Fluorescent Labelled Analogues of Neuropeptide Y for the Characterization of Cells Expressing NPY Receptor Subtypes

Abstract

Porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreatic polypeptide family, and subtype selective analogues have been synthesized by solid phase peptide synthesis. The peptides were labelled with Cy3^TM, a commercially available fluorescent marker based on a cyanine dye, by solid phase strategy. During the cleavage α partial fragmentation of the fluorescent marker occurred. This has been investigated by means of HPLC and electrospray mass spectrometry. The labelled analogues of NPY showed high affinity to the NPY receptor subtypes Y₁ and Y₂. Thus, Cy3-NPY. Y₁-selective Cy3-[Pro³⁴] NPY and Y₂ selective Cy3-[Ahx^5–24] NPY were used to label SK-N-MC- and SMS-KAN-cells, which are stably expressing the Y₁-(SK-N-MC) and the Y₂-receptor subtype (SMS-KAN). The binding of the labelled analogues to the receptors was reversible and specific. The photoactivatable analogue, [(Tmd)Phe²⁷] NPY, which showed high affinity to both receptor subtypes was labelled with Cy3 in solution. Whereas the fluorescent labelling of the cells with analogues without photoactivatable amino acid was reversible, successful photocrosslinking could be investigated by the irreversible staining of the cells using Cy3-[(Tmd)Phe²⁷] NPY. These subtype selective analogues are exciting tools to trace receptors in tissues and to identify the pharmacologically characterized subtypes without radioactivity.     

Synthesis of 3-(3-hydroxyphenyl)pyrrolidine dopamine D3 receptor ligands with extended functionality for probing the secondary binding pocket

A series of 3-(3-hydroxyphenyl)pyrrolidine analogues which incorporate N-alkyl groups and N-butylamide-linked benzamide functionality have been synthesized and their in vitro binding affinities at human dopamine receptors have been evaluated. Our ligand design strategy was to take the 3-(3-hydroxyphenyl)pyrrolidine scaffold and extend functionality from the orthosteric binding site to the secondary binding pocket for enhancing affinity and selectivity for the D₃ receptor. The N-alkyl analogues constitute a homologous series from N-pentyl to N-decyl to probe the length/bulk tolerance of the secondary binding pocket of the D₃ receptor. Enantiomeric 3-(3-hydroxyphenyl)pyrrolidine analogues were also prepared in order to test the chirality preference of the orthosteric binding site for this scaffold. Benzamide analogues were prepared to enhance affinity and/or selectivity based upon the results of the homologous series.     

A Novel Convergent Synthesis of the Antiglaucoma PGF2α Analogue Bimatoprost

The 17‐phenyl PGF_2α analogue bimatoprost ( 10a ) is the most efficacious ocular hypotensive agent currently available for the treatment of glaucoma or ocular hypertension. A novel convergent synthesis of 13,14‐en‐15‐ol prostamideF_2α analogues was developed employing Julia–Lythgoe olefination of the structurally advanced phenylsulfone (+)‐(5Z)‐ 15 with an enantiomerically pure aldehyde ω‐chain synthon (–)‐(S)‐ 16a . Subsequent hydrolysis of protecting groups and final amidation of the diol 26a yielded bimatoprost ( 10a ). The main advantage of the current strategy is the preparation of high‐purity bimatoprost ( 10a ). The novel convergent strategy allows the synthesis of a whole series of 13,14‐en‐15‐ol prostamideF_2α analogues with the desired C‐15 asymmetric center configuration from a common and structurally advanced prostaglandin intermediate (+)‐(5Z)‐ 15 . The preparation and identification of two synthetic impurities, 15‐epi isomer ( 10b ) of bimatoprost and a new prostaglandin related amide (+)‐(5Z)‐ 18 , are also described. Chirality 25:170–179, 2013. © 2013 Wiley Periodicals, Inc.     

