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Coordination compounds of lanthanides are indispensable in biomedical applications as MRI contrast agents and radiotherapeutics. However, since the introduction of the chelator DOTA four decades ago, there has been only limited progress on improving their thermodynamic stability and kinetic inertness, which are essential for safe in vivo use. Here, we present ClickZip, an innovative synthetic strategy employing a coordination-templated formation of a 1,5-triazole bridge that improves kinetic inertness up to a million-fold relative to DOTA, expanding utility of lanthanide chelates beyond traditional uses. Acting as unique mass tags, the ClickZip chelates can be released from (biological) samples by acidic hydrolysis, chromatographically distinguished from interfering lanthanide species, and sensitively detected by mass spectrometry. Lanthanides enclosed in ClickZip chelates are chemically almost indistinguishable, providing a more versatile alternative to chemically identical isotopic labels for multiplexed analysis. The bioanalytical potential is demonstrated on tagged cell-penetrating peptides in vitro, and anti-obesity prolactin-releasing peptides in vivo., Competing Interests: Competing interests M.P., T.D., M.S. and A.J. are co-inventors on a patent application no. PCT/CZ2022/050087 filed in the name of applicant Ustav Organicke Chemie a Biochemie AV CR V.V.I. The application covers some of the compounds and their use discussed in this work. M.P., T.D., M.S. and A.J. declare no other competing interests. The other authors declare no competing interests., (© 2024. The Author(s).)

Bioorthogonal reactions that enable switching molecular functions by breaking chemical bonds have gained prominence, with the tetrazine-mediated cleavage of trans-cyclooctene caged compounds (click-to-release) being particularly noteworthy for its high versatility, biocompatibility, and fast reaction rates. Despite several recent advances, the development of highly reactive tetrazines enabling quantitative elimination from trans-cyclooctene linkers remains challenging. In this study, we present the synthesis and application of sulfo-tetrazines, a class of derivatives featuring phenolic hydroxyl groups with increased acidity constants (pK a ). This unique property leads to accelerated elimination and complete release of the caged molecules within minutes. Moreover, the inclusion of sulfonate groups provides a valuable synthetic handle, enabling further derivatization into sulfonamides, modified with diverse substituents. Significantly, we demonstrate the utility of sulfo-tetrazines in efficiently activating fluorogenic compounds and prodrugs in living cells, offering exciting prospects for their application in bioorthogonal chemistry., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Ion channels represent a druggable family of transmembrane pore-forming proteins with important (patho)physiological functions. While electrophysiological measurement (manual patch clamp) remains the only direct method for detection of ion currents, it is a labor-intensive technique. Although automated patch clamp instruments have become available to date, their high costs limit their use to large pharma companies or commercial screening facilities. Therefore, fluorescence-based assays are particularly important for initial screening of compound libraries. Despite their numerous disadvantages, they are highly amenable to high-throughput screening and in many cases, no sophisticated instrumentation or materials are required. These features predispose them for implementation in early phases of drug discovery pipelines (hit identification), even in an academic environment. This review summarizes the advantages and pitfalls of individual methodological approaches for identification of ion channel modulators employing fluorescent probes (i. e., membrane potential and ion flux assays) with emphasis on practical aspects of their adaptation to high-throughput format., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

The emergence of SARS-CoV-2, the causative agent of COVID-19, has highlighted the need for advanced antiviral strategies. Targeting the coronaviral methyltransferase nsp14, which is essential for RNA capping, offers a promising approach for the development of small-molecule inhibitors. We designed and synthesized a series of adenosine 5'-carboxamide derivatives as potential nsp14 inhibitors and identified coumarin analogs to be particularly effective. Structural modifications revealed the importance of the 5'-carboxyl moiety for the inhibitory activity, showing superior efficacy compared to other modifications. Notably, compound 18l ( HK370 ) demonstrated high selectivity and favorable in vitro pharmacokinetic properties and exhibited moderate antiviral activity in cell-based assays. These findings provide a robust foundation for developing targeted nsp14 inhibitors as a potential treatment for COVID-19 and related diseases., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Yohimbine, a natural indole alkaloid and a nonselective adrenoceptor antagonist, possesses potential benefits in treating inflammatory disorders and sepsis. Nevertheless, its broader clinical use faces challenges due to its low receptor selectivity. A structure-activity relationship study of novel yohimbine analogues identified amino esters of yohimbic acid as potent and selective ADRA2A antagonists. Specifically, amino ester 4n , in comparison to yohimbine, showed a 6-fold higher ADRA1A/ADRA2A selectivity index (SI > 556 for 4n ) and a 25-fold higher ADRA2B/ADRA2A selectivity index. Compound 4n also demonstrated high plasma and microsomal stability, moderate-to-low membrane permeability determining its limited ability to cross the blood-brain barrier, and negligible toxicity on nontumor normal human dermal fibroblasts. Compound 4n represents an important complementary pharmacological tool to study the involvement of adrenoceptor subtypes in pathophysiologic conditions such as inflammation and sepsis and a novel candidate for further preclinical development to treat ADRA2A-mediated pathologies.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

