 cells, thereby resulting in a marked resistance to certain IAs. To identify the IA residues essential for substrate recognition by ABCG2, we here explored the ability of ABCG2 to extrude and confer resistance to a series of 23 IAs differing at defined residue(s) surrounding their common 10-azaanthracene structure. Taking advantage of the inherent fluorescent properties of IAs, ABCG2-dependent efflux and drug resistance were determined in A549/K1.5 cells using flow cytometry in the presence or absence of fumitremorgin C, a specific ABCG2 transport inhibitor. We find that a hydroxyl group at one of the R1, R2, or R3 positions in the proximal IA ring was essential for ABCG2-mediated efflux and consequent IA resistance. Moreover, elongation of the common distal aliphatic side chain attenuated ABCG2-dependent efflux, thereby resulting in the retention of parental cell sensitivity. Hence, the current study offers novel molecular insight into the structural determinants that facilitate ABCG2-mediated drug efflux and consequent drug resistance using a unique platform of fluorescent IAs. Moreover, these results establish that the IA determinants mediating cytotoxicity are precisely those that facilitate ABCG2-dependent drug efflux and IA resistance. The possible clinical implications for the future design of novel acridines that overcome ABCG2-dependent multidrug resistance are discussed.

Modulation of cellular response to anticancer treatment by caffeine: inhibition of cell cycle checkpoints, DNA repair and more.

Caffeine and other methylxanthines produce multiple physiologic effects throughout the human body, many of these effects could potentially modulate the activity of anticancer therapy. Caffeine may directly interfere with drug transport to tumor cells by formation of mixed stacking complexes with polyaromatic drugs. If formed in cells, these complexes may also prevent of intercalating drugs from DNA binding and, in this way, lower their antitumor activity. Since many of potent carcinogens are polyaromatic compounds, formation of stacking complexes with carcinogens could be associated with anti-genotoxic activity of caffeine and its use in cancer chemoprevention. Caffeine has also been reported to inhibit ATM and ATR kinases which leads to the disruption of multiple DNA damage-responsive cell cycle checkpoints and greatly sensitizes tumor cells to antitumor agents which induce genotoxic stress. Caffeine may inhibit repair of DNA lesions through a direct interference with DNA-PK activity and other repair enzymes. A number of in vitro and in vivo studies demonstrated that caffeine modulates both innate and adaptive immune responses via inhibition of cyclic adenosine monophosphate (cAMP)-phosphodiesterase. Finally, another group of effects induced by caffeine is mediated through its inhibitory action on adenosine receptors. This may modulate the stability of HIF1 alpha as well as VEGF and interleukin-8 expression in tumor cells, which could have a direct impact on neovascularization of human tumors. In this review, we present different molecular mechanisms by which caffeine and other methylxanthines may directly or indirectly modulate the effect of antitumor treatment in tumor cells and in cancer patients.

Dual inhibition of PI3K/Akt signaling and the DNA damage checkpoint in p53-deficient cells with strong survival signaling: implications for cancer therapy.

Natural (intrinsic) resistance of many tumor types to DNA damaging agents is closely associated with their capacity to undergo robust cell cycle arrest in G(2)/M. G(2) arrest is regulated by the DNA damage checkpoint and by survival signaling, with a potential role of PI3K/Akt in checkpoint function. In this work, we wanted to clarify if inhibition of multiple checkpoint/survival pathways may confer better efficacy in the potentiation of genotoxic agents compared to inhibition of either pathway alone. We compared the influence of UCN-01, which affects both the DNA damage checkpoint and PI3K/Akt-mediated survival signaling, with the PI3K inhibitors wortmannin and LY294002 in p53-deficient M1 acute myeloid leukemia cells treated with the DNA damaging agent cisplatin. Our results show that direct inhibition of PI3K/Akt in G(2)-arrested cells by wortmannin or LY294002 strongly enhanced the cytotoxicity of cisplatin without influencing the G(2) checkpoint. Unexpectedly, dual inhibition of both survival and checkpoint signaling by UCN-01, also increased the cytotoxicity of cisplatin, but to a lesser degree than wortmannin or LY294002. The differences in cytotoxicity were accompanied by differences in cell death pathways: direct inhibition of PI3K/Akt was accompanied by rapid apoptotic cell death during G(2), whereas cells underwent mitotic transit and cell division followed by cell death during G(1) when both checkpoint and survival signaling were inhibited. Our results elucidate a novel function for PI3K/Akt as a survival factor during DNA damage-induced G(2) arrest and could have important pharmacological consequences for the application of response modulators in p53-deficient tumors with strong survival signaling.