Synthesis and GABAA receptor activity of 2,19-sulfamoyl analogues of allopregnanolone

The synthesis of new analogues of allopregnanolone with a bridged sulfamidate ring over the β-face of ring A has been achieved from easily available precursors, using an intramolecular aziridination strategy. The methodology also allows the synthesis of 3α-substituted analogues such as the 3α-fluoro derivative. GABA_A receptor activity of the synthetic analogues was evaluated by assaying their effect on the binding of [³H]flunitrazepam and [³H]muscimol. The 3α-hydroxy-2,19-sulfamoyl analogue and its N-benzyl derivative were more active than allopregnanolone for stimulating binding of [³H]flunitrazepam. For the binding of [³H]muscimol, both synthetic analogues and allopregnanolone stimulated binding to a similar extent, with the N-benzyl derivative exhibiting a higher EC₅₀. The 3α-fluoro derivative was inactive in both assays.     

Efficient synthesis and antioxidant activities of N-heterocyclyl substituted Coenzyme Q analogues

A new strategy for the efficient synthesis of C-5 heterocyclyl substituted Coenzyme Q analogues was developed by N-alkylation of bromomethylated quinone 11 with a series of amines 12 under metal-free conditions. In vitro antioxidant activities of these Coenzyme Q analogues were evaluated and compared with commercial antioxidant Coenzyme Q₁₀ by employing DPPH assay. All these N-heterocyclyl substituted Coenzyme Q analogues are found to be exhibiting good antioxidant properties and may be used as potent antioxidants for combating oxidative stress.     

A sensitive mass spectrum assay to characterize engineered methionine adenosyltransferases with S-alkyl methionine analogues as substrates

Methionine adenosyltransferases (MATs) catalyze the formation of S-adenosyl-l-methionine (SAM) inside living cells. Recently, S-alkyl analogues of SAM have been documented as cofactor surrogates to label novel targets of methyltransferases. However, these chemically synthesized SAM analogues are not suitable for cell-based studies because of their poor membrane permeability. This issue was recently addressed under a cellular setting through a chemoenzymatic strategy to process membrane-permeable S-alkyl analogues of methionine (SAAMs) into the SAM analogues with engineered MATs. Here we describe a general sensitive activity assay for engineered MATs by converting the reaction products into S-alkylthioadenosines, followed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) quantification. With this assay, 40 human MAT mutants were evaluated against 7 SAAMs as potential substrates. The structure–activity relationship revealed that, besides better engaged SAAM binding by the MAT mutants (lower K_m value in contrast to native MATs), the gained activity toward the bulky SAAMs stems from their ability to maintain the desired linear S_N2 transition state (reflected by higher k_cat value). Here the I117A mutant of human MATI was identified as the most active variant for biochemical production of SAM analogues from diverse SAAMs.     

Design,synthesis, and biological activity of substituted 2-amino-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid derivatives as inhibitors of the inflammatory kinases TBK1 and IKKε for the treatment of obesity

The non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa-B kinase ε (IKKε) play a key role in insulin-independent pathways that promote energy storage and block adaptive energy expenditure during obesity. Utilizing docking calculations and the x-ray structure of TBK1 bound to amlexanox, an inhibitor of these kinases with modest potency, a series of analogues was synthesized to develop a structure activity relationship (SAR) around the A- and C-rings of the core scaffold. A strategy was developed wherein R₇ and R₈ A-ring substituents were incorporated late in the synthetic sequence by utilizing palladium-catalyzed cross-coupling reactions on appropriate bromo precursors. Analogues display IC₅₀ values as low as 210?nM and reveal A-ring substituents that enhance selectivity toward either kinase. In cell assays, selected analogues display enhanced phosphorylation of p38 or TBK1 and elicited IL-6 secretion in 3T3-L1 adipocytes better than amlexanox. An analogue bearing a R₇ cyclohexyl modification demonstrated robust IL-6 production in 3T3-L1 cells as well as a phosphorylation marker of efficacy and was tested in obese mice where it promoted serum IL-6 response, weight loss, and insulin sensitizing effects comparable to amlexanox. These studies provide impetus to expand the SAR around the amlexanox core toward uncovering analogues with development potential.     

