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Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that removes various adducts from the 3' end of DNA. Such adducts are formed by enzymes that introduce single-strand breaks in DNA during catalysis (for example, topoisomerase 1) and a number of anticancer drugs with different mechanisms of action. Poly(ADP-ribose) polymerase 1 (PARP1) is an enzyme that catalyzes posttranslational modification (PARylation) of various targets and thus controls many cell processes, including DNA repair. Tdp1 is a PARP1 target, and its PARylation attracts Tdp1 to the site of DNA damage. Olaparib is a PARP1 inhibitor used in clinical practice to treat homologous recombination-deficient tumors. Olaparib inhibits PARylation and, therefore, DNA repair. The Tdp1 inhibitor OL7-43 was used in combination with olaparib to increase the antitumor effect of the latter. Olaparib cytotoxicity was found to increase in the presence of OL7-43 in vitro. OL7-43 did not exert a sensitizing effect, but showed its own antitumor and antimetastatic effects in Lewis and Krebs-2 carcinoma models.

To date, various strategies have been proposed to increase the efficiency of cancer therapy. It is known that the action of DNA repair system can determine the resistance of cancer cells to DNA-damaging chemotherapy and radiotherapy, and one of these ways to increase therapeutic efficiency is the search for inhibitors of enzymes of the DNA repair system. Inhibition of the DNA repair enzyme tyrosyl-DNA phosphodiesterase1 (Tdp1) leads to an increase in the effectiveness of the topoisomerase 1 (Top1) inhibitor, the anticancer drug topotecan. Covalent complexes Top1-DNA, which are normally short-lived and are not a threat to the cell, are stabilized under the influence of topotecan and lead to cell death. Tdp1 eliminates such stabilized complexes and thus weaken the effect of topotecan therapy. We have previously shown that the use of the usnic acid hydrazonothiazole derivative OL9-119 in combination with topotecan increased the antitumor and antimetastatic efficacy of the latter in a mouse model of Lewis lung carcinoma. In this work, it was shown that the combined use of topotecan and Tdp1 inhibitor, the hydrazonothiazole derivative of usnic acid OL9-119, leads to an increase in the DNA-damaging effect of topotecan which is used in the clinic for the treatment of cancer. The study of the proapoptotic effect of the compound OL9-119 showed that the compound itself does not induce apoptosis, but increases the proapoptotic effect of topotecan. The results of the study could be used to improve the effectiveness of anticancer therapy and/or to reduce the therapeutic dose of topotecan and, therefore, the severity of side effects., (© 2023. The Author(s).)

Boron neutron capture therapy is a unique form of adjuvant cancer therapy for various malignancies including malignant gliomas. The conjugation of boron compounds and human serum albumin (HSA)-a carrier protein with a long plasma half-life-is expected to extend systemic circulation of the boron compounds and increase their accumulation in human glioma cells. We report on the synthesis of fluorophore-labeled homocystamide conjugates of human serum albumin and their use in thiol-'click' chemistry to prepare novel multimodal boronated albumin-based theranostic agents, which could be accumulated in tumor cells. The novelty of this work involves the development of the synthesis methodology of albumin conjugates for the imaging-guided boron neutron capture therapy combination. Herein, we suggest using thenoyltrifluoroacetone as a part of an anticancer theranostic construct: approximately 5.4 molecules of thenoyltrifluoroacetone were bound to each albumin. Along with its beneficial properties as a chemotherapeutic agent, thenoyltrifluoroacetone is a promising magnetic resonance imaging agent. The conjugation of bimodal HSA with undecahydro- closo -dodecaborate only slightly reduced human glioma cell line viability in the absence of irradiation (~30 μM of boronated albumin) but allowed for neutron capture and decreased tumor cell survival under epithermal neutron flux. The simultaneous presence of undecahydro- closo -dodecaborate and labeled amino acid residues (fluorophore dye and fluorine atoms) in the obtained HSA conjugate makes it a promising candidate for the combination imaging-guided boron neutron capture therapy.

In the milk of healthy women, antibodies were found with different catalytic activities (abzymes), which are absent in the sera of other healthy people. Moreover, it was previously shown that DNase antibodies-abzymes of patients with autoimmune diseases are cytotoxic to cancer cells. In this work, it was first shown that IgG and secretory IgA (sIgA) do not possess embryotoxicity; they practically do not affect the development of fertilized eggs of sea urchins but demonstrate sperm toxicity. After addition to the eggs of sperm preincubated with IgG and sIgA, the number of unfertilized eggs was increased, in the case of sIgA 1.6-fold higher than that for IgG. The suppression of the growth of MCF-7 breast cancer cells by sIgA was 2.2 times more effective than with IgG antibodies. The relative enzymatic activity of milk sIgA was higher than IgG (-fold): 1.9 (DNase), 4.6 (amylase), 1.7 (peroxidase), 1.3 (protease), 3.7 [hydrolysis of poly(C)], 3.3 [hydrolysis of poly(U)], and 1.7 (oxidation of 3,3'-diaminobenzidine). One of the possible reasons for the observed difference between sIgA and IgG could be that all 6 catalytic activities of sIgA were, on average, 2.6 times higher than that for IgG. Correlation coefficients between all the relative 6 enzymatic activities of IgG and sIgA and their toxicity to sea urchin sperm and to cancer cells were calculated. Maximum correlation coefficients were observed for DNase (+0.71), protease (+0.64) activities for sIgA, as well as protease (+0.59) and RNase (+0.77) of IgG with their toxicity toward sperm. The correlation coefficients were also high between peroxidase activity (+0.85) of sIgA and poly(U) hydrolysis by IgG (+0.58) with their suppression of tumor cell growth. It has been suggested that the catalytic activities of abzymes may be important in the manifestation of their sperm toxicity and inhibition of cancer cell growth., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising target for anticancer therapy due to its ability to counter the effects topoisomerase 1 (Top1) poison, such as topotecan, thus, decreasing their efficacy. Compounds containing adamantane and monoterpenoid residues connected via 1,2,4-triazole or 1,3,4-thiadiazole linkers were synthesized and tested against Tdp1. All the derivatives exhibited inhibition at low micromolar or nanomolar concentrations with the most potent inhibitors having IC 50 values in the 0.35-0.57 µM range. The cytotoxicity was determined in the HeLa, HCT-116 and SW837 cancer cell lines; moderate CC 50 (µM) values were seen from the mid-teens to no effect at 100 µM. Furthermore, citral derivative 20c , α-pinene-derived compounds 20f , 20g and 25c, and the citronellic acid derivative 25b were found to have a sensitizing effect in conjunction with topotecan in the HeLa cervical cancer and colon adenocarcinoma HCT-116 cell lines. The ligands are predicted to bind in the catalytic pocket of Tdp1 and have favorable physicochemical properties for further development as a potential adjunct therapy with Top1 poisons.

