Optimised approach to albumin-drug conjugates using monobromomaleimide-C-2 linkers.

Conjugation of therapeutics to human serum albumin (HSA) using bromomaleimides represents a promising platform for half-life extension. We show here that the Cys-34 crevice substantially reduces the rate of serum stabilising maleimide hydrolysis in these conjugates, necessitating reagent optimisation. This improved reagent design is applied to the construction of an HSA-paclitaxel conjugate, preventing drug loss during maleimide hydrolysis.

Microwave-assisted synthesis and biological evaluation of 3,4-diaryl maleic anhydride/N-substituted maleimide derivatives as combretastatin A-4 analogues.

A series of new CA-4 analogues bearing maleic anhydride/N-substituted maleimide moiety were synthesized via a microwave-assisted process. They were evaluated for the anti-proliferative activities against three tumor cell lines (SGC-7901, HT-1080 and KB). Most compounds showed moderate potencies in micromolar range, with the most promising analogue 6f showing active at submicromolar concentration against HT-1080 cancer cells which was selected to investigate the antitumor mechanisms. In addition, molecular docking studies within the colchicine binding site of tubulin were also in good agreement with the tubulin polymerization inhibitory data and provided a basis for further structure-guided design of novel CA-4 analogues.

N-(1-Pyrenyl) maleimide inhibits telomerase activity in a cell free system and induces apoptosis in Jurkat cells.

Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. Agents that interact selectively with telomerase are anticipated to exert specific action on cancer cells. In this study, we evaluated maleimide derivatives for their potency and selectivity of telomerase inhibition. Among the several N-substituted derivatives of maleimide tested, N-(1-Pyrenyl) maleimide was shown to exert the greatest inhibition of telomerase in a cell free system, with an IC50 value of 0.25 µM. Importantly, we demonstrated that N-(1-pyrenyl) maleimide induces apoptosis in Jurkat T cells and displays the greatest differential cytotoxicity against hematopoietic cancer cells. These results suggest that N-(1-pyrenyl) maleimide is an attractive maleimide to be tested and developed as anti-cancer drug.

Antifungal, cytotoxic and SAR studies of a series of N-alkyl, N-aryl and N-alkylphenyl-1,4-pyrrolediones and related compounds.

The synthesis, in vitro evaluation and SAR studies of 67 maleimides and derivatives acting as antifungal agents are reported. A detailed SAR study supported by theoretical calculations led us to determine that: an intact maleimido ring appears to be necessary for a strong antifungal activity, dissimilarly affected by the substituents in positions 2 and 3. The best activities were shown by 2,3-nonsubstituted followed by 2,3 dichloro- and 2-methyl-substituted maleimides. They all were fungicide rather than fungistatic enhancing the importance of their antifungal activity. 2,3-Dimethyl and 2,3-diphenyl-maleimides possessed marginal or null activity. The presence of a flexible connecting chain in N-phenylalkyl maleimides appears not to be essential for antifungal activity, although its length shows a correlation with the antifungal behavior, displaying maleimides with alkyl chains of n=3 and n=4 the best antifungal activities in most fungi. Different substituents on the benzene ring did not have a clear influence on the activity. Values of chemical potential properties as well as of energy do not sufficiently discriminate between active and inactive compounds. Nevertheless, it was found that, although logP alone is not strong enough to properly predict the antifungal activity, the comparison of its values for compounds within the same sub-type, showed an enhancement of antifungal activity along with an increment of lipophilicity. In addition, the LUMO's electronic clouds of the highly active compounds showed to be concentrated on the imido ring, indicating that their carbon atoms are potential sites for nucleophilic attack. Same results were obtained from MEPs. Most of the active compounds did not show cytotoxic activity against human cancer cell lines and no one possessed hemolytic activity, indicating that their activity is selective to pathogenic fungi and that they are not toxic at MIC concentrations.

Fabrication of self-healing pectin/chitosan hybrid hydrogel via Diels-Alder reactions for drug delivery with high swelling property, pH-responsiveness, and cytocompatibility.

