Allosteric drugs: the interaction of antitumor compound MKT-077 with human Hsp70 chaperones

Hsp70 (heat shock protein 70 kDa) chaperones are key to cellular protein homeostasis. However, they also have the ability to inhibit tumor apoptosis and contribute to aberrant accumulation of hyperphosphorylated tau in neuronal cells affected by tauopathies, including Alzheimer's disease. Hence, Hsp70 chaperones are increasingly becoming identified as targets for therapeutic intervention in these widely abundant diseases. Hsp70 proteins are allosteric machines and offer, besides classical active-site targets, also opportunities to target the mechanism of allostery. In this work, it is demonstrated that the action of the potent anticancer compound MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride) occurs through a differential interaction with Hsp70 allosteric states. MKT-077 is therefore an “allosteric drug.” Using NMR spectroscopy, we identify the compound's binding site on human HSPA8 (Hsc70). The binding pose is obtained from NMR-restrained docking calculations, subsequently scored by molecular-dynamics-based energy and solvation computations. Suggestions for the improvement of the compound's properties are made on the basis of the binding location and pose.     

The interaction of platinum and anthrapyrazole antitumor drugs with mouse thymocytes studied by terbium fluorescence

The fluorescent lanthanide, terbium has been employed to study the effect of a series of platinum and anthracycline drugs and an anthrapyrazole (oxanthrazole) on terbium binding to mouse thymocytes. It was observed that terbium fluorescence intensity was markedly decreased by two platinum drugs (cis-dichlorodiammine platinum(II) (cis-DDP) and cis-dichloro-trans-dihydroxybis(isopropylammine) platinum(IV) (CHIP)) and an anthrapyrazole (oxanthrazole), but that the lipophylic derivative cis-diammine-1,1-cyclobutanedicarboxylate platinum(II) had a small but significant effect and the anthracyclines (at low concentrations) had no effect. The calcium channel blocker, verapamil also had no effect. The effect of cis-DDP was markedly dependent on ionic strength in contradistinction to CHIP. The decreases in phosphorescence decay produced by cis-DDP also showed a marked dependence on ionic strength. It is proposed that cis-DDP interacts with the membrane primarily by a charge effect, but that CHIP may produce a conformational change in the membrane. These data are interesting, since the lipophylic platinum drugs (CHIP and CBDCA) also increased significantly the amount of bound intracellular calcium, but all the drugs decreased mitogen-stimulated calcium uptake into mouse thymocytes.     

DNA interaction with the antitumor agent thiophosphamide

DNA interaction with an alkylating antitumor drug N,N',N"-triethylenethiophosphoramide (thiotepa) in water-salt solutions at 37 degrees C has been studied by UV-spectroscopy, heat denaturation and electron microscopy methods. Changes of the DNA melting curve parameters provide information on the kinetics of alkylation. The dependence of the alkylation rate on DNA and thiotepa concentrations shows that the alkylation reaction is biomolecular. The increase of sodium chloride concentration from 10(-3) to 10(-1) M is accompanied by a drastic decrease of the alkylation rate. Thiotepa binding results in destabilization of the DNA secondary structure and formation of cross-links. An increased amount of bounded thiotepa results in DNA denaturation; prolonged alkylation causes breaks in the sugar-phosphate backbone. The results of the work are discussed in connection with the literature data on DNA interaction with thiotepa in vivo.     

Potential antitumor mechanisms of phenothiazine drugs

In this study, three kinds of phenothiazine drugs were analyzed to explore their potential antitumor mechanisms. First, target proteins that could interact with chlorpromazine, fluphenazine and trifluoperazine were predicted. Then, the target proteins of the three drugs were intersected. Cell signaling pathway enrichment and related disease enrichment were conducted for the intersected proteins to extract the enrichment categories associated with tumors. By regulation network analysis of the protein interactions, the mechanisms of action of these target proteins in tumor tissue were clarified, thus confirming the potential antitumor mechanisms of the phenothiazine drugs. The final results of cell signaling pathway enrichment and related disease enrichment showed that the categories with the highest score were all found in tumors. Target proteins belonging to the tumor category included signaling pathway members such as Wnt, MAPK and retinoic acid receptor. Moreover, another target protein, MAPK8, could indirectly act on target proteins CDK2, IGF1R, GSK3B, RARA, FGFR2 and MAPK10, thereby affecting tumor cell division and proliferation. Therefore, phenothiazine drugs may have potential antitumor effects, and tumor-associated target proteins play important roles in the process of cell signaling transduction cascades.     