Inhibition of monoamine oxidase by selected C5- and C6-substituted isatin analogues

Previous studies have shown that (E)-5-styrylisatin and (E)-6-styrylisatin are reversible inhibitors of human monoamine oxidase (MAO) A and B. Both homologues are reported to exhibit selective binding to the MAO-B isoform with (E)-5-styrylisatin being the most potent inhibitor. To further investigate these structure-activity relationships (SAR), in the present study, additional C5- and C6-substituted isatin analogues were synthesized and evaluated as inhibitors of recombinant human MAO-A and MAO-B. With the exception of 5-phenylisatin, all of the analogues examined were selective MAO-B inhibitors. The C5-substituted isatins exhibited higher binding affinities to MAO-B than the corresponding C6-substituted homologues. The most potent MAO-B inhibitor, 5-(4-phenylbutyl)isatin, exhibited an IC₅₀ value of 0.66 nM, approximately 13-fold more potent than (E)-5-styrylisatin and 18,500-fold more potent than isatin. The most potent MAO-A inhibitor was found to be 5-phenylisatin with an IC₅₀ value of 562 nM. The results document that substitution at C5 with a variety of substituents is a general strategy for enhancing the MAO-B inhibition potency of isatin. Possible binding orientations of selected isatin analogues within the active site cavities of MAO-A and MAO-B are proposed.     

Evolution-based strategy to generate non-genetically modified organisms Saccharomyces cerevisiae strains impaired in sulfate assimilation pathway

Aims: An evolution‐based strategy was designed to screen novel yeast strains impaired in sulfate assimilation. Specifically, molybdate and chromate resistance was used as selectable phenotype to select sulfate permease–deficient variants that unable to produce sulfites and hydrogen sulfide (H₂S). Methods and Results: Four Saccharomyces cerevisiae parent strains were induced to sporulate. After tetrad digestion, spore suspensions were observed under the microscope to monitor the conjugation of gametes. Then, the cell suspension was inoculated in tubes containing YPD medium supplemented with ammonium molybdate or potassium chromate. Forty‐four resistant strains were obtained and then tested in microvinifications. Three strains with a low sulfite production (SO₂ <10 mg l^?1) and with an impaired H₂S production in grape must without added sulfites were selected. Conclusions: Our strategy enabled the selection of improved yeasts with desired oenological characteristics. Particularly, resistance to toxic analogues of sulfate allowed us to detect strains that unable to assimilate sulfates. Significance and Impact of the Study: This strategy that combines the sexual recombination of spores and application of a specific selective pressure provides a rapid screening method to generate genetic variants and select improved wine yeast strains with an impaired metabolism regarding the production of sulfites and H₂S.     

Bradykinin antagonists with dehydrophenylalanine analogues at position 5

Continuing the studies on structural requirements of bradykinin antagonists, it has been found that analogues with dehydrophenylalanine (ΔPhe) or its ring‒substituted analogues (ΔPhe(X)) at position 5 act as antagonists on guinea pig pulmonary artery, and on guinea pig ileum. Because both organs are considered to be bradykinin B₂receptor tissues, the analogues with ΔPhe or ΔPhe(X) at position 5, but without any replacement at position 7, seem to represent a new structural type of B₂receptor antagonist. All the analogues investigated act as partial antagonists; they inhibit the bradykinin‒induced contraction at low concentrations and act as agonists at higher concentrations. Ring substitutions by methyl groups or iodine reduce both the agonistic and antagonistic activity. Only substitution by fluorine gives a high potency. Incorporation of ΔPhe into different representative antagonists with key modifications at position 7 does not enhance the antagonist activity of the basic structures, with one exception. Only the combination of ΔPhe at position 5 with D Phe at position 7 increases the antagonistic potency on guinea pig ileum by about one order of magnitude. Radio‒ligand binding studies indicate the importance of position 5 for the discrimination of B₂receptor subtypes. The binding affinity to the low‒affinity binding site (K_L) was not significantly changed by replacement of Phe by ΔPhe. In contrast, ring‒methylation of ΔPhe results in clearly reduced binding to K_L. The affinity to the high‒affinity binding site (K_H) was almost unchanged by the replacement of Phe in position 5 by ΔPhe, whereas the analogue with 2‒methyl‒dehydrophenylalanine completely failed to detect the K_H‒site. The peptides were synthesized on the Wang‒resin according to the Fmoc/Bu^tstrategy using Mtr protection for the side chain of Arg. The dehydrophenylalanine analogues were prepared by a strategy involving PyBop couplings of the dipeptide unit Fmoc‒Gly‒ΔPhe(X)‒OH to resin‒bound fragments. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Mevalonate analogues as substrates of enzymes in the isoprenoid biosynthetic pathway of Streptococcus pneumoniae