BODIPY dyes are photostable neutral derivatives of 4,4-difluoro-4-bora-3a,4a-diaza- s -indacene. These are widely used as chemosensors, laser materials, and molecular probes. At the same time, BODIPY dyes have small or moderate Stokes shifts like most other fluorophores. Large Stokes shifts are preferred for fluorophores because of higher sensitivity of such probes and sensors. The new boron containing BODIPY dye was designed and synthesized. We succeeded to perform an annulation of pyrrole ring with coumarin heterocyclic system and achieved a remarkable difference in absorption and emission maximum of obtained fluorophore up to 100 nm. This BODIPY dye was equipped with linker arm and was functionalized with a maleimide residue specifically reactive towards thiol groups of proteins. BODIPY residue equipped with a suitable targeting protein core can be used as a suitable imaging probe and agent for Boron Neutron Capture Therapy (BNCT). As the most abundant protein with a variety of physiological functions, human serum albumin (HSA) has been used extensively for the delivery and improvement of therapeutic molecules. Thiolactone chemistry provides a powerful tool to prepare albumin-based multimodal constructions. The released sulfhydryl groups of the homocysteine functional handle in thiolactone modified HSA were labeled with BODIPY dye to prepare a labeled albumin-BODIPY dye conjugate confirmed by MALDI-TOF-MS, UV-vis, and fluorescent emission spectra. Cytotoxicity of the resulting conjugate was investigated. This study is the basis for a novel BODIPY dye-albumin theranostic for BNCT. The results provide further impetus to develop derivatives of HSA for delivery of boron to cancer cells.

Multifunctional gold nanoparticles (AuNPs) may serve as a scaffold to integrate diagnostic and therapeutic functions into one theranostic system, thereby simultaneously facilitating diagnosis and therapy and monitoring therapeutic responses. Herein, albumin-AuNP theranostic agents have been obtained by conjugation of an anticancer nucleotide trifluorothymidine (TFT) or a boron-neutron capture therapy drug undecahydro- closo -dodecaborate (B 12 H 12 ) to bimodal human serum albumin (HSA) followed by reacting of the albumin conjugates with AuNPs. In vitro studies have revealed a stronger cytotoxicity by the AuNPs decorated with the TFT-tagged bimodal HSA than by the boronated albumin conjugates. Despite long circulation time, lack of the significant accumulation in the tumor was observed for the AuNP theranostic conjugates. Our unique labelling strategy allows for monitoring of spatial distribution of the AuNPs theranostic in vivo in real time with high sensitivity, thus reducing the number of animals required for testing and optimizing new nanosystems as chemotherapeutic agents and boron-neutron capture therapy drug candidates.

Para-Bromoanilides of deoxycholic acid with various functional groups on the steroid scaffold were designed as promising tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibitors. Tdp1 is a DNA repair enzyme, involved in removing DNA damage caused by topoisomerase I poisons; an important class of anticancer drugs. Thus, reducing the activity of Tdp1 can increase the efficacy of anticancer drugs in current use. Inhibitory activity in the low micromolar and submicromolar concentrations was observed with 3,12-dimethoxy para-bromoanilide 17 being the most active with an IC 50 value of 0.27 μM. The activity of N-methyl para-bromoanilides was 3-4.8 times lower than of the corresponding para-bromoanilides. Increased potency of the ligands was seen with higher molecular weight and log P values. The ligands were evaluated for their cytotoxic potential in a panel of tumor cell lines; all were nontoxic to the A549 pulmonary adenocarcinoma cell line. However, derivatives containing a hydroxyl group at the 12th position were more toxic than their 12-hydroxyl group counterparts (acetoxy-, oxo- and methoxy- group) against HCT-116 human colon and HepG2 hepatocellular carcinomas. In addition, an N-methyl substitution led to an increase in toxicity for the HCT-116 and HepG2 cell lines. The excellent activity as well as low cytotoxicity, derivative 17 can be considered as a lead compound for further development., (Copyright © 2020. Published by Elsevier Inc.)

Two novel structural types of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors with hexahydroisobenzofuran 11 and 3-oxabicyclo [3.3.1]nonane 12 scaffolds were discovered. These monoterpene-derived compounds were synthesized through preliminary isomerization of (+)-3-carene to (+)-2-carene followed by reaction with heteroaromatic aldehydes. All the compounds inhibit the TDP1 enzyme at micro- and submicromolar levels, with the most potent compound having an IC 50 value of 0.65 μM. TDP1 is an important DNA repair enzyme and a promising target for the development of new chemosensitizing agents. A panel of isogenic clones of the HEK293FT cell line knockout for the TDP1 gene was created using the CRISPR-Cas9 system. Cytotoxic effects of topotecan (Tpc) and non-cytotoxic compounds of the new structures were investigated separately and jointly in the TDP1 gene knockout cells. For two TDP1 inhibitors, 11h and 12k , a synergistic effect was observed with Tpc in the HEK293FT cells but was not found in TDP1 -/- cells. Thus, it is likely that the synergistic effect is caused by inhibition of TDP1. Synergy was also found for 11h in other cancer cell lines. Thus, sensitizing cancer cells using a non-cytotoxic drug can enhance the efficacy of currently used pharmaceuticals and, concomitantly, reduce toxic side effects.