Telomerase inhibitors as novel antitumor drugs

Telomerase activity is detected in most types of human tumors, but it is almost undetectable in normal somatic cells; therefore, telomerase is a promising therapeutic target. The present review describes various approaches to telomerase inhibition, namely, antisense therapy, RNA interference, and the use of ribozymes and agents interacting with the telomeric G-quadruplex. The use of these compounds in clinical research is analyzed in the review.     

DNA interaction with antitumor polyamine analogues: a comparison with biogenic polyamines

Biogenic polyamines, putrescine, spermidine, and spermine, are ubiquitous cellular cations and exert multiple biological functions. Polyamine analogues mimic biogenic polyamines at macromolecular level but are unable to substitute for natural polyamines and maintain cell proliferation, indicating biomedical applications. The mechanistic differences in DNA binding mode between natural and synthetic polyamines have not been explored. The aim of this study was to examine the interaction of calf thymus DNA with three polyamine analogues, 1,11-diamino-4,8-diazaundecane (333), 3,7,11,15-tetrazaheptadecane x 4 HCl (BE-333), and 3,7,11,15,19-pentazahenicosane x 5 HCl (BE-3333), using FTIR, UV-visible, and CD spectroscopy. Polyamine analogues bind with guanine and backbone PO2 group as major targets in DNA, whereas biogenic polyamines bind to major and minor grooves as well as to phosphate groups. Weaker interaction with DNA was observed for analogues with respect to biogenic polyamines, with K(333) = 1.90 (+/-0.5) x 10(4) M(-1), K(BE-333) = 6.4 (+/-1.7) x 10(4) M(-1), K(BE-3333) = 4.7 (+/-1.4) x 10(4) M(-1) compared to K(Spm) = 2.3 (+/-1.1) x 10(5) M(-1), K(Spd) = 1.4 (+/-0.6) x 10(5) M(-1), and K(Put) = 1.02 (+/-0.5) x 10(5) M(-1). A partial B- to A-DNA transition was also provoked by analogues. These data suggest distinct differences in the binding of natural and synthetic polyamines with DNA.     

Natural products as leads to antitumor drugs

The discussion in this short review emphasizes that the main and future source of novel natural products as leads to antitumor agents is probably in the areas of biology that cannot be seen, i.e. the microbial world. The review discusses the role of microbes in the production of secondary metabolites that were initially thought to be from marine invertebrates and goes on to discuss the potential for a number of well-known anticancer agents isolated from plant sources to actually be the products of a microbe-plant interaction and finishes with a discussion of the potential of microbial “cryptic clusters” as sources of novel agents/leads to anti-tumor treatments.     

Polyacrylates of noble metals as potential antitumor drugs

The antitumor activity of polyacrylates of the noble metals containing argentum (argacryl), aurum (auracryl) and platinum (platacryl) has been studied using experimental murine solid tumor models (Lewis lung carcinoma and Acatol adenocarcinoma). It has been found that polyacrylates of the noble metals are capable of inhibiting tumor development by 50–90% compared to control. Auracryl that inhibits the growth of Lewis lung carcinoma and Acatol adenocarcinoma by 80 and 90%, respectively, compared to control is the most efficient among the tested compounds and can be recommended for the further profound preclinical studies.     