Synthesis of isomeric, oxathiolone fused chalcones, and comparison of their activity toward various microorganisms and human cancer cells line.

Isomeric oxathiolone fused chalcones were prepared by condensation of appropriate acetylbenzo[1,3]oxathiol-2-ones with benzaldehydes under acidic conditions. The synthesized compounds were screened for cytotoxic activity using HeLa cells, as well as for antibacterial activity against Micrococcus luteus, Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, Proteus vulgaris, antifungal activity against Candida albicans, and tuberculostatic activity against Mycobacterium tuberculosis H(37)Rv and Mycobacterium kansasii strains.

Acid-catalyzed synthesis of oxathiolone fused chalcones. Comparison of their activity toward various microorganisms and human cancer cells line.

Substituted oxathiolone fused chalcones were prepared by condensation of 4-acetyl-5-methoxy-2-oxo-benz[1,3]oxathiole with benzaldehydes under acidic conditions. The compounds were tested for cytotoxic, antibacterial, antifungal and tuberculostatic activity. Three derivatives demonstrated weak activity against HeLa cells, two were slightly active against Micrococcus luteus and Staphylococcus aureus, and one was active against Mycobacterium tuberculosis H(37)Rv.

Cross-talk between DNA damage and cell survival checkpoints during G2 and mitosis: pharmacologic implications.

In this study, we wanted to clarify the role of survivin-mediated survival signaling during G2 and M in tumor cells treated with DNA-damaging agents. As a cellular model, we selected MOLT-4 human T-cell lymphoblastic leukemia cells that overexpress survivin and nonfunctional p53. Treatment with melphalan, a classic DNA-damaging agent, led to the induction of the DNA damage checkpoint and growth arrest in the G2 phase of the cell cycle. Checkpoint abrogation by caffeine was accompanied by mitotic entry and rapid apoptotic cell death, whereas cells remaining in G2 remained viable during the same time interval. Unexpectedly, when the spindle checkpoint was activated following G2 abrogation, two different effects could be observed. If the microtubules of the melphalan-treated cells were destabilized by nocodazole, cells became arrested in prometaphase with low survivin levels and entered apoptosis. In contrast, if the microtubules of the melphalan-treated cells were stabilized by taxol, cells were still arrested in prometaphase, but apoptotic execution was inhibited. This effect is, most likely, directly mediated by survivin itself given its well-established antiapoptotic functions. In conclusion, depending on the way the spindle checkpoint was activated in cells with damaged DNA, cells could be either protected by survivin or die during mitosis. We suggest that the efficacy of DNA damage checkpoint abrogators used in combination with DNA-damaging agents may critically depend on whether DNA damage is able to invoke spindle checkpoint response and to activate survivin-associated survival signaling during mitosis.

Down-regulation of DNA topoisomerase IIalpha leads to prolonged cell cycle transit in G2 and early M phases and increased survival to microtubule-interacting agents.

Microtubule binders are cell cycle-specific agents with preferential cytotoxicity toward mitotic cells. We have characterized vincristine-selected human leukemia cells to establish whether development of vincristine resistance was accompanied by changes in cell cycle kinetics and distribution. Our results indicate that vincristine resistance is accompanied by delayed G2 transit and prolonged early mitosis in both the absence and the presence of the microtubule binder nocodazole. The altered G2/M regulation is accompanied by resistance to short-term (12 h) but not continuous nocodazole exposure in agreement with the transient nature of the observed cell cycle alterations. Western blot analysis indicates that vincristine-selection is accompanied by down-regulation of topoisomerase IIalpha without detectable alterations of the other mitotic regulators studied, including Cdk1, p21, 14-3-3sigma, and 14-3-3epsilon. This was associated with at least 7-fold less chromosome-associated topoisomerase IIalpha, decreased catalytic activity, and cross-resistance to topoisomerase II inhibitors. Characterization of isogenic cell lines expressing different levels of topoisomerase II proteins shows that cellular levels of topoisomerase IIalpha, but not the closely related topoisomerase IIbeta, directly influence the cell cycle kinetics in G2 and early mitosis as well as the resistance to nocodazole. These results underline the importance of topoisomerase IIalpha in late G2 and early M phases and provide evidence for an as-yet-unsuspected interaction between topoisomerase II and microtubule-directed agents.