As the most abundant protein with a variety of physiological functions, albumin has been used extensively for the delivery of therapeutic molecules. Thiolactone chemistry provides a powerful tool to prepare spin-labeled albumin-based multimodal imaging probes and therapeutic agents. We report the synthesis of a tamoxifen homocysteine thiolactone derivative and its use in thiol-'click' chemistry to prepare multi-functionalized serum albumin. The released sulfhydryl group of the homocysteine functional handle was labeled with a nitroxide reagent to prepare a spin-labeled albumin-tamoxifen conjugate confirmed by MALDI-TOF-MS, EPR spectroscopy, UV-vis and fluorescent emission spectra. This is the basis for a novel multimodal tamoxifen-albumin theranostic with a significant (dose-dependent) inhibitory effect on the proliferation of malignant cells. The response of human glioblastoma multiforme T98G cells and breast cancer MCF-7 cells to tamoxifen and its albumin conjugates was different in tumor cells with different expression level of ERα in our experiments. These results provide further impetus to develop a serum protein for delivery of tamoxifen to cancer cells., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Spinocerebellar ataxia syndrome with axonal neuropathy (SCAN1) is a debilitating neurological disease that is caused by the mutation the Tyrosyl-DNA phosphodiesterase 1 (TDP1) DNA repair enzyme. The crucial His493 in TDP1's binding site is replaced with an arginine amino acid residue rendering the enzyme dysfunctional. A virtual screen was performed against the homology model of SCAN1 and seventeen compounds were identified and tested in a novel SCAN1 specific biochemical assay. Six compounds showed activity with IC 50 values between 3.5 and 25.1 µM. The most active ligand 5 (3.5 µM) is a dicoumarin followed by a close structural analogue 6 at 6.0 µM. A less potent series of β-carbolines (14 and 15) was found with potency in the mid-teens. According to molecular modelling an excellent fit for the active ligands into the binding pocket is predicted. To the best of our knowledge, data on inhibitors of the mutant form of TDP1 has not been reported previously. The virtual hits were also tested for wild type TDP1 activity and all six SCAN1 inhibitors are potent for the former, e.g., ligand 5 has a measured IC 50 at 99 nM. In the last decade, TDP1 is considered as a promising target for adjuvant therapy against cancer in combination with Topoisomerase 1 poisons. The active ligands are mostly non-toxic to cancer cell lines A-549, T98G and MCF-7 as well as the immortalized WI-38 human fetal lung cells. Furthermore, ligands 5 and 7, show promising synergy in conjunction with topotecan, a clinically used topoisomerase 1 anticancer drug. The active ligands 5, 7, 14 and 15 have a good balance of the physicochemical properties required for oral bioavailability making the excellent candidates for further development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme in humans, and a current and promising inhibition target for the development of new chemosensitizing agents due to its ability to remove DNA damage caused by topoisomerase 1 (Top1) poisons such as topotecan and irinotecan. Herein, we report our work on the synthesis and characterization of new Tdp1 inhibitors that combine the arylcoumarin (neoflavonoid) and monoterpenoid moieties. Our results showed that they are potent Tdp1 inhibitors with IC 50 values in the submicromolar range. In vivo experiments with mice revealed that compound 3ba (IC 50 0.62 µM) induced a significant increase in the antitumor effect of topotecan on the Krebs-2 ascites tumor model. Our results further strengthen the argument that Tdp1 is a druggable target with the potential to be developed into a clinically-potent adjunct therapy in conjunction with Top1 poisons., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising therapeutic target in cancer therapy. Combination chemotherapy using Tdp1 inhibitors as a component can potentially improve therapeutic response to many chemotherapeutic regimes. A new set of usnic acid derivatives with hydrazonothiazole pharmacophore moieties were synthesized and evaluated as Tdp1 inhibitors. Most of these compounds were found to be potent inhibitors with IC 50 values in the low nanomolar range. The activity of the compounds was verified by binding experiments and supported by molecular modeling. The ability of the most effective inhibitors, used at non-toxic concentrations, to sensitize tumors to the anticancer drug topotecan was also demonstrated. The order of administration of the inhibitor and topotecan on their synergistic effect was studied, suggesting that prior or simultaneous introduction of the inhibitor with topotecan is the most effective.

Lactoferrin (LF) is an Fe 3+ -binding glycoprotein first recognized in milk and then in other epithelial secretions and barrier body fluids to which many different functions have been attributed to LF, including protection from iron-induced lipid peroxidation, immunomodulation, cell growth regulation, DNA and RNA binding, as well as transcriptional activation, еtс. The polyfunctional physiological role of LF is still unclear, but it has been suggested to be responsible for primary defense against microbial and viral infections. Here, we present the first evidence that LF preparations isolated from milk of 18 cows of different breeds possess various levels of metal-dependent DNase and metal-independent RNase activities. For univocal assignment of DNase and RNase activities to cow LF, it was subjected to SDS-PAGE using gels with copolymerized calf thymus DNA or polymeric yeast RNA. In situ analysis was revealed DNase and RNase activities only in the gel zones corresponding to homogeneous LF. In contrast to human LF, cow LF possesses a relatively low cytotoxicity towards human tumor cells. The discovery that cow LF has these activities may contribute to understanding the multiple physiological functions of this extremely polyfunctional protein, including its protective role against microbial and viral infections. The computational spatial model of cow LF complex with DNA was obtained: according to the model positively charged residues of LF contact with DNA., (© 2019 John Wiley & Sons, Ltd.)

The druggability of the tyrosyl-DNA phosphodiesterase 1 (Tdp1) enzyme was investigated in conjunction with topoisomerase 1 inhibition. A novel class of thiazole, aminothiazole and hydrazonothiazole usnic acid derivatives was synthesized and evaluated as Tdp1 inhibitors and their ability to sensitize tumors to topotecan, a topoisomerase inhibitor in clinical use. Of all the compounds tested, four hydrazinothiazole derivatives, 20c, 20d, 20h and 20i, inhibited the enzyme in the nanomolar range. The activity of the compounds was verified by affinity experiments as well as supported by molecular modelling. The most effective Tdp1 inhibitor, 20d, was ton-toxic and increased the effect of topotecan both in vitro and in vivo in the Lewis lung carcinoma model. Furthermore, 20d showed significant increase in the antitumor and antimetastatic effect of topotecan in mice. The results presented here justify compound 20d to be considered as a drug lead for antitumor therapy., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background and Objective: The DNA repair enzyme tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a current inhibition target to improve the efficacy of cancer chemotherapy. Previous studies showed that compounds combining adamantane and monoterpenoid fragments are active against TDP1 enzyme. This investigation is focused on the synthesis of monoterpenoid derived esters of 1-adamantane carboxylic acid as TDP1 inhibitors., Methods: New esters were synthesized by the interaction between 1-adamantane carboxylic acid chloride and monoterpenoid alcohols. The esters were tested against TDP1 and its binding to the enzyme was modeling., Results: 13 Novel ester-based TDP1 inhibitors were synthesized with yields of 21-94%; of these, nine esters had not been previously described. A number of the esters were found to inhibit TDP1, with IC50 values ranging from 0.86-4.08 µM. Molecular modelling against the TDP1 crystal structure showed a good fit of the active esters in the catalytic pocket, explaining their potency. A non-toxic dose of ester, containing a 3,7- dimethyloctanol fragment, was found to enhance the cytotoxic effect of topotecan, a clinically used anti-cancer drug, against the human lung adenocarcinoma cell line A549., Conclusion: The esters synthesized were found to be active against TDP1 in the lower micromolar concentration range, with these findings being corroborated by molecular modeling. Simultaneous action of the ester synthesized from 3,7-dimethyloctanol-1 and topotecan revealed a synergistic effect., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