Differential scanning calorimetry of antitumor antibiotics-plasmid DNA interaction

Differential scanning calorimetry (DSC) can detect stepwise melting of plasmid DNA along the molecular chain with high resolution. This method was applied to study interaction of some antitumor antibiotics with the plasmid pJL3-TB5 DNA (5277 base-pairs in length). Analysis of DSC curves of the plasmid DNA in the presence of, for example, adriamycin, an antitumor antibiotics of anthracycline group, together with theoretical analysis of the DNA melting curves obtained by calculation from the entire base sequence, led to the conclusion that adriamycin bound preferentially to the four particular regions with high G + C content. The DSC method would thus be useful for the study of properties of drugs which bind to DNA.     

Intercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II

Many intercalative antitumor drugs have been shown to induce reversible protein-linked DNA breaks in cultured mammalian cells. Using purified mammalian DNA topoisomerase II, we have demonstrated that the antitumor drugs ellipticine and 2-methyl-9-hydroxyellipticine (2-Me-9-OH-E+) can produce reversible protein-linked DNA breaks in vitro. 2-Me-9-OH-E+ which is more cytotoxic toward L1210 cells and more active against experimental tumors than ellipticine is also more effective in stimulating DNA cleavage in vitro. Similar to the effect of 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) on topoisomerase II in vitro, the mechanism of DNA breakage induced by ellipticines is most likely due to the drug stabilization of a cleavable complex formed between topoisomerase II and DNA. Protein denaturant treatment of the cleavable complex results in DNA breakage and covalent linking of one topoisomerase II subunit to each 5'-end of the cleaved DNA. Cleavage sites on pBR322 DNA produced by ellipticine or 2-Me-9-OH-E+ treatment mapped at the same positions. However, many of these cleavage sites are distinctly different from those produced by the antitumor drug m-AMSA which also targets at topoisomerase II. Our results thus suggest that although mammalian DNA topoisomerase II may be a common target of these antitumor drugs, drug-DNA-topoisomerase interactions for different antitumor drugs may be different.     

Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II

Many intercalative antitumor drugs have been shown to cleave DNA indirectly through their specific effect on the stabilization of a cleavable complex formed between mammalian DNA topoisomerase II and DNA (Nelson, E.M., Tewey, K.M., and Liu, L.F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1361-1365). Antitumor epipodophyllotoxins (VP-16 and VM-26) which do not intercalate DNA can similarly induce protein-linked DNA breaks in cultured mammalian cells. In vitro studies using purified mammalian DNA topoisomerase II show that epipodophyllotoxins interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by stabilizing a cleavable complex. Treatment of this stabilized cleavable complex with protein denaturants results in DNA strand breaks and the covalent linking of a topoisomerase subunit to the 5'-end of the broken DNA. Furthermore, epipodophyllotoxins also inhibit the strand-passing activity of mammalian DNA topoisomerase II, presumably as a result of drug-enzyme interaction. The agreement between the in vivo and in vitro studies suggests that mammalian DNA topoisomerase II is a drug target in vivo. The similarity between the effect of epipodophyllotoxins on mammalian DNA topoisomerase II and the effect of nalidixic acid on Escherichia coli DNA gyrase suggests that the cytotoxic action of epipodophyllotoxins may be analogous to the bactericidal action of nalidixic acid.     

A protein interaction network associated with asthma

Identifying candidate genes related to complex diseases or traits and mapping their relationships require a system-level analysis at a cellular scale. The objective of the present study is to systematically analyze the complex effects of interrelated genes and provide a framework for revealing their relationships in association with a specific disease (asthma in this case). We observed that protein-protein interaction (PPI) networks associated with asthma have a power-law connectivity distribution as many other biological networks have. The hub nodes and skeleton substructure of the result network are consistent with the prior knowledge about asthma pathways, and also suggest unknown candidate target genes associated with asthma, including GNB2L1, BRCA1, CBL, and VAV1. In particular, GNB2L1 appears to play a very important role in the asthma network through frequent interactions with key proteins in cellular signaling. This network-based approach represents an alternative method for analyzing the complex effects of candidate genes associated with complex diseases and suggesting a list of gene drug targets. The full list of genes and the analysis details are available in the following online supplementary materials: http://biosoft.kaist.ac.kr:8080/resources/asthma_ppi.     