In human cells, receptor-interacting protein kinase 2 (RIPK2) is mainly known to mediate downstream enzymatic cascades from the nucleotide-binding oligomerization domain-containing receptors 1 and 2 (NOD1/2), which are regulators of pro-inflammatory signaling. Thus, the targeted inhibition of RIPK2 has been proposed as a pharmacological strategy for the treatment of a variety of pathologies, in particular inflammatory and autoimmune diseases. In this work, we designed and developed novel thieno[2,3d]pyrimidine derivatives, in order to explore their activity and selectivity as RIPK2 inhibitors. Primary in vitro evaluations of the new molecules against purified RIPKs (RIPK1-4) demonstrated outstanding inhibitory potency and selectivity for the enzyme RIPK2. Moreover, investigations for efficacy against the RIPK2-NOD1/2 signaling pathways, conducted in living cells, showed their potency could be tuned towards a low nanomolar range. This could be achieved by solely varying the substitutions at position 6 of the thieno[2,3d]pyrimidine scaffold. A subset of lead inhibitors were ultimately evaluated for selectivity against 58 human kinases other than RIPKs, displaying great specificities. We therefore obtained new inhibitors that might serve as starting point for the preparation of targeted tools, which could be useful to gain a better understanding of biological roles and clinical potential of RIPK2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Receptor-interacting protein kinases 2 and 3 (RIPK2 and RIPK3) are considered attractive therapeutic enzyme targets for the treatment of a multitude of inflammatory diseases and cancers. In this study, we developed three interrelated series of novel quinazoline-based derivatives to investigate the effects of extensive modifications of positions 6 and 7 of the central core on the inhibitory activity and the selectivity against these RIPKs. The design of the derivatives was inspired by analyses of available literary knowledge on both RIPK2 and RIPK3 in complex with known quinazoline or quinoline inhibitors. Enzymatic investigations for bioactivity of the prepared molecules against purified RIPKs (RIPK1-4) shed light on multiple potent and selective RIPK2 and dual RIPK2/3 inhibitors. Furthermore, evaluations in living cells against the RIPK2-NOD1/2-mediated signaling pathways, identified as the potential primary targets, demonstrated nanomolar inhibition for a majority of the compounds. In addition, we have demonstrated overall good stability of various lead inhibitors in both human and mouse microsomes and plasma. Several of these compounds also were evaluated for selectivity across 58 human kinases other than RIPKs, exhibiting outstanding specificity profiles. We have thus clearly demonstrated that tuning appropriate substitutions at positions 6 and 7 of the developed quinazoline derivatives may lead to interesting potency and specificities against RIPK2 and RIPK3. This knowledge might therefore be employed for the targeted preparation of new, highly potent and selective tools against these RIPKs, which could be of utility in biological and clinical research., Competing Interests: Declaration of competing interest Authors declare no competing interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Adefovir based acyclic nucleoside phosphonates were previously shown to modulate bacterial and, to a certain extent, human adenylate cyclases (mACs). In this work, a series of 24 novel 7-substituted 7-deazaadefovir analogues were synthesized in the form of prodrugs. Twelve analogues were single-digit micromolar inhibitors of Bordetella pertussis adenylate cyclase toxin with no cytotoxicity to J774A.1 macrophages. In HEK293 cell-based assays, compound 14 was identified as a potent (IC 50  = 4.45 μM), non-toxic, and selective mAC2 inhibitor (vs. mAC1 and mAC5). Such a compound represents a valuable addition to a limited number of small-molecule probes to study the biological functions of individual endogenous mAC isoforms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The development of reagents that can selectively react in complex biological media is an important challenge. Here we show that N1-alkylation of 1,2,4-triazines yields the corresponding triazinium salts, which are three orders of magnitude more reactive in reactions with strained alkynes than the parent 1,2,4-triazines. This powerful bioorthogonal ligation enables efficient modification of peptides and proteins. The positively charged N1-alkyl triazinium salts exhibit favorable cell permeability, which makes them superior for intracellular fluorescent labeling applications when compared to analogous 1,2,4,5-tetrazines. Due to their high reactivity, stability, synthetic accessibility and improved water solubility, the new ionic heterodienes represent a valuable addition to the repertoire of existing modern bioorthogonal reagents., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

FLT3 kinase is a potential drug target in acute myeloid leukemia (AML). Patients with FLT3 mutations typically have higher relapse rates and worse outcomes than patients without FLT3 mutations. In this study, we investigated the suitability of various heterocycles as central cores of FLT3 inhibitors, including thieno[3,2- d ]pyrimidine, pyrazolo[1,5- a ]pyrimidine, imidazo[4,5- b ]pyridine, pyrido[4,3- d ]pyrimidine, and imidazo[1,2- b ]pyridazine. Our assays revealed a series of imidazo[1,2- b ]pyridazines with high potency against FLT3. Compound 34f showed nanomolar inhibitory activity against recombinant FLT3-ITD and FLT3-D835Y (IC 50 values 4 and 1 nM, respectively) as well as in the FLT3-ITD-positive AML cell lines MV4-11, MOLM-13, and MOLM-13 expressing the FLT3-ITD-D835Y mutant (GI 50 values of 7, 9, and 4 nM, respectively). In contrast, FLT3-independent cell lines were much less sensitive. In vitro experiments confirmed suppression of FLT3 downstream signaling pathways. Finally, the treatment of MV4-11 xenograft-bearing mice with 34f at doses of 5 and 10 mg/kg markedly blocked tumor growth without any adverse effects.

The search for new drugs against COVID-19 and its causative agent, SARS-CoV-2, is one of the major trends in the current medicinal chemistry. Targeting capping machinery could be one of the therapeutic concepts based on a unique mechanism of action. Viral RNA cap synthesis involves two methylation steps, the first of which is mediated by the nsp14 protein. Here, we rationally designed and synthesized a series of compounds capable of binding to both the S -adenosyl-l-methionine and the RNA-binding site of SARS-CoV-2 nsp14 N 7 -methyltransferase. These hybrid molecules showed excellent potency, high selectivity toward various human methyltransferases, nontoxicity, and high cell permeability. Despite the outstanding activity against the enzyme, our compounds showed poor antiviral performance in vitro. This suggests that the activity of this viral methyltransferase has no significant effect on virus transcription and replication at the cellular level. Therefore, our compounds represent unique tools to further explore the role of the SARS-CoV-2 nsp14 methyltransferase in the viral life cycle and the pathogenesis of COVID-19., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Purine nucleoside phosphorylase (PNP) is a well-known molecular target with potential therapeutic applications in the treatment of T-cell malignancies and/or bacterial/parasitic infections. Here, we report the design, development of synthetic methodology, and biological evaluation of a series of 30 novel PNP inhibitors based on acyclic nucleoside phosphonates bearing a 9-deazahypoxanthine nucleobase. The strongest inhibitors exhibited IC 50 values as low as 19 nM (human PNP) and 4 nM ( Mycobacterium tuberculosis ( Mt ) PNP) and highly selective cytotoxicity toward various T-lymphoblastic cell lines with CC 50 values as low as 9 nM. No cytotoxic effect was observed on other cancer cell lines (HeLa S3, HL60, HepG2) or primary PBMCs for up to 10 μM. We report the first example of the PNP inhibitor exhibiting over 60-fold selectivity for the pathogenic enzyme ( Mt PNP) over hPNP. The results are supported by a crystallographic study of eight enzyme-inhibitor complexes and by ADMET profiling in vitro and in vivo .