Survival of the human pathogen Streptococcus pneumoniae requires a functional mevalonate pathway, which produces isopentenyl diphosphate, the essential building block of isoprenoids. Flux through this pathway appears to be regulated at the mevalonate kinase (MK) step, which is strongly feedback-inhibited by diphosphomevalonate (DPM), the penultimate compound in the pathway. The human mevalonate pathway is not regulated by DPM, making the bacterial pathway an attractive antibiotic target. Since DPM has poor drug characteristics, being highly charged, we propose to use unphosphorylated, cell-permeable prodrugs based on mevalonate that will be phosphorylated in turn by MK and phosphomevalonate kinase (PMK) to generate the active compound in situ. To test the limits of this approach, we synthesized a series of C₃-substituted mevalonate analogues to probe the steric and electronic requirements of the MK and PMK active sites. MK and PMK accepted substrates with up to two additional carbons, showing a preference for small substituents. This result establishes the feasibility of using a prodrug strategy for DPM-based antibiotics in S. pneumoniae and identified several analogues to be tested as inhibitors of MK. Among the substrates accepted by both enzymes were cyclopropyl, vinyl, and ethynyl mevalonate analogues that, when diphosphorylated, might be mechanism-based inactivators of the next enzyme in the pathway, diphosphomevalonate decarboxylase.     

A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes

Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC₅₀ values of 5.8 (meta isomer) and 3.0 μM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K_I of 0.46 μM. Radiolabeled forms of both analogues selectively labeled the β-subunit of ScPFTase. The para isomer was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes.     

Further properties of the polyamine oxidase of barley leaves

Polyamine analogues have been studied as potential inhibitors or substrates of barley leaf polyamine oxidase. NH₂(CH₂)₃NH(CH₂)₁₀NH₂ was particularly effective as an inhibitor of spermine oxidation at pH 4·5 (K_i = 5 × 10^?6 M). Methylglyoxal-bis(guanylhydrazone) inhibited spermine oxidation only slightly (K_i = 10^?4 M). Activity with the polyamine analogues as substrates was generally 10% or less of the activity with spermine. The K_m for oxygen was 3 × 10^?4 M. The K_m for spermine oxidation was independent of oxygen concentration. Using the N-methyl-2-benzothiazolone hydrazine reagent, 1-(3-aminopropyl)pyrroline was shown to be formed stoichiometrically by the enzyme on oxidation of spermine. The enzyme will not function as a dehydrogenase in the presence of oxygen with either potassium ferricyanide or dichlorophenolindophenol as electron acceptors. Activity in the leaves increased with age, up to 4 weeks. In the leaves of 11-week-old plants activity was lower than in leaves of 1-week-old plants. The enzyme was mainly associated with an easily-sedimented particulate fraction, and relatively small proportions were found in the cell wall or soluble fractions.     

Production of Resting Forms by the Gram-Negative Chemolithoautotrophic Bacteria Thioalkalivibrio versutusand Thioalkalimicrobium aerophilum

The haloalkaliphilic chemolithoautotrophic gram-negative bacteria Thioalkalivibrio versutus, strain AL2, and Thioalkalimicrobium aerophilum, strain AL3, were shown to possess the capacity to produce resting forms, namely cystlike refractile cells (CRC), whose production was controlled by the level of the d₁ extracellular factors exhibiting the function of anabiosis autoinducers. The conditions were elucidated that promote the formation of CRC in the developmental cycles of the cultures studied, in condensed cell suspensions undergoing autolysis, and under the action of exogenously introduced chemical analogues of anabiosis autoinducers (alkylhydroxybenzenes). The peculiarities of the fine structure of the resting cells obtained were studied. Distinctions were revealed (with respect to viability and thermotolerance) between the CRC formed under different conditions. The relationship between the growth strategy and survival strategy of extremophilic bacteria is discussed taking into account the effect of the d₁ autoregulatory factors. A new model of CRC formation is proposed: CRC production in the life cycle of bacteria developing under conditions of increased concentration of anabiosis autoinducers.     