Self-healing hydrogels with pH-responsiveness could protect loaded drugs from being destroyed till it arrives to the target. The pectin-based hydrogel is a candidate due to the health benefit, anti-inflammation, antineoplastic activity, nontoxicity, and biospecific degradation, et al. However, the abundant existence of water-soluble branched heteropolysaccharide chains influenced its performance resulting in limitation of the potential. In the present study, we prepared a series of self-healing pectin/chitosan hydrogels via the Diels-Alder reaction. Moreover, pectin/chitosan composite hydrogel was prepared as a contrast. By comparison, it can be seen that the Diels-Alder reaction greatly improved the cross-linking density of hydrogels. The self-healing experiments showed excellent self-healing performance. In different swelling mediums, significant transformation in the swelling ratio was shown, indicating well-swelling property, pH- and thermo-responsiveness. The drug loading and release studies presented high loading efficiency and sustained release performance. The cytotoxicity assay that showed a high cell proliferation ratio manifested great cytocompatibility.

In vivo irreversible albumin-binding near-infrared dye conjugate as a naked-eye and fluorescence dual-mode imaging agent for lymph node tumor metastasis diagnosis.

Tumor metastases account for about 90% of cancer-related death, among which lymphatic metastases play a pivotal role. Therefore, high-efficiency sentinel lymph node (SLN) identification is significant for lymph node (LN) metastasis diagnosis in clinic. Herein, a novel in vivo covalent albumin-binding near-infrared (NIR) fluorescent IR820-maleimide conjugate (IR-Mal) is firstly designed as a SLN dual-mode imaging agent. The IR-Mal conjugate exhibits bright blue appearance and its large Stokes shift (over 100 nm) increases the fluorescent imaging resolution effectively. The fluorescence intensity of covalent albumin-binding IR-Mal (BSA-IR-Mal) complex is considerably stronger than that of IR-Mal. In vivo, IR-Mal could rapidly covalently bind the tissue interstitial albumin following subcutaneous administration and BSA-IR-Mal complexes could specifically accumulate on LN, and detect both normal and metastatic SLN through naked-eye and fluorescence imaging with high resolution. Moreover, the light stability and enhanced fluorescence intensity of BSA-IR-Mal complex facilitates its diagnosis accuracy. These findings suggest that such in vivo irreversible albumin-binding fluorescence conjugates could serve as a new agent for dual-mode imaging and have a great potential to be applied in the SLNs imaging and diagnosis.

Cornel iridoid glycoside induces autophagy to protect against tau oligomer neurotoxicity induced by the activation of glycogen synthase kinase-3ß.

Tau oligomers are the etiologic molecules of Alzheimer's disease, and correlate strongly with neuronal loss and exhibit neurotoxicity. Recent evidence indicates that small tau oligomers are the most relevant toxic aggregate species. The aim of the present study was to investigate the mechanisms of cornel iridoid glycoside (CIG) on tau oligomers and cognitive functions. We injected wortmannin and GF-109203X (WM/GFX, 200 µM each) into the lateral ventricles to induce tau oligomer and memory impairment in rats. When orally administered with CIG at 60 and 120 mg/kg/day for 14 days, CIG decreased the escape latency in the Morris water maze test. We also found that CIG restored the expression of presynaptic p-synapsin, synaptophysin, and postsynaptic density-95 (PSD-95) decreased by WM/GFX in rat cortex. CIG reduced the accumulation of tau oligomers in the brain of WM/GFX rats and in cells transfected with wild type glycogen synthase kinase-3ß (wtGSK-3ß). In addition, CIG up-regulated the levels of ATG7, ATG12, Beclin-1, and LC3II in vivo and in vitro, suggesting the restoration of autophagy function. These results suggest that CIG could ameliorate memory deficits and regulate memory-associated synaptic proteins through the clearance of tau oligomers accumulation. Moreover, CIG clears tau oligomers by restoring autophagy function.

Rottlerin causes pulmonary edema in vivo: a possible role for PKCdelta.