Novel 4-aminoquinazoline-6-carboxamide derivatives bearing differently substituted aryl or heteroaryl groups at position 7 in the core were rationally designed, synthesized and evaluated for biological activity in vitro as phosphatidylinositol 4-kinase IIα (PI4K2A) inhibitors. The straightforward approach described here enabled the sequential, modular synthesis and broad functionalization of the scaffold in a mere six steps. The SAR investigation reported here is based on detailed structural analysis of the conserved binding mode of ATP and other adenine derivatives to the catalytic site of type II PI4Ks, combined with extensive docking studies. Several compounds exhibited significant activity against PI4K2A. Moreover, we solved a crystal structure of PI4K2B in complex with one of our lead ligand candidates, which validated the ligand binding site and pose predicted by our docking-based ligand model. These discoveries suggest that our structure-based approach may be further developed and employed to synthesize new inhibitors with optimized potency and selectivity for this class of PI4Ks., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The goal of this study was to evaluate mixed cortical and hippocampal primary rat postnatal neuronal culture as in vitro tool for identification of N-methyl-D-aspartate receptor (NMDAR) antagonists and to find out, whether this model is comparable with other commonly used primary rat neuronal models differing in their origin (pure cortical vs. mixed cortical and hippocampal) and differentiation state (embryonal vs. postnatal). Induced pluripotent stem cell (iPSC) - derived human glutamatergic neurons have been included in this study as well. First, the cultures were characterized by their neuron/astrocyte composition, the mRNA expression of NR2B/NR2A NMDAR subunit ratios, and the expression of glutamate transporters (GLT1, GLAST). Then, selected endogenous steroids and synthetic neuroactive steroids that have been previously identified as negative allosteric modulators of recombinant GluN1/GluN2B NMDA receptors, were evaluated for their ability to prevent an NMDA or glutamate-induced Ca 2+ influx (acute effect) and excitotoxicity over 24 h. Though the neuroprotective potential against excitotoxic stimuli varied among the models studied, postnatal mixed cortical and hippocampal culture proved to be a convenient and robust tool for NMDAR antagonist screening. The most widely used embryonal (E18) cultures offered higher cell yields but at the expense of a higher sensitivity to compounds' cytotoxicity. iPSC-derived neurons were not found to be superior to rat cultures for screening purposes., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A series of acyclic nucleoside phosphonates (ANPs) was designed as inhibitors of bacterial adenylate cyclases (ACs), where adenine was replaced with 2-amino-4-arylthiazoles. The target compounds were prepared using the halogen dance reaction. Final AC inhibitors were evaluated in cell-based assays (prodrugs) and cell-free assays (phosphono diphosphates). Novel ANPs were potent inhibitors of adenylate cyclase toxin (ACT) from Bordetella pertussis and edema factor (EF) from Bacillus anthracis, with substantial selectivity over mammalian enzymes AC1, AC2, and AC5. Six of the new ANPs were more potent or equipotent ACT inhibitors (IC 50 =9-18 nM), and one of them was more potent EF inhibitor (IC 50 =12 nM), compared to adefovir diphosphate (PMEApp) with IC 50 =18 nM for ACT and IC 50 =36 nM for EF. Thus, these compounds represent the most potent ACT/EF inhibitors based on ANPs reported to date. The potency of the phosphonodiamidates to inhibit ACT activity in J774A.1 macrophage cells was somewhat weaker, where the most potent derivative had IC 50 =490 nM compared to IC 50 =150 nM of the analogous adefovir phosphonodiamidate. The results suggest that more efficient type of phosphonate prodrugs would be desirable to increase concentrations of the ANP-based active species in the cells in order to proceed with the development of ANPs as potential antitoxin therapeutics., (© 2021 Wiley-VCH GmbH.)

This study describes the discovery of novel prodrugs bearing tyrosine derivatives instead of the phenol moiety present in FDA-approved tenofovir alafenamide fumarate (TAF). The synthesis was optimized to afford diastereomeric mixtures of novel prodrugs in one pot (yields up to 86%), and the epimers were resolved using a chiral HPLC column into fast-eluting and slow-eluting epimers. In human lymphocytes, the most efficient tyrosine-based prodrug reached a single-digit picomolar EC 50 value against HIV-1 and nearly 300-fold higher selectivity index (SI) compared to TAF. In human hepatocytes, the most efficient prodrugs exhibited subnanomolar EC 50 values for HBV and up to 26-fold higher SI compared to TAF. Metabolic studies demonstrated markedly higher cellular uptake of the prodrugs and substantially higher levels of released tenofovir inside the cells compared to TAF. These promising results provide a strong foundation for further evaluation of the reported prodrugs and their potential utility in the development of highly potent antivirals.