Backbone Cyclization of the C-terminal Part of Substance P. Part 1: The Important Role of the Sulphur in Position 11

Novel backbone-to-side chain and backbone-to-backbone cyclic analogues of substance P (SP) were prepared by solid-phase synthesis and screened for biological activity. An analogue containing a thioether- lactam ring between positions 9 and 11 showed an EC₅₀ value of 20nM toward the neurokinin 1 (NK-1) and was inactive toward the NK-2 and NK-3 receptors. On the other hand, in a multiple backbone cyclic peptide library of similar analogues, in which the sulphur was excluded from the ring, very low activity was detected. The activity was re-evaluated and was found to be even lower (EC₅₀=0.11 mM ) than the previously published data. These results indicate that the thioether moiety has a crucial role in receptor activation. The results also show tolerance of the NK-1 receptor, but not NK-2 or NK-3, to cyclization of the C-terminal portion of the SP_6–11 hexapeptide.     

Effects of epoxymethano analogues of prostaglandin endoperoxides on aggregation,on release of 5-hydroxytryptamine and on the metabolism of 3′,5′-cyclic AMP and cyclic GMP in human platelets

The effects on human platelets of two synthetic analogues of prostaglandin endoperoxides were examined in order to explore the relationship between aggregation and prostaglandin and cyclic nucleotide metabolism, and to help elucidate the role of the natural endoperoxide intermediates in regulating platelet function.Both analogues (Compound I, (15S)-hydroxy-9α,11α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid, and Compound II, (15S)-hydroxy-11α,9α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid) caused platelets to aggregate, an effect which could be inhibited by prostaglandin E₁ but not by indomethacin. Compound II produced primary, reversible aggregation at concentrations which did not induce release of 5-hydroxytryptamine. Production of thromboxane B₂ and malonyldialdehyde was monitored as an index of endogenous production of prostaglandin endoperoxides and thromboxane A₂ and were increased after incubation of human platelets with thrombin, collagen or arachidonic acid. However, neither malonydialdehyde nor thromboxane B₂ levels were significantly influenced by the endoperoxide analogues. Both analogues produced a small elevation of adenylate cyclase activity in platelet membranes and of cyclic AMP content in intact platelets, but neither had any modifying effect on the much greater stimulation of adenylate cyclase and cyclic AMP levels by prostaglandin E₁. Of all the aggregating agents tested, only arachidonic acid produced any significant increase in platelet cyclic GMP levels.These results suggest that the epoxymethano analogues of prostaglandin endoperoxides induce platelet aggregation independently of thromboxane biosynthesis and without inhibiting adenylate cyclase or lowerin platelet cyclic AMP levels. They therefore differ from better known aggregating agents such as ADP, epinephrine and collagen, which increase thromboxane A₂ production and reduce cyclic AMP levels, at least in platelets previously exposed to prostaglandin E₁.     

