Development of a Potent and Selective G2A (GPR132) Agonist.

G protein-coupled receptor G2A was postulated to be a promising target for the development of new therapeutics in neuropathic pain, acute myeloid leukemia, and inflammation. However, there is still a lack of potent, selective, and drug-like G2A agonists to be used as a chemical tool or as the starting matter for the development of drugs. In this work, we present the discovery and structure-activity relationship elucidation of a new potent and selective G2A agonist scaffold. Systematic optimization resulted in (3-(pyridin-3-ylmethoxy)benzoyl)-d-phenylalanine (T-10418) exhibiting higher potency than the reference and natural ligand 9-HODE and high selectivity among G protein-coupled receptors. With its favorable activity, a clean selectivity profile, excellent solubility, and high metabolic stability, T-10418 qualifies as a pharmacological tool to investigate the effects of G2A activation.

Antiviral activity of natural phenolic compounds in complex at an allosteric site of SARS-CoV-2 papain-like protease.

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host's innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

High-Throughput Screening for CEBPD-Modulating Compounds in THP-1-Derived Reporter Macrophages Identifies Anti-Inflammatory HDAC and BET Inhibitors.

This study aimed to identify alternative anti-inflammatory compounds that modulate the activity of a relevant transcription factor, CCAAT/enhancer binding protein delta (C/EBPδ). C/EBPδ is a master regulator of inflammatory responses in macrophages (Mϕ) and is mainly regulated at the level of CEBPD gene transcription initiation. To screen for CEBPD-modulating compounds, we generated a THP-1-derived reporter cell line stably expressing secreted alkaline phosphatase (SEAP) under control of the defined CEBPD promoter (CEBPD::SEAP). A high-throughput screening of LOPAC®1280 and ENZO®774 libraries on LPS- and IFN-γ-activated THP-1 reporter Mϕ identified four epigenetically active hits: two bromodomain and extraterminal domain (BET) inhibitors, I-BET151 and Ro 11-1464, as well as two histone deacetylase (HDAC) inhibitors, SAHA and TSA. All four hits markedly and reproducibly upregulated SEAP secretion and CEBPD::SEAP mRNA expression, confirming screening assay reliability. Whereas BET inhibitors also upregulated the mRNA expression of the endogenous CEBPD, HDAC inhibitors completely abolished it. All hits displayed anti-inflammatory activity through the suppression of IL-6 and CCL2 gene expression. However, I-BET151 and HDAC inhibitors simultaneously upregulated the mRNA expression of pro-inflammatory IL-1ß. The modulation of CEBPD gene expression shown in this study contributes to our understanding of inflammatory responses in Mϕ and may offer an approach to therapy for inflammation-driven disorders.

A SARS-CoV-2 cytopathicity dataset generated by high-content screening of a large drug repurposing collection.

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic, in which acute respiratory infections are associated with high socio-economic burden. We applied high-content screening to a well-defined collection of 5632 compounds including 3488 that have undergone previous clinical investigations across 600 indications. The compounds were screened by microscopy for their ability to inhibit SARS-CoV-2 cytopathicity in the human epithelial colorectal adenocarcinoma cell line, Caco-2. The primary screen identified 258 hits that inhibited cytopathicity by more than 75%, most of which were not previously known to be active against SARS-CoV-2 in vitro. These compounds were tested in an eight-point dose response screen using the same image-based cytopathicity readout. For the 67 most active molecules, cytotoxicity data were generated to confirm activity against SARS-CoV-2. We verified the ability of known inhibitors camostat, nafamostat, lopinavir, mefloquine, papaverine and cetylpyridinium to reduce the cytopathic effects of SARS-CoV-2, providing confidence in the validity of the assay. The high-content screening data are suitable for reanalysis across numerous drug classes and indications and may yield additional insights into SARS-CoV-2 mechanisms and potential therapeutic strategies.

Identification of Inhibitors of SARS-CoV-2 3CL-Pro Enzymatic Activity Using a Small Molecule in Vitro Repurposing Screen.

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro and have identified 62 additional compounds with IC50 values below 1 µM and profiled their selectivity toward chymotrypsin and 3CL-Pro from the Middle East respiratory syndrome virus. A subset of eight inhibitors showed anticytopathic effect in a Vero-E6 cell line, and the compounds thioguanosine and MG-132 were analyzed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Å, showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.