A series of novel acyclic nucleoside phosphonates (ANPs) was synthesized as potential adenylate cyclase inhibitors, where the adenine nucleobase of adefovir (PMEA) was replaced with a 5-substituted 2-aminothiazole moiety. The design was based on the structure of MB05032, a potent and selective inhibitor of fructose 1,6-bisphosphatase and a good mimic of adenosine monophosphate (AMP). From the series of eighteen novel ANPs, which were prepared as phosphoroamidate prodrugs, fourteen compounds were potent (single digit micromolar or submicromolar) inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT), mostly without observed cytotoxicity in J774A.1 macrophage cells. Selected phosphono diphosphates (nucleoside triphosphate analogues) were potent inhibitors of ACT (IC 50 as low as 37 nM) and B. anthracis edema factor (IC 50 as low as 235 nM) in enzymatic assays. Furthermore, several ANPs were found to be selective mammalian AC1 inhibitors in HEK293 cell-based assays (although with some associated cytotoxicity) and one compound exhibited selective inhibition of mammalian AC2 (only 12% of remaining adenylate cyclase activity) but no observed cytotoxicity. The mammalian AC1 inhibitors may represent potential leads in development of agents for treatment of human inflammatory and neuropathic pain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Schistosomiasis, a parasitic disease caused by blood flukes of the genus Schistosoma , is a global health problem with over 200 million people infected. Treatment relies on just one drug, and new chemotherapies are needed. Schistosoma mansoni cathepsin B1 (SmCB1) is a critical peptidase for the digestion of host blood proteins and a validated drug target. We screened a library of peptidomimetic vinyl sulfones against SmCB1 and identified the most potent SmCB1 inhibitors reported to date that are active in the subnanomolar range with second order rate constants ( k 2nd ) of ∼2 × 10 5 M -1 s -1 . High resolution crystal structures of the two best inhibitors in complex with SmCB1 were determined. Quantum chemical calculations of their respective binding modes identified critical hot spot interactions in the S1' and S2 subsites. The most potent inhibitor targets the S1' subsite with an N -hydroxysulfonic amide moiety and displays favorable functional properties, including bioactivity against the pathogen, selectivity for SmCB1 over human cathepsin B, and reasonable metabolic stability. Our results provide structural insights for the rational design of next-generation SmCB1 inhibitors as potential drugs to treat schistosomiasis.

In analogy to antiviral acyclic nucleoside phosphonates, a series of 5-amino-3-oxo-1,2,4-thiadiazol-3(2H)-ones bearing a 2-phosphonomethoxyethyl (PME) or 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) group at the position 2 of the heterocyclic moiety has been synthesized. Diisopropyl esters of PME- and HPMP-amines have been converted to the N-substituted ureas and then reacted with benzoyl, ethoxycarbonyl, and Fmoc isothiocyanates to give the corresponding thiobiurets, which were oxidatively cyclized to diisopropyl esters of 5-amino-3-oxo-2-PME- or 2-HPMP- 1,2,4-thiadiazol-3(2H)-ones. The phosphonate ester groups were cleaved with bromotrimethylsilane, yielding N 5 -protected phosphonic acids. The subsequent attempts to remove the protecting group from N 5 under alkaline conditions resulted in the cleavage of the 1,2,4-thiadiazole ring. Similarly, compounds with a previously unprotected 5-amino-1,2,4-thiadiazolone base moiety were stable only in the form of phosphonate esters. The series of twenty-one newly prepared 1,2,4-thiadiazol-3(2H)-ones were explored as potential inhibitors of cysteine-dependent enzymes - human cathepsin K (CatK) and glycogen synthase kinase 3β (GSK-3β). Several compounds exhibited an inhibitory activity toward both enzymes in the low micromolar range. The inhibitory potency of some of them toward GSK-3β was similar to that of the thiadiazole GSK-3β inhibitor tideglusib, whereas others exhibited more favorable toxicity profile while retaining good inhibitory activity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

All four isomeric series of novel 4-substituted pyrido-fused 7-deazapurine ribonucleosides possessing the pyridine nitrogen atom at different positions were designed and synthesized. The total synthesis of each isomeric fused heterocycle through multistep heterocyclization was followed by glycosylation and derivatization at position 4 by cross-coupling reactions or nucleophilic substitutions. All compounds were tested for cytostatic and antiviral activity. The most active were pyrido[4',3':4,5]pyrimidine nucleosides bearing MeO, NH 2 , MeS, or CH 3 groups at position 4, which showed submicromolar cytotoxic effects and good selectivity for cancer cells. The mechanism involved activation by phosphorylation and incorporation to DNA where the presence of the modified ribonucleosides causes double-strand breaks and apoptosis., (© 2020 Wiley-VCH GmbH.)

Bioorthogonal cleavage reactions are gaining popularity in chemically inducible prodrug activation and in the control of biomolecular functions. Despite similar applications, these reactions were developed and optimized on different substrates and under different experimental conditions. Reported herein is a side-by-side comparison of palladium-, ruthenium- and tetrazine-triggered release reactions, which aims at comparing the reaction kinetics, efficiency and overall advantages and limitations of the methods. In addition, we disclose the possibility of mutual combination of the cleavage reactions. Finally, we compare the efficiency of the bioorthogonal deprotections in cellular experiments, which revealed that among the three methods investigated, the palladium- and the tetrazine-promoted reaction can be used for efficient prodrug activation, but only the tetrazine-triggered reactions proceed efficiently inside cells., (© 2020 Wiley-VCH GmbH.)

Human cathepsin D (CatD), a pepsin-family aspartic protease, plays an important role in tumor progression and metastasis. Here, we report the development of biomimetic inhibitors of CatD as novel tools for regulation of this therapeutic target. We designed a macrocyclic scaffold to mimic the spatial conformation of the minimal pseudo-dipeptide binding motif of pepstatin A, a microbial oligopeptide inhibitor, in the CatD active site. A library of more than 30 macrocyclic peptidomimetic inhibitors was employed for scaffold optimization, mapping of subsite interactions, and profiling of inhibitor selectivity. Furthermore, we solved high-resolution crystal structures of three macrocyclic inhibitors with low nanomolar or subnanomolar potency in complex with CatD and determined their binding mode using quantum chemical calculations. The study provides a new structural template and functional profile that can be exploited for design of potential chemotherapeutics that specifically inhibit CatD and related aspartic proteases.