Selective pharmacological modulation of renal peripheral-type benzodiazepine binding by treatment with diuretic drugs

We have assessed the effects of in vivo administration of different classes of diuretic drugs on the expression of the peripheral-type benzodiazepine binding site (PBBS) in crude membranes derived from the cortex and outer medulla of rat kidney by saturation analysis with the PBBS-selective ligands [3H]RO5-4864 and [3H]PK 11195 in cortex and [3H]RO5-4864 in outer medulla. Administration for 14-15 days of furosemide, a drug that blocks NaCl-KCl coupled transport in the thick ascending limb of the loop of Henle, produced a significant doubling in the PBBS density (Bmax) in outer medulla, a region of the kidney rich in thick ascending limbs, and produced a lesser but significant increase in PBBS density in the cortex. Conversely, administration for 14-15 days of the carbonic anhydrase inhibitor acetazolamide, which acts predominantly in the proximal tubule, and hydrochlorothiazide, which acts predominantly in the early distal tubule, elicited statistically significant increases in PBBS density in renal cortex but not in renal outer medulla. Furthermore, all drug treatments were without effect on the equilibrium dissociation constants (Kds) of [3H]RO5-4864 and [3H]PK 11195 binding to cortical and outer medullary membrane preparations. These findings demonstrate that the PBBS can be selectively "up-regulated" in different regions of the kidney by diuretic drugs with different modes/sites of action.     

Microbial natural products: molecular blueprints for antitumor drugs

Microbes from two of the three domains of life, the Prokarya, and Eukarya, continue to serve as rich sources of structurally complex chemical scaffolds that have proven to be essential for the development of anticancer therapeutics. This review describes only a handful of exemplary natural products and their derivatives as well as those that have served as elegant blueprints for the development of novel synthetic structures that are either currently in use or in clinical or preclinical trials together with some of their earlier analogs in some cases whose failure to proceed aided in the derivation of later compounds. In every case, a microbe has been either identified as the producer of secondary metabolites or speculated to be involved in the production via symbiotic associations. Finally, rapidly evolving next-generation sequencing technologies have led to the increasing availability of microbial genomes. Relevant examples of genome mining and genetic manipulation are discussed, demonstrating that we have only barely scratched the surface with regards to harnessing the potential of microbes as sources of new pharmaceutical leads/agents or biological probes.     

Analytical methodologies for metallomics studies of antitumor Pt-containing drugs

Pt-containing drugs are nowadays essential components in cancer chemotherapy. However, drug resistance and side effects limit the efficiency of the treatments. In order to improve the response to Pt-based drugs, different administration strategies or new Pt-compounds have been developed with little success. The reason for this failure could be that the mechanism of action of these drugs is not completely understood. In this way, metallomics studies may contribute to clarify the interactions of Pt-containing drugs within the organism. This review is mainly focused on the role of Analytical Chemistry on the study of the interactions between Pt-based drugs and biomolecules. A summary of the analytical techniques and the most common sample treatment procedures currently used in metallomics studies of these drugs is presented. Both are of paramount importance to study these complex samples preserving the drug-biomolecule interaction. Separation and detection techniques must be carefully selected in order to achieve the intended goals. The use of multidimensional hyphenated techniques is usually necessary for a better understanding of the Pt-based drugs interactions in the organism. An overview of Pt-drugs biological interactions is presented, considering the different sample matrices and the drugs course through the organism. Samples analysed in the included studies are blood, urine, cell cytosol, DNA as well as the drugs themselves and their derivatives. However, most of these works are based on in vitro experiments or incubations of standards, leading in some cases to contradictory results depending on the experimental conditions used. Though in vivo experiments represent a great challenge due to the high complexity and the low concentrations of the Pt-adducts in real samples, these studies must be undertaken to get a deeper understanding of the real interactions concerning Pt-containing drugs.