A new class of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors based on disaccharide nucleosides was identified. TDP1 plays an essential role in the resistance of cancer cells to currently used antitumour drugs based on Top1 inhibitors such as topotecan and irinotecan. The most effective inhibitors investigated in this study have IC 50 values (half-maximal inhibitory concentration) in 0.4-18.5 µM range and demonstrate relatively low own cytotoxicity along with significant synergistic effect in combination with anti-cancer drug topotecan. Moreover, kinetic parameters of the enzymatic reaction and fluorescence anisotropy were measured using different types of DNA-biosensors to give a sufficient insight into the mechanism of inhibitor's action.

Objective: Since during pregnancy and lactation women tend to develop autoimmune processes, characterization of placenta immunoglobulins and their possible catalytic functions is a very important step towards understanding the function of placenta., Methods: ELISA, affinity chromatography, SDS-PAGE and MALDI mass spectrometry were used., Results: It was shown, that ten placentas contain in average 1.1 ± 0.33 mg IgGs/g of placenta and 0.11 ± 0.1 mЕ anti-DNA antibodies/g of placenta. All ten individual IgG preparations were characterized by a specific ratio and various combinations of heavy (47.5-51.7 kDa) and light (22.2-33.3 kDa) chains with different molecular masses. All intact IgGs demonstrate DNase but have not amylase activity. Several strict criteria have been used to show that the DNase activity is an intrinsic property of placenta IgGs. The heavy chains of antibodies were catalytically inactive. The light chains of 22-24 kDa of all IgGs demonstrated maximal DNase activity, while the chains of approximately 25-33.2 kDa were significantly less active. IgGs were completely inactive after dialysis against EDTA. The optimal external cofactors of placenta DNase abzymes are Mg 2+ and Cu 2+ ions, while, activity in the presence of Mn 2+ and Ca 2+ is approximately 3-4-fold lower. Activation of DNase abzymes by Cu 2+ ions was revealed for the first time. Placental antibodies possess selective cytotoxicity against cancer cells., Conclusion: It was shown previously, that appearance of serum DNase abzymes is the good indicator of the beginning and obvious spontaneous or induced autoimmune diseases. The detection of placenta DNase antibodies indicates that during pregnancy, autoimmune processes can affect this organ., (Copyright © 2018. Published by Elsevier Ltd.)

An Important task in the treatment of oncological and neurodegenerative diseases is the search for new inhibitors of DNA repair system enzymes. Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is one of the DNA repair system enzymes involved in the removal of DNA damages caused by topoisomerase I inhibitors. Thus, reducing the activity of Tdp1 can increase the effectiveness of currently used anticancer drugs. We describe here a new class of semisynthetic small molecule Tdp1 inhibitors based on the bile acid scaffold that were originally identified by virtual screening. The influence of functional groups of bile acids (hydroxy and acetoxy groups in the steroid framework and amide fragment in the side chain) on inhibitory activity was investigated. In vitro studies demonstrate the ability of the semisynthetic derivatives to effectively inhibit Tdp1 with IC 50 up to 0.29 µM. Furthermore, an excellent fit is realized for the ligands when docked into the active site of the Tdp1 enzyme., Competing Interests: The authors declare no conflict of interest.