Cytostatic resistance profile of the sulfur mustard resistant keratinocyte cell line HaCaT/SM.

BACKGROUND: The cell line HaCaT/SM was developed as a sulfur mustard (SM) resistant cell line from the human keratinocyte cell line HaCaT. This cell line was established to learn more about the effect of SM and possible therapeutic approaches to counteract the cytotoxic effects of SM. The aim of this study was to clarify whether the SM-resistant cell line HaCaT/SM exhibit also resistance to other alkylating agents or cytotoxic drugs with different mechanism of action. MATERIAL AND METHOD: The chemosensitivity of SM-resistant human keratinocyte cell line HaCaT/SM and the original cell line HaCaT were tested using the XTT assay. Nine cytotoxic drugs from five different substance groups were investigated. RESULTS: HaCaT/SM showed a significant increase in resistance against all tested drugs. From the substance class of the alkylating agents, HaCaT/SM showed the strongest resistance increase against chlorambucil (1.7 fold increase). Whereas over all substances strongest increase was observed against cisplatin (5.1 fold increase). DISCUSSION: The highest resistance was observed for cisplatin. The SM resistant cells revealed changes in the miRNA profile as described before. The resistance to cisplatin is also connected to a specific miRNA profile. Interestingly, changes of miRNA-203 and miRNA-21 levels were found in HaCaT/SM as well as in cisplatin resistant cells. It is therefore conceivable that the same resistance pathways are involved for both substances.

Sulfur mustard resistant keratinocytes obtained elevated glutathione levels and other changes in the antioxidative defense mechanism.

BACKGROUND: Sulfur mustard (SM) is a potent blistering chemical warfare agent, which was first used in 1917. Despite the Chemical Weapons Convention, a use was recently reported in Syria in 2015. This emphasizes the importance to develop countermeasures against chemical warfare agents. Despite intensive research, there is still no antidote or prophylaxis available against SM. METHODS: The newly developed SM-resistant keratinocyte cell line HaCaT/SM was used to identify new target structures for drug development, particularly the adaptations in protective measures against oxidative stress. For this purpose, glutathione (GSH) and NAD(P)H levels, the effect of glutathione S-transferase (GST) inhibition as well as activation and expression of Nrf2, GST, glutamate cysteine ligase (GCL) and glutathione-disulfide reductase (GSR) as well as multi-drug resistance (MDR) proteins 1, 3 and 5 were investigated. RESULTS: The HaCaT/SM cells showed not only a better survival after treatment with SM or cytostatic drugs, but also hydrogen peroxide (H2O2). They exhibit more GSH even after SM treatment. Nrf2 levels were significantly lower. Inhibition of GST led to significantly decreased, activation to slightly higher IC50 values after SM treatment and a lower expression of GST was observed. The cells also expressed less GCLC and GSR. Expression of MDR1, MDR3 and MDR5 was higher under control conditions, but less stimulated by SM treatment. An increased NADP+/NADPH ratio as well as higher NAD+ levels were shown. CONCLUSION: In summary, an improved response of the resistant cell line to oxidative stress was observed. The underlying mechanisms are elevated GSH levels as well as lower expression of Nrf2 and its targets GCLC and GST as well as GSR and MDR1, MDR3 and MDR5. GST is an especially interesting target because its inhibition already induced a significant SM sensitivity. SM resistance also caused redox equivalent level differences. Taken together, these findings provide further insight into the mechanism of SM resistance and may open a window for novel therapeutic targets in SM therapy.

Development and implementation of a cell-based assay to discover agonists of the nuclear receptor REV-ERBα.

The nuclear receptors are transcription factors involved in the regulation of a variety of physiological processes whose activity can be modulated by binding to relevant small molecule ligands. Their dysfunction has been shown to play a role in disease states such as diabetes, cancer, inflammatory diseases, and hormonal resistance ailments, which makes them interesting targets for drug discovery. The nuclear receptor REV-ERBα is involved in regulating the circadian rhythm and metabolism. Its natural ligand is heme and there is significant interest in identifying novel synthetic modulators to serve as tools to characterize its function and to serve as drugs in treating metabolic disorders. To do so, we established a mammalian cell-based two-hybrid assay system capable of measuring the interaction between REV-ERBα and its co-repressor, nuclear co-repressor 1. This assay was validated to industry standard criteria and was used to screen a subset of the LOPAC®1280 library and 29568 compounds from a diverse compound library. Profiling of the primary hits in a panel of counter and selectivity assays confirmed that REV-ERBα activity can be modulated pharmacologically and chemical scaffolds have been identified for optimization.