In the present study, we assessed the effects of chemical inhibitors shown to be selective for protein kinase C (PKC) isoforms on lung barrier function both in vitro and in vivo. Rottlerin, a purported inhibitor of PKCdelta, but not other chemical inhibitors, dose dependently promoted barrier dysfunction in lung endothelial cells in vitro. This barrier dysfunction correlated with structural changes in focal adhesions and stress fibers, which were consistent with functional changes in cell stiffness. To determine whether the effects noted in vitro correlated with changes in intact lungs, we tested the effects of rottlerin in the formation of pulmonary edema in rats using both ex vivo and in vivo models. Isolated, perfused lungs demonstrated a significant increase in filtration coefficients on exposure to rottlerin, compared with vehicle-treated lungs, an effect that correlated with increased extravasation of Evan's blue dye (EBD)-conjugated albumin. Additionally, compared with vehicle, the ratio of the wet lung weights to dry lung weights was significantly greater on exposure of animals to rottlerin; rottlerin also produced a dose-dependent increase in EBD extravasation into the lungs. These effects on lung edema occurred without any increase in right ventricular pressures. Microscopic assessment of edema in the ex vivo lungs demonstrated perivascular cuffing, with no evidence of septal capillary leak, in rottlerin-exposed lungs. Taken together, rottlerin increases barrier dysfunction in pulmonary endothelial cell monolayers and causes pulmonary edema in rats; results suggestive of an important role for PKCdelta in maintaining lung endothelial barrier function.

High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure.

BACKGROUND: Our objective was to test neural active compounds in a human developmental neurotoxicity (DNT) model that represents neural tube stages of vulnerability. Previously we showed that 14 days in vitro (DIV 14) was sufficient to generate cryopreserved neuronal cells for post thaw neurite recovery assays. However, short exposure and assessment may not detect toxicants that affect an early neurogenesis continuum, from a mitotic human neural progenitor (hNP) cell population through the course of neurite outgrowth in differentiating neurons. Therefore, we continuously exposed differentiating hNP cells from DIV 0 through DIV 14 to known toxicants and endocrine active compounds in order to assess at DIV 14 effects of these compounds in a human DNT maturation model for neurogenesis. METHODS: The Human DNT continuum (DIV 0 to DIV 14) was determined using immunocytochemistry for SOX1+ (proliferating hNP) and HuC/D+ (post mitotic neurons). The cumulative effects of five compounds was observed on neurite outgrowth in (ßIII-tubulin+) and (HuC/D+) cells using high content imaging. All data were analyzed using a one-way ANOVA with a significance threshold of p < 0.05. RESULTS: During maturation in vitro, the neural cultures transitioned from uniform hNP cells (DIV 0) to predominantly mature post mitotic neuronal neurons (HuC/D+, 65%; DIV14) but also maintained a smaller population of hNP cells (SOX1+). Using this DNT maturation model system, Bis-1, testosterone, and ß-estradiol inhibited neuronal maturation at micromolar levels but were unaffected by acetaminophen. ß-estradiol also disrupted neurite extension at 10 µM. Treating cells in this window with Bisphenol A (BPA) significantly inhibited neurite outgrowth and branching in these continuum cultures but only at the highest concentrations tested (10 µM). CONCLUSIONS: Cumulative effects of neurotoxicant exposure during a maturation continuum altered human neurogenesis at lower exposure levels than observed in acute exposure of static cryopreserved neurite recovery neurons cultures. Unlike prior acute studies, ß-estradiol was highly toxic when present throughout the continuum and cytotoxicity was manifested starting early in the continuum via a non-estrogen receptor α (ER α) mechanism. Therefore, the effect of neural developmental neurotoxins can and should be determined during the dynamic process of human neural maturation.

A study on peroxidative damage of the porcine intestinal brush-border membranes using a fluorogenic thiol reagent, N-(1-pyrene)maleimide.