The ability of a number of nitrogen-containing compounds that simultaneously carry the adamantane and monoterpene moieties to inhibit Tdp1, an important enzyme of the DNA repair system, is studied. Inhibition of this enzyme has the potential to overcome chemotherapeutic resistance of some tumor types. Compound (+)-3c synthesized from 1-aminoadamantane and (+)-myrtenal, and compound 4a produced from 2-aminoadamantane and citronellal were found to be most potent as they inhibited Tdp1 with IC 50 values of 6 and 3.5 µM, respectively. These compounds proved to have low cytotoxicity in colon HCT-116 and lung A-549 human tumor cell lines (CC 50  > 50 µM). It was demonstrated that compound 4a at 10 µM enhanced cytotoxicity of topotecan, a topoisomerase 1 poison in clinical use, against HCT-116 more than fivefold and to a lesser extent of 1.5 increase in potency for A-549., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anticancer fluorinated nucleotide conjugated with a dual-labeled albumin. A fluorine-labeled homocysteine thiolactone has been used as functional handle to synthesize the fluorinated albumin and couple it with a chemotherapeutic agent 5-trifluoromethyl-2'-deoxyuridine 5'-monophosphate (pTFT). The conjugate allows for direct optical and 19 F magnetic resonance cancer imaging and release of the drug upon addition of glutathione. Interestingly, the pTFT release from albumin conjugate could only be promoted by the increased acidity (pH 5.4). The in vitro study and primary in vivo investigations showed stronger antitumor activity than free pTFT., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A possible ability of twelve new derivatives of known antioxidants trolox (TroH), trolox succinate (TroS), α-tocopheryl succinate (α-TOS) containing nitroxyl radicals (1-12) to protect bacterial cells from spontaneous and peroxide-induced mutagenesis and their cytotoxicity against six different tumor cells as well as two normal cells were investigated and compared with that for TroH, TroS, α-TOH, and α-TOS for the first time. In contrast to TroH and TroS, all nitroxide derivatives 1-12 demonstrated not only antioxidant properties, but also suppress the growth of human tumor cells: myeloma, mammary adenocarcinoma, hepatocarcinoma, T cells leukemia, histiocytic lymphoma, and T-cellular leucosis. The IC50 values (24 - ≥ 300 μM) depend significantly on the compounds and type of tumor cells. Some compounds were capable to inhibit the growth of normal mouse (LMTK) and hamster (AG17) fibroblast cells and demonstrate very different ratios in inhibition of various tumor and normal cell lines. Some nitroxide conjugates showed pronounced selectivity in suppressing the growth of several cancer cells. Overall, several compounds may be promising in parallel as antioxidants and as specific inhibitors of some tumor cells growth. Among considered spin labeled conjugates the most perspective derivatives as antioxidants and as antitumor agents are the compounds containing pyrrolidine nitroxides. In contrast to spin labeled TroH, TroS and α-TOS conjugates 1-12 succinyl derivatives 13-15 were inactive in inhibiting the growth of any tumor cells. It means that for suppressing the cancer cells the compounds should contain in their structures fragments of TroH, TroS or α-TOS., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Straightforward and reliable tools for in vivo imaging of tumors can benefit the studies of cancer development, as well as contribute to successful diagnosis and treatment of cancer. (19)F NMR offers an exceptional quantitative way of in vivo imaging of the infused agents because of the lack of (19)F signals from the endogenous molecules in the body. The purpose of this study is to develop molecular probes with appropriate NMR characteristics and the biocompatibility for in vivo applications using (19)F MRI. We have studied the reaction between perfluorotoluene and homocysteine thiolactone resulting in the formation of N-substituted homocysteine thiolactone derivative. It has been shown that the reaction occurs selectively at the para position. This fluorine-labeled homocysteine thiolactone has been employed for the introduction of a perfluorotoluene group as a (19)F-containing tag into human serum albumin. The modified protein has been studied in terms of its ability to aggregate and promote the formation of free radicals. By comparing the properties of N-perfluorotoluene-homocystamide of albumin with N-homocysteinylated albumin, it has been revealed that blocking of the alpha-amino group of the homocysteine residue in the fluorinated albumin conjugate inhibits the dangerous aggregation process, as well as free radical formation. A dual-labeled albumin-based molecular probe for (19)F MRI and fluorescence microscopy has been obtained by functionalizing the protein with both maleimide of a fluorescent dye and a fluorinated thiolactone derivative. The incubation of cells with this conjugate did not reveal any significant reduction in cell viability with respect to the parent albumin. The perfluorotoluene-labeled albumin has been demonstrated to act as a promising agent for in vivo (19)F MRI., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Herein, we report a novel strategy to engineer an acid-sensitive anticancer theranostic agent using a vector-drug ensemble. The ensemble was synthesized by directly conjugating the linoleic acid (LA)-modified branched polyethyleneimine with a chemotherapeutic drug trifluorothymidine. Linoleic acid residues were grafted onto 25 kDa polyethyleneimine (PEI) by treating PEI with linoleic acid chloroanhydride. 5-Trifluoromethyl-2'-deoxyuridine (trifluorothymidine, TFT) was introduced into LA-PEI conjugate by phosphorylating the conjugate with amidophosphate of trifluorothymidine 5'-monophosphate (pTFT), which had been activated by its conversion into the N,N-dimethylaminopyridine derivative. The extent of mononucleotide analog incorporation in the polymer was regulated by the ratio of pTFT to the polymer during the synthesis. Samples containing 20-70 TFT residues per PEI molecule were obtained. The cytotoxicity of PEI-LA-pTFT conjugates decreased with increasing nucleotide content, as examined using the MTT method. Due to the presence of fluorine atoms, TFT-based conjugates could be detected directly in the animals by (19)F magnetic resonance imaging. In addition, the presence of the amidophosphate group in PEI-LA-pTFT conjugates allowed their detection by in vivo(31)P NMR spectroscopy. Indeed, the (31)P NMR signal of a phosphoramide (δ ~ 12 ppm) was observed in the mouse muscle tissue treated with PEI-LA-pTFT conjugate along with the signals from endogenous phosphorus-containing compounds. At the same time, the use of PEI-LA-pTFT conjugate for chemotherapeutic drug delivery is limited due to the low release of pTFT from the carrier. To enhance the release of the drug from the conjugate in the endosomes, PEI-LA polymer was coupled with urocanic acid (UA), which bears imidazole ring and thus can form an acid-labile P-N bond with pTFT. The PEI-LA-UA-pTFT conjugate containing 30 residues of UA and 40 residues of pTFT was tested against the murine Krebs-II ascites carcinoma, grown as an ascetic tumor. The intraperitoneal injection of the conjugates resulted in prolongation of the animals' life and to the complete disappearance of the tumor after three injections.

It was previously shown that small fractions of IgGs and IgMs from the sera of AIDS patients specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Here we present evidence showing that these IgGs and IgMs are extreme catalytically heterogeneous. Affinity chromatography on IN-Sepharose using elution of IgGs (or IgMs) with different concentration of NaCl and acidic buffer separated catalytic antibodies (ABs) into many AB subfractions demonstrating different values of K(m) for IN and k(cat). Nonfractionated IgGs and IgMs possess serine-, thiol-, acidic-like, and metal-dependent proteolytic activity. Metal-dependent activity of abzymes increases in the presence of ions of different metals. In contrast to canonical proteases having one pH optimum, initial nonfractionated IgGs and IgMs demonstrate several optima at pH from 3 to 10. The data obtained show that IN-hydrolyzing polyclonal IgG and IgM of HIV-infected patients are cocktails of anti-IN ABs with different structure of the active centers possessing various affinity to IN, pH optima, and relative rates of the specific substrate hydrolysis., (© Pleiades Publishing, Ltd., 2011.)

Human immunodeficiency virus type 1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. It was shown previously that IN preincubation with various oligodeoxynucleotides (ODNs) induces formation of dimers and oligomers of different gyration radii; only specific ODNs stimulate the formation of catalytically active dimers. Here we have shown that preincubation of IN with specific and nonspecific ODNs leads to a significant and comparable decrease in its hydrolysis by chymotrypsin, while nonspecific ODNs protect the enzyme from the hydrolysis by trypsin worse than specific ODNs; all ODNs had little effect on the IN hydrolysis by proteinase K. In contrast to canonical proteweases, IgGs from HIV-infected patients specifically hydrolyze only IN. While d(pT)(n) markedly decreased the IgG-dependent hydrolysis of IN, d(pA)(n) and d(pA)(n) •d(pT)(n) demonstrated no detectable protective effect. The best protection from the hydrolysis by IgGs was observed for specific single- and especially double-stranded ODNs. Although IN was considerably protected by specific ODNs, proteolytic IgGs and IgMs significantly suppressed both 3'-processing and integration reaction catalyzed by IN. Since anti-IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded., (Copyright © 2011 John Wiley & Sons, Ltd.)