Microbiological activities of nucleotide loop-modified analogues of vitamin B12

Novel vitamin B₁₂ analogues in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analogue containing imidazole instead of 5,6-dimethylbenzimidazole as well as cobinamide methyl phosphate were tested for biological activities with Escherichia coli 215, a B₁₂- or methionine-auxotroph, and Lactobacillus leichmannii ATCC 7830 as test organisms. A cyano form of 5,6-dimethylbenzimidazolyl tetramethylene, trimethylene and hexamethylene analogues supported the growth of L. leichmannii in this order. 5.6-Dimethylbenzimidazolyl dimethylene and imidazolyl trimethylene analogues did not show B₁₂ activity and behaved as weak B₁₂ antagonists when added together with cyanocobalamin. An adenosyl form of the biologically active analogues served as coenzymes for ribonucleotide reductase of this bacterium, whereas that of the inactive analogues did not. The latter acted as weak competitive inhibitors against adenosylcobalamin. ON the contrary, all the analogues did not support the growth of E. coli 215 at all by themselves and inhibited the growth when added with a suboptimum level of cyanocobalamin. A methyl form of the analogues also did not support the growth of E. coli 215, although they served as active coenzymes for methionine synthase of the bacterium. Since unlabeled analogues strongly inhibited the uptake of [³H]cyanocobalamin by this bacterium, it seems likely that the analogues exert their anti-B₁₂ activity toward E. coli 215 by blocking the B₁₂-transport systemAbbreviations AdoCbl adenosylcobalamin - MeCbl methylcobalamin - CN-Cbl cyanocobalamin or vitamin B₁₂ - Cbl cobalamin - (CN, aq)Cbi cyanoaquacobinamide - MeCbi methylcobinamide - Cbi cobinamide - (CN, aq)Cbi-PMe cyanoaquacobinamide methyl phosphate - Cbi-PMe cobinamide methyl phosphate - DBI 5,6-dimethylbenzimidazole - DBIyl 5,6-dimethylbenzimidazolyl - FMNH₂ fully reduced form of riboflavin 5-phosphate     

Growth-promoting activity of some selectively modified gibberellins

Synthetic gibberellin analogues derived mainly from GA₃ were tested for their growth-promoting activity in three standard bioassay systems (dwarf pea, cucumber and lettuce). The highest potency in all the tests was displayed by 13-O-methyl-GA₃, while in the case of 7-homo-GA₃ a large decrease in activity was observed. No obvious correlation could be found between the partitioning of acidic analogues between ethyl acetate and water and their biological potencies. The results of the bioassays appear to be compatible with the assumption that it is the spatial correspondence between an active gibberellin (such as GA₃ or GA₇) and a specific receptor that plays the prime role in the growth response.     

Substituents at the C13 Position of Retinal and Their Influence on the Function of Bacteriorhodopsin

Retinal analogues in which the 13-methyl group is replaced by H, C₂H₅, CF₃, and OCH₃ residues are studied by means of quantumchemical modified neglect of diatomic overlap-correlated version (MNDOC) calculations. The analogues are suitable to test the stereochemical mechanism of proton pumping in bacteriorhodopsin. The results explain the proton-pumping activities of bacterio-opsin reconstituted with these analogues and elucidate the decisive role of retinal's ground-state intramolecular properties in the pump cycle of bacteriorhodopsin.     

Cellular Resistance Against Troxacitabine in Human Cell Lines and Pediatric Patient Acute Myeloid Leukemia Blast Cells

Troxacitabine is a cytotoxic deoxycytidine analogue with an unnatural L-configuration, which is activated by deoxycytidine kinase (dCK). The configuration is responsible for differences in the uptake and metabolism of troxacitabine compared to other deoxynucleoside analogues. To determine whether troxacitabine has an advantage over other nucleoside analogues several cell lines resistant to cladribine and gemcitabine were exposed to troxacitabine, while blast cells from pediatric leukemia patients were tested for cross-resistance with other deoxynucleoside analogues. The gemcitabine resistant AG6000 (IC₅₀: >3000 nM), and the cladribine resistant CEM (IC₅₀: 150 nM) and HL-60 (IC₅₀: >3000 nM) cell lines, all with no or decreased dCK expression, were less sensitive to troxacitabine than their wild type counterparts (IC₅₀; A2780: 410, CEM: 71 and HL-60: 158 nM). dCK protein expression in CEM was higher than in HL-60, which, in turn, was higher than in A2780. Catalytically inactive p53 seems to increase the sensitivity to troxacitabine. The patient samples showed a large range of sensitivity to troxacitabine, similar to other deoxynucleoside analogues. Cross-resistance with all other deoxynucleoside analogues was observed.