Alteration of miRNA expression in a sulfur mustard resistant cell line.

BACKGROUND: MicroRNAs (miRNAs) are responsible for post-transcriptional control of protein expression. Numerous miRNAs have been identified to be responsible for the resistance of tumor cells to cytostatic drugs. Possibly, the same miRNAs also play a role in the sulfur mustard (SM)-resistance of the keratinocyte cell line HaCaT/SM as alkylating cytostatics exhibit similar cytotoxic effects as SM. METHODS: Basal expression levels of 1920 miRNAs in total were analyzed in HaCaT/SM compared to the origin human keratinocyte cell line HaCaT. The effect for selected miRNAs on cell survival was analyzed using antagomirs for ectopic miRNA level decrease or miRNA mimics for increase. Cell survival was calculated as SM dose-dependent-curves. RESULTS: Out of 1920 miRNAs analyzed, 49 were significantly up- and 29 were significantly downregulated in HaCaT/SM when compared to HaCaT controls. Out of these, 36 could be grouped in miRNA families. Most of the 15 miRNA family members showed either a common increase or decrease. Only the members of miR-10, miR-154, miR-430 and miR-548 family showed an inconsistent picture. The ectopic increase of miR-181 in HaCaT/SM had a positive effect on cell survival in the presence of SM. CONCLUSION: In summary, the extensive differences in miRNA expression pattern between these cell lines indicate that specific miRNAs may play a role in the resistance mechanism against sulfur mustard. The miR-125b-2 and miR-181b alone are not responsible for the resistance development against SM, but an ectopic increase of miR-181 even enhances the SM resistance of HaCaT/SM. Improving the resistance in normal keratinocytes by treatment with either both miRNAs together or a different combination might be used as an initial step in development of an innovative new drug or prophylactic agent against SM.

Establishing the Secondary Metabolite Profile of the Marine Fungus: Tolypocladium geodes sp. MF458 and Subsequent Optimisation of Bioactive Secondary Metabolite Production.

As part of an international research project, the marine fungal strain collection of the Helmholtz Centre for Ocean Research (GEOMAR) research centre was analysed for secondary metabolite profiles associated with anticancer activity. Strain MF458 was identified as Tolypocladium geodes, by internal transcribed spacer region (ITS) sequence similarity and its natural product production profile. By using five different media in two conditions and two time points, we were able to identify eight natural products produced by MF458. As well as cyclosporin A (1), efrapeptin D (2), pyridoxatin (3), terricolin A (4), malettinins B and E (5 and 6), and tolypocladenols A1/A2 (8), we identified a new secondary metabolite which we termed tolypocladenol C (7). All compounds were analysed for their anticancer potential using a selection of the NCI60 cancer cell line panel, with malettinins B and E (5 and 6) being the most promising candidates. In order to obtain sufficient quantities of these compounds to start preclinical development, their production was transferred from a static flask culture to a stirred tank reactor, and fermentation medium development resulted in a nearly eight-fold increase in compound production. The strain MF458 is therefore a producer of a number of interesting and new secondary metabolites and their production levels can be readily improved to achieve higher yields.

Methoxylated 2'-hydroxychalcones as antiparasitic hit compounds.

Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2'-hydroxy methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Among the synthesized library, compounds 1, 3, 4, 7 and 8 were the most potent and selective anti-T. brucei compounds (EC50 = 1.3-4.2 µM, selectivity index >10-fold). Compound 4 showed the best early-tox and antiparasitic profile. The pharmacokinetic studies of compound 4 in BALB/c mice using hydroxypropil-ß-cyclodextrins formulation showed a 7.5 times increase in oral bioavailability.

Patent Foramen Ovale and Cryptogenic Strokes in the Stroke in Young Fabry Patients Study.