To examine the effects of lipid peroxidation on the protein conformation in the porcine intestinal brush-border membranes, a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM) was employed. By treatment of NPM-labeled membranes with 100 microM ascorbic acid/10 microM Fe2+ in the presence of various concentrations of tert-butyl hydroperoxide (t-BuOOH), the fluorescence intensity of the complex decreased with the formation of conjugated diene, depending on the hydroperoxide concentration. The temperature dependence profile of the fluorescence intensity of NPM-labeled control membranes showed a thermal transition of the NPM fluorescence at 27-28 degrees C. The transition phenomenon of the NPM fluorescence in the membranes around this temperature disappeared by treatment of the labeled membranes with 100 microM ascorbic acid/10 microM Fe2+/0.6 mM t-BuOOH. The difference in response of the fluorescence characteristics of the bound NPM for temperature variation between the control and peroxidized membranes was also observed in the quenching efficiency with acrylamide. Measurement of the fluorescence polarization revealed that the harmonic mean of the rotational relaxation times of the bound NPM molecules to the membrane proteins increased from 1.96 to 4.93 microseconds by lipid peroxidation of the membranes. This indicates that the movement of the region containing NPM-labeled SH groups in the membrane proteins is restricted by lipid peroxidation. Treatment of NPM-labeled peroxidized membranes with sodium dodecyl sulfate (SDS) resulted in a restoration of the intensity of the NPM fluorescence to the level of the control ones. In addition, the temperature dependence profile of the fluorescence intensity of NPM-labeled peroxidized membranes in the presence of SDS also showed an appearance of a transition phenomenon around 30 degrees C. The result of SDS-polyacrylamide gel electrophoresis of the peroxidized membranes revealed that high-molecular-weight aggregates of the membrane proteins were not formed by lipid peroxidation. On the basis of these results, changes in the environmental properties around NPM-labeled SH groups in the membrane proteins by lipid peroxidation are discussed.

Novel indolylindazolylmaleimides as inhibitors of protein kinase C-beta: synthesis, biological activity, and cardiovascular safety.

Novel indolylindazolylmaleimides were synthesized and examined for kinase inhibition. We identified low-nanomolar inhibitors of PKC-beta with good to excellent selectivity vs other PKC isozymes and GSK-3beta. In a cell-based functional assay, 8f and 8i effectively blocked IL-8 release induced by PKC-betaII (IC(50) = 20-25 nM). In cardiovascular safety assessment, representative lead compounds bound to the hERG channel with high affinity, potently inhibited ion current in a patch-clamp experiment, and caused a dose-dependent increase of QT(c) in guinea pigs.

Reducible, dibromomaleimide-linked polymers for gene delivery.

Polycations have been successfully used as gene transfer vehicles both in vitro and in vivo; however, their cytotoxicity has been associated with increasing molecular weight. Polymers that can be rapidly degraded after internalization are typically better tolerated by mammalian cells compared to their non-degradable counterparts. Here, we report the use of a dibromomaleimide-alkyne (DBM-alkyne) linking agent to reversibly bridge cationic polymer segments for gene delivery and to provide site-specific functionalization by azide-alkyne cycloaddition chemistry. A panel of reducible and non-reducible, statistical copolymers of (2-dimethylamino)ethyl methacrylate (DMAEMA) and oligo(ethylene glycol)methyl ether methacrylate (OEGMA) were synthesized and evaluated. When complexed with plasmid DNA, the reducible and non-reducible polymers had comparable DNA condensation properties, sizes, and transfection efficiencies. When comparing cytotoxicity, the DBM-linked, reducible polymers were significantly less toxic than the non-reducible polymers. To demonstrate polymer functionalization by click chemistry, the DBM-linked polymers were tagged with an azide-fluorophore and were used to monitor cellular uptake. Overall, this polymer system introduces the use of a reversible linker, DBM-alkyne, to the area of gene delivery and allows for facile, orthogonal, and site-specific functionalization of gene delivery vehicles.

Consistency of QSAR models: Correct split of training and test sets, ranking of models and performance parameters.