Three series of isomeric pyrrolo- and furo-fused 7-deazapurine ribonucleosides were synthesized and screened for cytostatic and antiviral activity. The synthesis was based on heterocyclizations of hetaryl-azidopyrimidines to form the tricyclic heterocyclic bases, followed by glycosylation and final derivatizations through cross-coupling reactions or nucleophilic substitutions. The pyrrolo[2',3':4,5]pyrrolo[2,3- d]pyrimidine and furo[2',3':4,5]pyrrolo[2,3- d]pyrimidine ribonucleosides were found to be potent cytostatics, whereas the isomeric pyrrolo[3',2',4,5]pyrrolo[2,3- d]pyrimidine nucleosides were inactive. The most active were the methyl, methoxy, and methylsulfanyl derivatives exerting submicromolar cytostatic effects and good selectivity toward cancer cells. We have shown that the nucleosides are activated by intracellular phosphorylation and the nucleotides get incorporated to both RNA and DNA, where they cause DNA damage. They represent a new type of promising candidates for preclinical development toward antitumor agents.

A series of 13 acyclic nucleoside phosphonates (ANPs) as bisamidate prodrugs was prepared. Five compounds were found to be non-cytotoxic and selective inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT) in J774A.1 macrophage cell-based assays. The 8-aza-7-deazapurine derivative of adefovir (PMEA) was found to be the most potent ACT inhibitor in the series (IC 50 =16 nm) with substantial selectivity over mammalian adenylate cyclases (mACs). AC inhibitory properties of the most potent analogues were confirmed by direct evaluation of the corresponding phosphonodiphosphates in cell-free assays and were found to be potent inhibitors of both ACT and edema factor (EF) from Bacillus anthracis (IC 50 values ranging from 0.5 to 21 nm). Moreover, 7-halo-7-deazapurine analogues of PMEA were discovered to be potent and selective mammalian AC1 inhibitors (no inhibition of AC2 and AC5) with IC 50 values ranging from 4.1 to 5.6 μm in HEK293 cell-based assays., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Inhibition of Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF), key virulence factors with adenylate cyclase activity, represents a potential method for treating or preventing toxemia related to whooping cough and anthrax, respectively. Novel α-branched acyclic nucleoside phosphonates (ANPs) having a hemiaminal ether moiety were synthesized as potential inhibitors of bacterial adenylate cyclases. ANPs prepared as bisamidates were not cytotoxic, but did not exhibit any profound activity (IC 50 >10 μm) toward ACT in J774A.1 macrophages. The apparent lack of activity of the bisamidates is speculated to be due to the inefficient formation of the biologically active species (ANPpp) in the cells. Conversely, two 5-haloanthraniloyl-substituted ANPs in the form of diphosphates were shown to be potent ACT and EF inhibitors with IC 50 values ranging from 55 to 362 nm., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is indispensable for the replication of various positive-sense single stranded RNA viruses, which hijack this cellular enzyme to remodel intracellular membranes of infected cells to set up the functional replication machinery. Therefore, the inhibition of this PI4K isoform leads to the arrest of viral replication. Here, we report on the synthesis of novel PI4KB inhibitors, which were rationally designed based on two distinct structural types of inhibitors that bind in the ATP binding side of PI4KB. These "hybrids" not only excel in outstanding inhibitory activity but also show high selectivity to PI4KB compared to other kinases. Thus, these compounds exert selective nanomolar or even subnanomolar activity against PI4KB as well as profound antiviral effect against hepatitis C virus, human rhinovirus, and coxsackievirus B3. Our crystallographic analysis unveiled the exact position of the side chains and explains their extensive contribution to the inhibitory activity.

Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF) are key virulence factors with adenylate cyclase (AC) activity that substantially contribute to the pathogenesis of whooping cough and anthrax, respectively. There is an urgent need to develop potent and selective inhibitors of bacterial ACs with prospects for the development of potential antibacterial therapeutics and to study their molecular interactions with the target enzymes. Novel fluorescent 5-chloroanthraniloyl-substituted acyclic nucleoside phosphonates (Cl-ANT-ANPs) were designed and synthesized in the form of their diphosphates (Cl-ANT-ANPpp) as competitive ACT and EF inhibitors with sub-micromolar potency (IC 50 values: 11-622 nm). Fluorescence experiments indicated that Cl-ANT-ANPpp analogues bind to the ACT active site, and docking studies suggested that the Cl-ANT group interacts with Phe306 and Leu60. Interestingly, the increase in direct fluorescence with Cl-ANT-ANPpp having an ester linker was strictly calmodulin (CaM)-dependent, whereas Cl-ANT-ANPpp analogues with an amide linker, upon binding to ACT, increased the fluorescence even in the absence of CaM. Such a dependence of binding on structural modification could be exploited in the future design of potent inhibitors of bacterial ACs. Furthermore, one Cl-ANT-ANP in the form of a bisamidate prodrug was able to inhibit B. pertussis ACT activity in macrophage cells with IC 50 =12 μm., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: 9-Norbornyl-6-chloropurine (NCP) is a representative of a series of antienteroviral bicycle derivatives with selective cytotoxicity towards leukemia cell lines. In this work we explored the mechanism of the antileukemic activity of NCP in T-cell lymphoblast cells (CCRF-CEM). Specifically, we searched for a potential link between its ability to induce cell death on the one hand and to modulate intracellular glutathione (GSH) that is necessary to its metabolic transformation via glutathione-S-transferase on the other hand. We have observed that GSH levels decreased rapidly in NCP-treated cells. Despite a complete regeneration following 24h of incubation with NCP, this profound drop in cellular GSH content triggered ER stress, ROS production and lipid peroxidation leading to the loss of mitochondrial membrane potential (MMP). These events induced concentration-dependent cell cycle arrest in G2/M phase and apoptosis. Both MMP loss and apoptosis were reversed by sulfhydryl-containing compounds (GSH, N-acetyl-l-cysteine). Furthermore, we have also shown that NCP-induced GSH decrease activated the Nrf2 pathway and its downstream targets, Nad(p)h: quinone oxidoreductase (NQO-1) and glutamate cysteine ligase modifier subunit (GCLm), thus explaining the fast restoration of GSH pool and ROS decrease. Importantly, we confirmed that the cell death-inducing properties of the compounds were co-dependent on their ability to diminish cellular GSH level by analyzing the relationships between the GSH-depleting potency and cytotoxicity in a series of other norbornylpurine analogs. Altogether, the results demonstrated that in CCRF-CEM cells NCP triggered apoptosis through GSH depletion-associated oxidative and ER stress and mitochondrial depolarization., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