HIV-1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. In contrast to canonical proteases (trypsin, chymotrypsin and proteinase K), IgGs and IgMs isolated from HIV-infected patients by affinity chromatography on immobilized IN specifically hydrolyzed only IN but not many other tested intact globular proteins. The sites of IN cleavage determined by MALDI mass spectrometry were localized mainly within seven known immunodominant regions of IN. Thin layer chromatography analysis has shown that the abzymes (Abzs) could also cleave 17 to 22-mer oligopeptides (OPs) corresponding to the immunodominant regions of IN sequence with a much higher rate than non-specific long peptides or three- and tetrapeptides of various sequence. Therefore, a prolonged incubation of IN with AIDS IgGs and IgMs having high catalytic activity usually produces many OPs of different length. Since anti-IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of the Abzs in counteracting the infection is possible.

Fluorinated derivatives of 1,4-naphthoquinones are highly potent inhibitors of Cdc25A and Cdc25B phosphatases and growth of tumor cells. Eight new derivatives of polyfluoro-1,4-naphthoquinone were synthesized and their cytotoxicity in human myeloma, human mammary adenocarcinoma, mouse fibroblasts and primary mouse fibroblast cells as well as their mutagenic and antioxidant properties in a Salmonella tester strain were studied. The efficiency of suppressing the growth of two lines of tumor cells decreased in the order: 2-(2-hydroxy-ethylamino)-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (1), 2,3-dimethoxy-5,6,7,8-tetrafluoro-1,4-naphthoquinone (2), 2-[2-hydroxyethyl(methyl)amino]-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (3), 2-morpholino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (4), 2-[bis-(2-hydroxyethyl)amino]-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (5), 2-[(2-hydroxy)ethylsulfanyl)]-5,6,7,8-tetrafluoro-1,4-naphthoquinone (6), 2-methoxy-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (7), and 1,4-dioxo-3-(1-pyridinio)-1,4-dihydro-5,6,7,8-tetrafluoronaphthalene-2-olate (8). Taking into account these data together with the better cytotoxic effect against cancer cells as compared with normal mammalian cells, protecting of bacterial cells from spontaneous and H(2)O(2)-dependent mutagenesis, and lower general toxicity of the compounds towards different cells, one can propose that compounds 3-5 may be considered as useful potential inhibitors of growth of tumor cells., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

IgG abzymes (Abzs) with different catalytic activities are a distinctive feature of various autoimmune (AI) diseases. At the same time, data concerning IgMs with catalytic activities are very limited. Electrophoretically and immunologically homogeneous IgMs were isolated from the sera of acquired immunodeficiency syndrome (AIDS) patients by chromatography on several affinity sorbents. Several rigid criteria have been applied to show that the integrase (IN)-hydrolyzing activity is an intrinsic property of IgMs from HIV-infected patients but not from healthy donors. We present evidence showing that 22 of 24 (91.7%) IgMs purified from the sera of HIV-infected patients specifically hydrolyze only HIV IN but not many other tested proteins. Usually, proteolytic antibodies of AI patients are serine protease-like or metal dependent. Only 30% of IN-hydrolyzing IgMs were inhibited by specific inhibitors of serine proteases and 60% by inhibitors of metal-dependent proteases. Unusually, a significant reduction of the activity by specific inhibitors of acidic (in 20% of IgM preparations) and thiol proteases (in 100% of IgM preparations) was observed. Although HIV infection leads to formation of antibodies to many viral and human antigens, possible biological roles for most of them are unknown. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of Abzs in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for assessment of the immune status in AIDS patients.

Fluorinated derivatives of 1,4-naphthoquinone are highly potent inhibitors of Cdc25A and Cdc25 phosphatases and growth of tumor cells. Five new N-substituted polyfluorinated derivatives of 2-amino-1,4-naphthoquinone were synthesized and their mutagenic and antioxidant properties in Salmonella cells, as well as cytotoxicity in human myeloma (RPMI 8226), human mammary adenocarcinoma (MCF-7), mouse fibroblasts (LMTK) and primary mouse fibroblast cells (PMF) were studied. 2-tert-Butylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (1) inhibited the growth of normal control and tumor cells at the same concentration. Three compounds: 2-diethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (2), 2-ethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (3), 2-phenylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (4) exhibited a 50% decrease in the growth of cancer cells at low and comparable concentrations (2.4-8.6 microM) while being remarkably less cytotoxic toward normal LMTK and PMF cells. Quinones (1)-(4), but not 2-phenylamino-3-methyl-5,6,7,8-tetrafluoro-1,4-naphthoquinone (5), efficiently suppressed spontaneous mutagenesis in Salmonella cells, while all compounds 1-5 decreased the mutagenic effect of H2O2 on bacterial cells. Their possible perspectives as anticancer drugs are shortly discussed., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

HIV-1 integrase (IN) catalyses integration of a DNA copy of the viral genome into the host genome. Specific interactions between retroviral IN and long terminal repeats (LTR) are required for this insertion. To characterize quantitatively the influence of the determinants of DNA substrate specificity on the oligomerization status of IN, we used the small-angle X-ray scattering (SAXS) technique. Under certain conditions in the absence of ODNs IN existed only as monomers. IN preincubation with specific ODNs led mainly to formation of dimers, the relative amount of which correlated well with the increase in the enzyme activity in the 3'-processing reaction. Under these conditions, tetramers were scarce. Non-specific ODNs stimulated formation of catalytically inactive dimers and tetramers. Complexes of monomeric, dimeric and tetrameric forms of IN with specific and non-specific ODNs had varying radii of gyration (R(g)), suggesting that the specific sequence-dependent formation of IN tetramers can probably occur by dimerization of two dimers of different structure. From our data we can conclude that the DNA-induced oligomerization of HIV-1 IN is probably of importance to provide substrate specificity and to increase the enzyme activity.