Recent implementations of QSAR modelling software provide the user with numerous models and a wealth of information. In this work, we provide some guidance on how one should interpret the results of QSAR modelling, compare and assess the resulting models, and select the best and most consistent ones. Two QSAR datasets are applied as case studies for the comparison of model performance parameters and model selection methods. We demonstrate the capabilities of sum of ranking differences (SRD) in model selection and ranking, and identify the best performance indicators and models. While the exchange of the original training and (external) test sets does not affect the ranking of performance parameters, it provides improved models in certain cases (despite the lower number of molecules in the training set). Performance parameters for external validation are substantially separated from the other merits in SRD analyses, highlighting their value in data fusion.

Down-regulation of gap junctional intercellular communication between osteoblastic MC3T3-E1 cells by basic fibroblast growth factor and a phorbol ester (12-O-tetradecanoylphorbol-13-acetate).

To address the relation between osteoblast growth and cell-to-cell communication, we examined the effects of basic fibroblast growth factor (bFGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), both potent stimulators of osteoblastic proliferation, on gap junctional intercellular communication between osteoblastic MC3T3-E1 cells. The level of intercellular communication was estimated by a photobleaching method. TPA inhibited the degree of intercellular communication in two different time-dependent manners. The early (< 1 h) inhibition by TPA was consistent with an increase in the phosphorylation of connexin 43 (Cx43). The later inhibition was caused by reduction in the total amount of Cx43 on the plasma membrane, due to the decrease in the level of Cx43 transcripts. These qualitative and quantitative modulations by TPA were inhibited by a selective inhibitor of protein kinase C, GF109203X. bFGF also attenuated the gap junctional intercellular communication. However, short exposure (< 5 h) to bFGF did not affect the communication. The fact that the growth factor immediately stimulated the phosphorylation of Cx43 indicates that the phosphorylation site(s) affected by bFGF was not involved in the inhibition of communication. The decrease in the intercellular communication level was detected by the longer exposure (> 8 h) to bFGF and paralleled the decline in the Cx-mRNA level. This inhibitory effect of bFGF was abolished by the addition of a tyrosine kinase inhibitor, herbimycin A. Thus, gap junctional intercellular communication between osteoblasts was down-regulated by osteoblastic mitogens through different mechanisms of the modulation of Cx43.

Synthesis and in vitro efficacy of transferrin conjugates of the anticancer drug chlorambucil.

One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which differ in the stability of the chemical link between drug and spacer. The maleimide ester derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic hydrazone bonds were as active as or more active than chlorambucil in both cell lines. Especially, the conjugate in which chlorambucil was bound to transferrin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18-fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil. The structure-activity relationships of the transferrin conjugates are discussed with respect to their pH-dependent acid sensitivity, their serum stability, and their cytotoxicity.

Influence of N-(o-methoxyphenyl)-maleimide on 5-fluorouracil cytotoxicity in Ehrlich (ascites) carcinoma.

N-(o-methoxyphenyl)-maleimide (I), an intermediate obtained during the synthesis of pyrrolidinedione-N-mustards, did not exhibit antitumor activity against Ehrlich (ascites) carcinoma. The effect of co-administration of (I), with established anticancer drugs was studied against P388 leukemia, S180 (ascites) and Ehrlich (ascites) carcinoma. A significant potentiation in the activity of 5-Fluorouracil (5-FU) against Ehrlich (ascites) carcinoma by (I) was observed. The possible mechanisms responsible for potentiation of the activity of 5-FU are presented.

Comparative pathogenesis of haloacetic acid and protein kinase inhibitor embryotoxicity in mouse whole embryo culture.