We report on an extensive structure-activity relationship study of novel PI4K IIIβ inhibitors. The purine derivative of the potent screening hit T-00127-HEV1 has served as a suitable starting point for a thorough investigation of positions 8 and 2. While position 8 of the purine scaffold can only bear a small substituent to maintain the inhibitory activity, position 2 is opened for extensive modification and can accommodate even substituted phenyl rings without the loss of PI4K IIIβ inhibitory activity. These empirical observations nicely correlate with the results of our docking study, which suggests that position 2 directs towards solution and can provide the necessary space for the interaction with remote residues of the enzyme, whereas the cavity around position 8 is strictly limited. The obtained compounds have also been subjected to antiviral screening against a panel of (+)ssRNA viruses., (Copyright © 2016. Published by Elsevier Ltd.)

Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L-phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2-fluoro PMEA derivative (IC50 =0.145 μM). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phosphatidylinositol 4-kinase IIIβ is a cellular lipid kinase pivotal to pathogenesis of various RNA viruses. These viruses hijack the enzyme in order to modify the structure of intracellular membranes and use them for the construction of functional replication machinery. Selective inhibitors of this enzyme are potential broad-spectrum antiviral agents, as inhibition of this enzyme results in the arrest of replication of PI4K IIIβ-dependent viruses. Herein, we report a detailed study of novel selective inhibitors of PI4K IIIβ, which exert antiviral activity against a panel of single-stranded positive-sense RNA viruses. Our crystallographic data show that the inhibitors occupy the binding site for the adenine ring of the ATP molecule and therefore prevent the phosphorylation reaction.

6-Chloropurines substituted at the position 9 with variously modified bicyclic skeletons represent promising antiviral and anticancer agents. This work aimed to investigate the transport mechanisms of 9-[(1R*,2R*,4S*)-bicyclo[2.2.1]hept-2-yl]-6-chloro-9H-purine (9-norbornyl-6-chloropurine, NCP) and their relationship to the metabolism and biological activity of the compound. Transport experiments were conducted in CCRF-CEM cells using radiolabeled compound ([(3)H]NCP). The pattern of the intracellular uptake of [(3)H]NCP in CCRF-CEM cells pointed to a combination of passive and facilitated diffusion as prevailing transport mechanisms. NCP intracellular metabolism was found to enhance its uptake by modifying NCP concentration gradient. The transport kinetics reached steady state under the conditions of MRP and MDR proteins blockade, indicating that NCP is a substrate for these efflux pumps. Their inhibition also increased the cytotoxicity of NCP. Our findings suggest that the novel nucleoside analog NCP has potential to become a new orally available antileukemic agent due to its rapid membrane permeation.

The synthesis of a novel library of purine derivatives bearing various bicyclic and polycylic substituents at the N-9 position is described. The series includes norbornanes, bicyclo[2.2.2]octanes, and bicyclo[3.2.1]octanes attached at the bridgehead position as well as bicyclo[3.1.1]heptanes, tetrahydro-1-naphthalenes, and adamantanes bonded either directly or via a linear chain to the 6-chloropurine nucleobase. A number of prepared derivatives exerted significant activity against the enterovirus. Despite attempts to correlate the activity against picornaviruses with their phosphatidylinositol 4-kinase KIIIβ inhibitory activity, it is clear that the inhibition of this host factor cannot explain the observed antiviral potency., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Adenylate cyclase toxin (ACT) is the key virulence factor of Bordetella pertussis that facilitates its invasion into the mammalian body. 9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the active metabolite of the antiviral drug bis(POM)PMEA (adefovir dipivoxil), has been shown to inhibit ACT. The objective of this study was to evaluate six novel amidate prodrugs of PMEA, both phenyloxy phosphonamidates and phosphonodiamidates, for their ability to inhibit ACT activity in the J774A.1 macrophage cell line. The two phenyloxy phosphonamidate prodrugs exhibited greater inhibitory activity (50% inhibitory concentration [IC50] = 22 and 46 nM) than the phosphonodiamidates (IC50 = 84 to 3,960 nM). The inhibitory activity of the prodrugs correlated with their lipophilicity and the degree of their hydrolysis into free PMEA in J774A.1 cells. Although the prodrugs did not inhibit ACT as effectively as bis(POM)PMEA (IC50 = 6 nM), they were significantly less cytotoxic. Moreover, they all reduced apoptotic effects of ACT and prevented an ACT-induced elevation of intracellular [Ca(2+)]i. The amidate prodrugs were less susceptible to degradation in Caco-2 cells compared to bis(POM)PMEA, while they exerted good transepithelial permeability in this assay. As a consequence, a large amount of intact amidate prodrug is expected to be available to target macrophages in vivo. This feature makes nontoxic amidate prodrugs attractive candidates for further investigation as novel antimicrobial agents.