BACKGROUND AND PURPOSE: A patent foramen ovale (PFO) is disproportionately prevalent in patients with cryptogenic stroke. Without alternative explanations, it is frequently considered to be causative. A detailed stratification of these patients may improve the identification of incidental PFO. METHODS: We investigated the PFO prevalence in 3497 transient ischemic attack and ischemic stroke patients aged 18 to 55 years in the prospective multicenter SIFAP1 study (Stroke in Young Fabry Patients 1) using the ASCO classification. Patients without an obvious cause for transient ischemic attack/stroke (ASCO 0) were divided into subgroups with and without vascular risk factors (ASCO 0+ and 0-). In addition, we looked for PFO-related magnetic resonance imaging lesion patterns. RESULTS: PFO was identified in 25% of patients. Twenty percent of patients with a definite or probable cause of transient ischemic attack/stroke (≥1 grade 1 or 2 ASCO criterion; n=1769) had a PFO compared with 29% of cryptogenic stroke patients (ASCO 0 and 3; n=1728; P<0,001); subdivision of cryptogenic strokes revealed a PFO in 24% of 978 ASCO 3 patients (n.s. versus ASCO 1 and 2) and a higher prevalence of 36% in 750 ASCO 0 cases (P<0.001 versus ASCO 3 and versus ASCO 1 and 2). PFO was more commonly observed in ASCO 0- (n=271) than in ASCO 0+ patients (n=479; 48 versus 29%; P<0.001). There was no PFO-associated magnetic resonance imaging lesion pattern. CONCLUSIONS: Cryptogenic stroke patients demonstrate a heterogeneous PFO prevalence. Even in case of less conclusive diseases like nonstenotic arteriosclerosis, patients should preferentially be considered to have a non-PFO-mediated stroke. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00414583.

VCP/p97 Extracts Sterically Trapped Ku70/80 Rings from DNA in Double-Strand Break Repair.

During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.

Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs.

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 µM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 µM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

The potential use of spectral electromyographic fatigue as a screening and outcome monitoring tool of sarcopenic back muscle alterations.

BACKGROUND: To examine whether or not median frequency surface electromyographic (MF-EMG) back muscle fatigue monitoring would be able to identify alterations in back muscle function in elderly muscles, if a protocol was used that allowed optimum standardization of the processes underlying electromyographic fatigue, and whether these tests were reliable from day to day. METHODS: A total of 42 older (21 females; 67 (±10.5) years old) and 44 younger persons (19 females; 33 (±10) years) performed maximum isometric back extensions which were followed by one 30 s lasting 80% submaximum extension. Participants were seated on a dynamometer with their trunks 30° anteflexed, and they repeated all tests after 1-2 days and 6 weeks. SEMG was recorded bilaterally from the L1 (iliocostalis lumborum), L2 (longissimus), and L5 (multifidus) recording sites. Outcome variables included maximum back extension torque, initial MF-EMG (IMF-EMG), MF-EMG slope declines, and individual MF-EMG muscular imbalance scores. Two-factorial ANOVAs served to examine the age and gender-specific effects, and models from Generalizability Theory (G-Theory) were used for assessing retest-reliability. RESULTS: Maximum back extension moment was non-significantly smaller in elders. IMF-EMG was overall higher in elders, with significant differences at the L5 recordings sites. In the elderly, MF-EMG fatigue declines were significantly smaller in L5, in the recording with the most negative slope, or if the slope of all electrodes was considered. Retest reliability was unanimous in young and older persons. ICC-type measurements from G-Theory of both the IMF and the fatigue slopes ranged from 0.7 to 0.85. Absolute SEM values were found clinically acceptable for the IMF-EMG, but relatively high for the fatigue slope declines. CONCLUSIONS: The MF-EMG fatigue method is able to elucidate alterations of aging back muscles. This method, thus, might be suggested as a potential biomarker to objectively identify persons at risk for sarcopenia. Considering the clinical relevance of the IMF-EMG relative to the MF-EMG slope declines, spectral EMG may also be used as an outcome monitoring tool in elderly populations.

Optimisation and validation of a high throughput screening compatible assay to identify inhibitors of the plasma membrane calcium ATPase pump--a novel therapeutic target for contraception and malaria.