The influence of new non-natural regular minor groove binders (MGB), containing 2-4 imidazole, pyrrole or thiazole residues, and their conjugates with oligonucleotides, on the polymerization reaction catalyzed by HIV-1 reverse transcriptase was analyzed. Various model template-primer complexes: poly(A)-oligo(U), poly(A)-oligo(dT), poly(dA)-oligo(U), poly(dA)-oligo(dT) and activated DNA were used. The concentration of oligopeptides, giving 50% inhibition (I50) of the RT-dependent polymerization reaction, was shown to depend strongly on the structure of template-primer complexes, number and type of the heterocycle rings in the MGBs analyzed. The range of I50 for the most of the compounds studied is 7.7 x 10(-3)-1.0 x 10(-5) M. The affinity of MGB is minimal for poly(A)-oligo(U). However, some of imidazole and pyrrole-containing MGBs demonstrated unusually high affinity (I50 = 3 x 10(-9)-4 x 10(-8) M) to the above template-primer in complex with RT. The affinity of conjugates of thiazolecarboxamides with oligonucleotides complementary or partially complementary to the template, is 1-4 orders higher compared to free thiazolecarboxamides. The possible reasons of the dependence of I50 values upon the structure of the template-primer complexes, the structure of MGB, and their conjugates with oligonucleotides are discussed.

New compounds having tri- or pentamethylenamine linker functions were synthesized. These derivatives were covalently attached through the 5'-phosphoramide linkage to heptanucleotide pd(CCAAACA). Complementary complexes of the octanucleotide pd(TGTTTGGC) and above oligonucleotide conjugates were tested for their thermodynamic response. The T(m) data and thermodynamic parameters for complex formation confirmed the ability of chromone (gamma-pyrone) derivatives to stabilize strongly the 7-mer/8-mer complementary complex. Moreover, benzochromone (naphthopyrane) and, surprisingly, tetrahydropyrimidinethanone derivatives showed the capacity of stabilizing this 7-mer/8-mer complementary complex. The effect of all these compounds on the stability of the oligonucleotide complexes (DeltaDeltaG at 37 degrees C ranged from -1.2 to -2.0 kcal/mol) was shown to be comparable to the effect of one nucleotide base pair and similar to the effect (DeltaDeltaG at 37 degrees C ranged from -1.5 to -2.0 kcal/mol) found for acridine-oligonucleotide conjugates, which served as a reference in this study.

Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3'-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn(2+), the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide approximately 8 orders of magnitude in the affinity (Delta G degrees approximately equal to -10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to approximately 1.5 orders of magnitude (Delta G degrees approximately equal to - 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the k(cat) term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.

N9-Polymethylene derivatives of adenine and hypoxanthine with various functional groups in the omega-position of the alkyl substituent were synthesized. Their physico-chemical properties and effect on the HIV reverse transcriptase and DNA topoisomerase I were studied.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the retrovirus, responsible for catalysing the insertion of the viral genome into the host cell chromosome. For this reason it provides an attractive target for antiviral drug design. We synthesized a series of novel thiazole (Tz)-containing oligopeptides (TCOs; oligo-1,3-thiazolecarboxamides), specifically interacting within the minor groove of DNA. The oligocarboxamide derivatives contained 1-4 Tz rings and different N- and C-terminal groups. The effect of these oligocarboxamides on the HIV-1 IN-catalysed reaction was investigated. Some of the compounds were able to inhibit the reaction. The inhibitory effect of the TCOs increased with the number of Tz units. The structure of various additional positively and/or negatively charged groups attached to the N- and C-termini of TCOs had a pronounced effect on their interaction with the DNA substrate complexed to IN. Modified TCOs having a better affinity for this complex should provide a rationale for the design of drugs targeting the integration step.

N1-Acyclic derivatives of pyrimidine bases (uracil, thymine, and cytosine) with hydrophobic polymethylene chains containing various functional groups in an omega-position of the alkyl substituent were synthesized. Their physicochemical properties and inhibitory effect on the HIV reverse transcriptase and human DNA topoisomerase I were studied.

A set of oligo-1,3-thiazolecarboxamide derivatives able to interact with the minor groove of nucleic acids was synthesized. These oligopeptides contained different numbers of thiazole units presenting dimethylaminopropyl or EDTA moieties on the C-terminus, and aminohexanoyl or EDTA moieties on the N-terminus. The inhibition of such compounds on HIV-1 reverse transcriptase activity was evaluated using different model template primer duplexes: DNA x DNA, RNA x DNA, DNA x RNA and RNA x RNA. The biological properties of the thiazolecarboxamide derivatives were compared to those of distamycin, another minor groove binder which contains three pyrrole rings. Similar to distamycin, the thiazole containing oligopeptides were good inhibitors of the reverse transcription reaction in the presence of DNA x DNA. But in contrast to distamycin, the oligothiazolide derivatives were able to inhibit reverse transcription in the presence of RNA x DNA or DNA x RNA template primers. Both distamycin and oligothiazolecarboxamides had low affinity for RNA x RNA duplexes. The inhibition obtained with the newly synthesized thiazolecarboxamides showed that these compounds were more powerful and versatile inhibitors of the RT-dependent polymerization than the natural minor groove binder distamycin.

Ten different pyranone-related substituents (chromones or coumarins) were covalently linked to the 5' end of various oligonucleotides (ODN). The interaction of these compounds with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was analyzed. A different behavior was found to depend on the structure of the oligonucleotide derivatives. Some compounds activated the enzyme at relatively low concentrations (0.1-0.5 microM), followed by an inhibition of the activity at higher concentrations (5-20 microM), whereas others behave just as inhibitors. Because the presence of some coumarin or chromone derivatives conjugated to ODNs enhanced the interaction with the reverse transcriptase, we analyzed the capacity of such ODN derivatives to be used as primers. The introduction of substituent I, a chromone derivative, the 2-[(3-(aminopropyl)amino]-8-isopropyl-5-methyl-4-oxo-4H-1-benzopyran-3-c arbaldehyde], and II, a coumarin derivative, the 1-(3-aminopropoxy)-2-ethyl-3H-naphto[2,1-b]pyran-3-one, into the 5' end of a noncomplementary ODN allowed these compounds to be used as primers. In the case of complementary primers, the presence of conjugated derivatives enhanced the affinity with Km values that were two to three orders of magnitude lower than that of a complementary primer of the same length. After addition of a ddT-unit to the 3'-terminal end of the ODN, some of these primers became very effective inhibitors of RT with Ki values in the nanomolar range.