Aim: 6-Chloropurines substituted at position 9 with bicyclic skeletons represent promising chemotherapeutic agents. We explored the metabolism and membrane transport of 9-norbornyl-6-chloropurine (NCP) aiming to understand its mechanism of action., Materials and Methods: The metabolism of NCP was studied in vitro in whole cells (CCRF-CEM), cellular extracts, subcellular fractions and purified enzymes. Transport experiments were conducted in Caco-2 cell monolayers., Results: Three metabolites were identified, a glutathione conjugate (NCP-GS), NCP-cysteinylglycine and NCP-cysteine. Both glutathione-S-transferase inhibition and glutathione (GSH) depletion prevented metabolite formation and increased the cytotoxicity of NCP. Transepithelial transport (Caco-2) indicated good permeability, with Papp (12.6±0.3) ×10(-5) cm/s. Importantly, the drug induced glutathione depletion in treated cells and affected the activity of several GSH-dependent enzymes., Conclusion: The novel nucleoside analog NCP represents a promising orally available antileukemic agent, acting through lowering of GSH levels in tumor cells.

The oxidation reactions of 5-aminopyrimidine derivatives in dimethyl sulphoxide (DMSO) were studied. The DMSO solutions of the studied compounds became deeply coloured within a few hours or days. The oxidation products can undergo further condensation reactions with the starting pyrimidines to yield bipyrimidines and/or pyrimidopteridines. The reaction mechanism of the oxidation-condensation reaction was also supported by reactions of the 5-aminopyrimidines with alloxan (2,4,5,6-tetraoxopyrimidine). DMSO is often used as the solvent in in vitro tests of biological activities, but it is also an oxidising agent and may react with solute molecules and significantly affect the quality of the generated biochemical data., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: 9-[2-(phosphonomethoxy)ethyl] guanine (PMEG) is a nucleotide analogue with anticancer activity. Here we investigate the role of ERK, p38, JNK and AKT kinases in PMEG-induced apoptosis., Materials and Methods: CCRF-CEM and HL-60 leukemia cells were used to assess MAPK mRNA and protein expression in PMEG-treated cells. MAPK activation was measured using phospho-specific antibodies. Apoptosis was evaluated by caspase-3 and PARP cleavage., Results: Up-regulation of p38β, γ and δ mRNA were observed following PMEG treatment of CCRF-CEM cells, however, the total protein expression remained unchanged. Neither PMEG nor its analogue 9-[2-(phosphonomethoxy) ethyl]-2,6-diaminopurine (PMEDAP) induced p38 kinase phosphorylation in CCRF-CEM cells, whereas increased p38 phosphorylation was observed in HL-60 cells. The ERK pathway was also activated by these compounds. Pretreatment of the cells with the p38 inhibitor SB203580 diminished drug-induced apoptosis whereas inhibition of ERK, JNK or AKT pathways did not. [corrected]., Conclusion: PMEG- and PMEDAP-induced. [corrected].

The aminopyrimidine structural motif can be found in diverse biologically active compounds. This study aimed to describe the antioxidant activity of a series of di- and tri-substituted 5-aminopyrimidines using in vitro (TEAC, LPO) and cell-based assays. 2,4,6-trisubstituted 5-aminopyrimidines displayed the highest activity in the TEAC and LPO assays whereas compounds with protected 5-aminogroup were active in the cellular assay. This is most likely because of their better membrane permeability and intracellular metabolic activation. In summary, we have identified the antioxidant activity of a series of substituted 5-aminopyrimidines and their potential prodrugs which may have implications in the treatment of oxidative stress-related diseases., (© 2012 Informa UK, Ltd.)

Coxsackievirus and related enteroviruses are important human pathogens that cause various diseases with clinical manifestations ranging from trivial flu-like syndromes to dangerous or even fatal diseases such as myocarditis, meningitis and encephalitis. Here, we report on our continuous SAR study focused on 9-(bicyclo[2.2.1]hept-2-yl)-9H-purines as anti-enteroviral inhibitors. The purine moiety was modified at positions 2, 6 and 8. Several analogues inhibited Coxsackievirus B3 as well as other enteroviruses at low-micromolar concentrations. The 6-chloropurine derivative was confirmed as the most active compound in this series., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Acyclic nucleotide analogue PMEG represents promising drug candidate against lymphomas. In the present work we describe the ability of PMEG to induce resistance and we elucidate the mechanisms involved in this process. CCRF-CEM T-lymphoblastic cells resistant to either PMEG or its 6-amino congener PMEDAP were prepared and assayed for the expression of membrane transporters, PMEG and PMEDAP uptake and intracellular metabolism. Genes for guanylate kinase (GUK) and adenylate kinase (AK) isolated from PMEG- and PMEDAP-resistant cells were sequenced and cloned into mammalian expression vectors. PMEG-resistant cells were transfected with GUK vectors and catalytic activities of GUKs isolated from PMEG-sensitive and resistant cells were compared. PMEG phosphorylation to PMEG mono- and diphosphate was completely impaired in resistant cells. GUK obtained from PMEG-resistant cells revealed two point mutations S(35)N V(168)F that significantly suppressed its catalytic activity. Transfection of resistant cells with wtGUK led to the recovery of phosphorylating activity as well as sensitivity towards PMEG cytotoxicity. No differences in PMEG uptake have been found between sensitive and resistant cells. In contrast to GUK no changes in primary sequence of AK isolated from PMEDAP resistant cells were identified. Therefore, resistance induced by PMEDAP appears to be conferred by other mechanisms. In conclusion, we have identified GUK as the sole molecular target for the development of acquired resistance to the cytotoxic nucleotide PMEG. Therefore, PMEG is unlikely to cause cross-resistance in combination therapeutic protocols with most other commonly used anticancer drugs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Restoration of transcriptionally silenced genes by means of methyltransferases inhibitors plays a crucial role in the current therapy of myelodysplastic syndromes and certain types of leukemias. A comparative study of hypomethylating activities of a series of 5-azacytidine nucleosides: 5-azacytidine (AC), 2'-deoxy-5-azacytidine (DAC) and its α-anomer (α-DAC), 5,6-dihydro-5-azacytidine (DHAC), 2'-deoxy-5,6-dihydro-5-azacytidine (DHDAC, KP-1212) and its α-anomer (α-DHDAC), and of a 2-pyrimidone ribonucleoside (zebularine) was conducted. Methylation-specific PCR was employed to detect the efficiency of individual agents on cyclin-dependent kinase inhibitor 2B and thrombospondin-1 hypermethylated gene loci. Overall changes in DNA methylation level were quantified by direct estimation of 5-methyl-2'-deoxycytidine-5'-monophosphate by HPLC using digested genomic DNA. Flow cytometric analysis of cell cycle progression and apoptotic markers was used to determine cytotoxicity of the compounds. mRNA expression was measured using qRT-PCR. 2'-deoxy-5,6-dihydro-5-azacytidine was found to be less cytotoxic and more stable than 2'-deoxy-5-azacytidine at the doses that induce comparable DNA hypomethylation and gene reactivation. This makes it a valuable tool for epigenetic research and worth further investigations to elucidate its possible therapeutic potential.