PURPOSE: ATPases, which constitute a major category of ion transporters in the human body, have a variety of significant biological and pathological roles. However, the lack of high throughput assays for ATPases has significantly limited drug discovery in this area. We have recently found that the genetic deletion of the ATP dependent calcium pump PMCA4 (plasma membrane calcium/calmodulin dependent ATPase, isoform 4) results in infertility in male mice due to a selective defect in sperm motility. In addition, recent discoveries in humans have indicated that a single nucleotide polymorphism (SNP) in the PMCA4 gene determines the susceptibility towards malaria plasmodium infection. Therefore, there is an urgent need to develop specific PMCA4 inhibitors. In the current study, we aim to optimise and validate a high throughput screening compatible assay using recombinantly expressed PMCA4 and the HTRF® Transcreener® ADP (TR-FRET) assay to screen a drug library. METHODS AND RESULTS: PMCA4 membrane microsomes were prepared from HEK293 cells overexpressing PMCA4. Western blot quantification revealed nearly nine-fold increased expression of PMCA4 compared to LacZ (control virus)-infected cells. Maximal PMCA4 microsomal activity was achieved in the TR-FRET assay with 15ng/µl microsomal concentration, 30-minute pre-incubation with compounds at 37°C, and calcium buffering with 1mM EGTA providing 1µM free-calcium. Finally a dose-response curve for carboxyeosin (a non-specific PMCA inhibitor) under optimised conditions showed significant PMCA4 inhibition. Upon confirmation that the assay was suitable for high-throughput screening, we have screened the ChemBioNet small molecule library (~21,000 compounds) against the PMCA4 assay to identify those that are its apparent inhibitors. This screening yielded 1,494 primary hits. CONCLUSIONS: We have optimised the HTRF® Transcreener® ADP assay for high-throughput screening to identify PMCA4 inhibitors. The output of the screening campaign has provided preliminary chemical starting points that could be further developed to specific PMCA4 inhibitors for non-hormonal contraception or anti-malaria therapy.

Influence of matrigel on biodistribution studies in cancer research.

Matrigel matrix is widely used for supporting tumour implantation. However, some tumour cells are unable to degrade matrigel matrix resulting in residual matrigel at the time point of the biodistribution study or therapy experiment. In vitro cell uptake of tumour affine compounds into tumour cells embedded in matrigel was compared with matrigel free tumour cells. Matrigel accumulation exceeded cellular uptake of the tumour affine peptides. This suggests that matrigel might have an influence on the acquired biodistribution data when it is still present at the time point of the study. A quantitation of residual matrigel in tumour explants fourteen days after tumour implantation with a matrigel-tumour cell mixture showed that the overall matrigel content in the case of MCF-7 and AR42J tumours was about 23%. In order to evaluate the extent of accumulation of compounds in matrigel, nude mice bearing either tumours, tumour-matrigel-mixtures or matrigel alone received intravenous injections of fluorophor tagged tumour specific peptides. Fluorescence microscopy of cryosectioned matrigel, matrigel-tumour mixtures and tumour explants showed that the labelled compounds were matrigel associated and tumour cell associated with a higher fluorescence intensity in matrigel. In summary, matrigel matrix can influence biodistribution studies. It leads to believe in a higher tumour accumulation. Therefore, either a number of control experiments or the separation of matrigel from the tumour is necessary in order to obtain correct biodistribution data.

Role of acidic cell organelles in the higher nonmelanoma retention of melanoma markers based on N-(2-dialkylaminoethyl)benzamides and the cytotoxicity of alkylating benzamides.