Retroviral integrase (IN) catalyzes the integration of double-stranded viral DNA into the host cell genome. The reaction can be divided in two steps: 3'-end processing and DNA strand transfer. Here we studied the effect of short oligonucleotides (ODNs) on human immunodeficiency virus type 1 (HIV-1) IN. ODNs were either specific, with sequences representing the extreme termini of the viral long terminal repeats, or nonspecific. All ODNs were found to competitively inhibit the processing reaction with Ki values in the nM range for the best inhibitors. Our studies on the interaction of IN with ODNs also showed that: (i) besides the 3'-terminal GT, the interaction of IN with the remaining nucleotides of the 21-mer specific sequence was also important for an effective interaction of the enzyme with the substrate; (ii) in the presence of specific ODNs the activity of the enzyme was enhanced, a result which suggests an ODN-induced conformational change of HIV-1 IN.

While the molecular basis of HIV-1 AZT resistance has been widely studied, a biochemical explanation of this process is not well known. No significant changes in the binding affinity of reverse transcriptase (RT) mutants for AZT-triphosphate has been found. Here we analyzed the interaction of wild type and AZT-resistant mutant forms of HIV-1 RT with different primers. Site-directed mutagenesis was used to introduce point mutations on the retroviral enzyme. Primers were either synthetic oligonucleotides or tRNA(Lys3) derivatives containing d(pT)n or r(pU)n at the 3' end. In all cases, determination of kinetic parameters was done in the presence or absence of compounds known to modify protein conformation, such as dimethyl sulfoxide (DMSO), urea, and Triton X-100. Although we found similar K(m) values for all RTs, there was generally an increase in the affinity when enzymes were tested in the presence of DMSO, urea, and Triton X-100. Then, we analyzed the nucleation and elongation steps of the polymerization process. The efficiency of formation of the first base pair was determined by measuring K(m1), the affinity between RT and the 3' terminal nucleotide of the primer. An important difference was found: in the presence of DMSO, urea, and Triton X-100, the K(m1) values for mutated enzymes were higher than those of wild type RTs. Thus, the presence of compounds able to change protein conformation led to a marked destabilization of the interaction of mutated RTs with the 3' terminal nucleotide of the primer. From these results, it can be hypothesized that resistance to AZT is not due to the direct influence of mutations on RT, but rather to conformational changes of the mutated RT in complex with the template-primer altering the ability of the enzyme to select or reject an incoming dNTP.

The topography and functional implications of the complex formed in vitro between human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and its primer tRNALys3 were studied in this work. On the basis of previous results showing the high affinity both of the native primer, tRNALys3, as well as that of mismatched short oligonucleotide primers for HIV-1 RT, we synthesized chimeric primers containing tRNALys3 linked to U and T residues of different lengths. We found that the affinity of the oligonucleotide primers for HIV-1 RT is dramatically increased when linked to primer tRNA. Our results also show that in the tRNA.RT complex, before annealing tRNALys3 to the retroviral RNA genome, the 3'-terminal nucleotide of tRNALys3 is positioned at a distance of one nucleotide unit away from the template in the active polymerization site of the enzyme.

We have carried out a comparison of KM and Vmax values for various primers in the polymerization reaction catalyzed by the HIV-1 RT. The affinity of RT for complementary d(pT)6 containing two different 5'-end pyranone derivatives was 2-3 orders of magnitude higher (KM = 3-15 nM) than that of d(pT)6 (KM = 12.6 mM). Oligodeoxynucleotides (ODNs) noncomplementary to poly(A) template were not elongated by RT. However, derivatives of d(CAGGTG) containing the 5'-terminal chromone and coumarin related groups were efficient primers showing KM (30-300 nM) and Vmax (75-93%) values comparable with that for d(pT)10 (800 nM; 100%). The [d(CAGGTG)]ddT ODN derivatives were effective inhibitors of RT. The primer function of derivatives of noncomplementary ODNs appears to be due to the additional interactions of their 5'-terminal groups with the enzyme tRNA-binding site.

DNA synthesis with various deoxyribo homo- and heterooligoprimers in the presence of complementary templates was investigated. The lengthening of d(pN)n, primers (n = 1-10) by a unit resulted in an increase of the primer affinity and a maximal rate of polymerization. The coefficient of the affinity enhancement of primers due to formation of one hydrogen bond between primer and template was found to be 1.35. The dependence of the primer affinity and polymerization rate on template-primer structure in solution was analyzed and the objective laws of the changes of the KM and Vmax values were revealed.

The recognition of primer tRNA by retroviral reverse transcriptase is a crucial step in the replication of retroviruses. In the complex formed by HIV-1 reverse transcriptase and its natural primer tRNALys3, the heterodimeric enzyme, p66/p51, binds two molecules of tRNALys3 with different affinities. The same complex but in the presence of a non-complementary template, poly(A), gave higher Kd values. Preincubation of the reverse transcriptase with tRNA at concentrations comparable to the Kd2 value results in different levels of stimulation of the DNA polymerase activity: 300% in the absence and 70-80% in the presence of poly(A). The activation of the catalytically active p66 subunit is most probably mediated through tRNA interaction with the site of reverse transcriptase presenting the lower affinity. In this article, we describe the results obtained with new chemically reactive derivatives of tRNA bearing three or seven hydrophobic residues. Incubation of reverse transcriptase with tRNA derivatives, in the presence or absence of poly(A), leads to covalent binding of the reagents and inactivation of the enzymatic activity. However, during the initial step of the modification reaction, in the absence of poly(A), a slight stimulation of reverse transcriptase by tRNA derivatives took place, followed by a decrease in the enzymatic activity due to the covalent binding of tRNA derivatives to reverse transcriptase. In the presence of poly(A), enzyme inactivation occurs according to pseudo-first-order reaction kinetics. The affinities of tRNA derivatives for the p66/p51 heterodimer estimated from affinity modification data (Kd values) and from the inhibition of polymerization reaction (Ki values) were determined. Each analog of tRNA presented two Kd and two Ki values.