The clinical usefulness of anthracycline antineoplastic drugs is limited by their cardiotoxicity. Its mechanisms have not been fully understood, although the induction of oxidative stress is widely believed to play the principal role. Glutathione is the dominant cellular antioxidant, while glutathione peroxidase (GPx) together with glutathione reductase (GR) constitutes the major enzymatic system protecting the cardiac cells from oxidative damage. Therefore, this study aimed to assess their roles in anthracycline cardiotoxicity. Ten-week intravenous administration of daunorubicin (DAU, 3 mg/kg weekly) to rabbits induced heart failure, which was evident from decreased left ventricular ejection fraction and release of cardiac troponins to circulation. However, no significant changes in either total or oxidized glutathione contents or GR activity were detected in left ventricular tissue of DAU-treated rabbits when compared with control animals. GPx activity in the cardiac tissue significantly increased. In H9c2 rat cardiac cells, 24-h DAU exposure (0.1-10 μM) induced significant dose-dependent toxicity. Cellular content of reduced glutathione was insignificantly decreased, oxidized glutathione and GR activity were unaffected, and GPx activity was significantly increased. Neither buthionine sulfoximine (BSO, glutathione biosynthesis inhibitor) nor 2-oxo-4-thiazolidine-carboxylic acid (OTC, glutathione biosynthetic precursor) had significant effects on DAU cytotoxicity. This contrasted with model oxidative injury induced by hydrogen peroxide, which cytotoxicity was increased by BSO and decreased by OTC. In conclusion, our results suggest that the dysfunction of glutathione antioxidant system does not play a causative role in anthracycline cardiotoxicity.

We have previously shown that PMEG diphosphate (PMEGpp) and PMEDAP diphosphate (PMEDAPpp) inhibit the enzymatic activity of human telomerase in a cell-free assay. Here, we investigated the ability of PMEG and PMEDAP to induce telomere shortening and telomerase inhibition at both transcriptional and activity level in T-lymphoblastic leukemia cells CCRF-CEM and MOLT-4. At defined time points (3days and 9weeks), the telomerase activity and relative levels of hTERT and c-myc mRNA were determined using real-time RT-PCR. Telomere length was measured by the flow-FISH method. Both PMEDAP and PMEG induced telomere shortening in CCRF-CEM cells after 9weeks of exposure by 50% and 20%, respectively, without major impairment of telomerase activity. The effect of the tested compounds on telomere length in MOLT-4 cells was the opposite, with telomere elongation by 50% and 40% after 9-week treatment with PMEDAP and PMEG, respectively. At this time point, telomerase activity in MOLT-4 cells appeared to be slightly higher than that of CCRF-CEM cells, nevertheless no correlation between telomerase activity and telomere length was found. Both compounds down-regulated the expression of hTERT and c-myc mRNA in CCRF-CEM and MOLT-4 cells at 72h in a concentration-dependent manner while prolonged exposure to PMEG or PMEDAP for 9weeks had weaker effects. In conclusion, PMEDAP and PMEG are able to modulate telomere length in leukemic cells and this effect is cell-type specific. It is neither due to direct telomerase inhibition nor impairment of hTERT expression and it is likely to be telomerase-independent., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Background/aim: 9-[2-(phosphonomethoxy)ethyl] guanine (PMEG) is a guanine acyclic nucleotide analog whose targeted prodrugs are being investigated for chemotherapy of lymphomas. Its antiproliferative effects have been attributed to cell cycle arrest and induction of apoptosis, however, the underlying mechanisms remain poorly understood. The objective of this study was to determine the requirements for caspase and CD95/Fas activation in PMEG-induced apoptosis. Additionally, the influence of PMEG on cell cycle regulatory proteins was explored., Materials and Methods: CCRF-CEM cells were exposed to PMEG with/without caspase inhibitor or anti-Fas blocking antibody and assayed for phosphatidyl serine externalization, mitochondrial depolarization and the cleavage of procaspase 3 and the nuclear protein poly (ADP-ribose) polymerase (PARP)., Results: Despite an observed increase of caspase 3, 8 and 9 proteolytic activity, neither pretreatment of the cells with cell-permeable caspase inhibitors nor blocking the death receptor with anti-Fas antibody did prevent apoptosis induced by PMEG., Conclusion: PMEG-induced apoptosis is caspase- and CD95/Fas-independent.