Melanoma markers based on both N-(2-dialkylaminoethyl)benzamides and lysosomotropic agents comprise a N-(2-dialkylaminoethyl)aminocarbamoyl pharmacophore, suggesting that benzamides and lysosomotropic probes should show affinity to melanoma and acidic cell organelles. We prepared novel fluorescent N-(2-dialkylaminoethyl)benzamides to prove this presumption. Lysosomotropic probes showed a melanin affinity comparable to benzamides. Lysosomal markers and benzamides colocalized in acidic organelles. Various nonmelanoma cell lines showed equal benzamide uptake and retention compared with melanoma cells. In nonmelanoma cells the amount of retained benzamides correlates with the number of acidic cell organelles. Benzamides almost completely failed to accumulate in melanoma cells with neutralized acidic organelles but normal melanin content. In melanoma retention of benzamides, acidic cell organelles are the main determinant. N-(2-dialkylaminoethyl)benzamides are lysosomotropic probes with high accumulation in nonmelanoma tumors with many acidic cell organelles. Alkylating benzamides were reported previously to show a melanoma unselective, in general enhanced cytotoxicity. Alkylating benzamides can act as lysosomotropic detergents or as DNA alkylators. The ability of alkylating benzamides to disrupt the membrane of lysosomes and cause liberation of lysosomal-trapped fluorescent dyes was demonstrated by fluorescence microscopy. Whether they act as an alkylating agent or a lysosomotropic detergent in a specific cell line is dependent on the amount of acidic cell organelles. In cell lines with small amounts of acidic cell organelles alkylating benzamides act as alkylating agents, whereas in cell lines with many acidic cell organelles they act as lysosomotropic detergents. In cell lines with high amounts of acidic cell organelles they do not reach the nucleus.

Polyamine-substituted gadolinium chelates: a new class of intracellular contrast agents for magnetic resonance imaging of tumors.

A new class of intracellular contrast agents (CA) for magnetic resonance imaging has been developed, based on Gd(DTPA) with two positively charged amide-linked substituents. Uptake of Gd(DTPA) into cultured tumor cell lines (B16 mouse melanoma, MH3924A Morris hepatoma) was below the detection limit while CA with the melanin-binding pharmacophore 2-(diethylamino)ethylamine reached intracellular concentrations of ca. 0.03 fmol/cell (ca. 20 microM) for melanoma and 0.02 fmol/cell for hepatoma (24 h at 10 microM CA). With the polyamine substituents bis(2-aminoethyl)amine or spermidine, CA uptake increased up to 3-fold for melanoma (0.083 fmol/cell) and 9-fold for hepatoma (0.18 fmol/cell). Uptake of polyamine-substituted CA was reduced by the polyamine transport inhibitor benzyl viologen. Molar relaxivities for three Gd-DTPA-polyamine complexes were in the range 5.6-6.9 for the free complex in solution and 7.7-23.5 s-1 mM-1 for Morris hepatoma cell pellets. T1-weighted magnetic resonance imaging at 2.35 T of rats with MH3924A tumors showed contrast enhancement in tumor at 1 and 24 h postinjection of polyamine-substituted CA.

Alkylating benzamides with melanoma cytotoxicity: experimental chemotherapy in a mouse melanoma model.

The in-vivo antineoplastic potential of the alkylating N-(2-dialkylaminoethyl)benzamides BZA1 and BZA2, novel melanoma targeted anticancer drugs, was evaluated in a mouse melanoma model with nude mice bearing subcutaneous SkMel28, B16 or WM266-4. The maximal tolerated dose (MTD) for the intraperitoneal application of both agents was found to be 24 mg/kg. Treatment was initiated with an intraperitoneal injection of 8 mg/kg of BZA1 or BZA2 on days 0, 2 and 4 in the case of B16 melanoma on days 0, 1 and 2 after the onset of the experiment, when the mean tumor diameter ranged within 4-6 mm. The experiment was terminated when the mean tumor diameter in the control group had reached a value of 12 mm. Tumor growth delay of these agents was compared with dacarbazine (3x250 mg/kg), chlorambucil (3x5 mg/kg) and an untreated control group. Significant tumor growth delay was observed under BZA1, BZA2 and dacarbazine treatment compared with the untreated control group in all three evaluated melanomas with insignificant differences among BZA1, BZA2 and dacarbazine. The insignificant effect of chlorambucil and the strong improvement on growth delay achieved with BZA1 and BZA2 demonstrated melanoma targeting characteristics of the N-(2-dialkylaminoethyl)benzamide structure element. Dacarbazine was more effective in the in-vivo antineoplastic assay compared with the in-vitro cytotoxicity studies, probably due to hepatic bioactivation. Similar side effect intensity of BZA2 and dacarbazine was observed, whereas BZA1 was more toxic. BZA2 might represent an alternative antimelanoma drug, especially in patients not responding to dacarbazine.