Haloacetic acids (HAs) are embryotoxic contaminants commonly found in drinking water. The mechanism of HA embryotoxicity has not been defined, but may be mediated in part by protein kinase C (PKC) inhibition. This study was conducted to evaluate the pathogenesis of HA embryotoxicity, and to compare these data with those from specific (Bis I) and non-specific (staurosporine) inhibitors of PKC. Embryos were incubated for varying times with several HAs, Bis I, staurosporine, or Bis V (a negative control). Cell cycle analysis was performed by flow cytometry following nuclear staining with propidium iodide; apoptosis was evaluated by fluorescence microscopy following LysoTracker staining. At concentrations producing 100% embryotoxicity with no embryolethality, only staurosporine perturbed the cell cycle. However, flow cytometry revealed accumulation of sub-G1 events (an apoptotic indicator) across time with bromochloroacetic acid, dichloroacetic acid, and staurosporine, but not dibromoacetic acid, Bis I, or Bis V. Sub-G1 events were particularly prominent in the head region, and remained at control levels in the heart. LysoTracker staining confirmed a similar pattern of apoptosis in the intact embryo; BCA and DCA produced intense staining in the prosencephalon, with virtually no staining in the heart. These data indicate that while cell-cycle perturbation may not mediate the pathogenesis of HA embryotoxicity, these agents do induce embryonic apoptosis. In addition, the lack of Bis I-induced apoptosis indicates that PKC inhibition is unlikely to be the sole mediator of HA embryotoxicity.

Aneuploidy induction in Saccharomyces cerevisiae by two solvent compounds, 1-methyl-2-pyrrolidinone and 2-pyrrolidinone.

A number of solvent compounds that were tested in Saccharomyces cerevisiae were potent inducers of aneuploidy, although they did not induce any other genetic effects. As an extention of these earlier findings, 1-methyl-2-pyrrolidinone was tested and was found to induce aneuploidy. Several structurally related compounds were also tested; 2-pyrrolidinone induced aneuploidy, but succinimide, pyrrolidine, 1-methylpyrrolidine, 1-methyl-3-pyrrolidinol, and 2-pyrrolidineethanol did not. Maleimide and its N-hydroxy, N-methyl, and N-ethyl derivatives were also negative for aneuploidy induction.

4-Maleimidohippuric acid--a tailor-made, direct, site-specific nephrotoxin: effects on renal function and ultrastructure in pentobarbital-anesthetized dogs.

A model has been proposed to explain at least one of the possible pathways through which a xenobiotic might produce proximal tubule necrosis. The model is formulated on the idea that a compound must possess two structural features: (i) a carboxyl or amino acid moiety that would allow for selective uptake into proximal tubule cells via the strategically located antiluminal membrane-bound organic anion transport system or the luminal membrane-bound amino acid transport system(s), respectively, and (ii) a highly reactive moiety that can directly alkylate proximal tubular components, or a moiety that can be biotransformed within proximal tubular cells to such a substance. In an attempt to validate the proposed structural features as prerequisites for xenobiotic induction of proximal tubular necrosis, a novel compound, 4-maleimidohippuric acid (4-MHA), was synthesized which possesses an anionic group and a reactive moiety. Following the administration of 4-MHA directly into the renal artery of pentobarbital-anesthetized dogs, specific unilateral ultrastructural damage was noted only in the S1 and S2 cell types of the proximal tubule; the most notable renal function changes included proteinuria and glucosuria. Anionic, but non-alkylating, relatives of 4-MHA failed to alter renal function or ultrastructure. The specific proximal tubular toxicity of 4-MHA validates the proposed structural requirements for induction of proximal tubular necrosis.

Toxicity of N-(3,5-dichlorophenyl)succinimide and metabolites to rat renal proximal tubules and mitochondria.

The acute nephrotoxicity caused by N-(3,5-dichlorophenyl) succinimide (NDPS) has been shown to be due to a metabolite(s) of the parent compound. This study examined the toxicity of NDPS, its known metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA), N-(3,5-dichlorophenyl)malonamic acid (DMA), N-(3,5-dichlorophenyl)succinamic acid (NDPSA), and two postulated metabolites N-(3,5-dichlorophenyl)maleamic acid (NDPMA) and N-(3,5-dichlorophenyl)maleimide (NDPM) to suspensions of renal proximal tubules (RPT) prepared from male Fischer 344 rats. Tubule viability and mitochondrial function were not adversely affected by exposure of RPT to either 1 mM NDPS, NDHS, NDHSA, DMA, NDPSA, or NDPMA for 4 h. However, NDPM caused a concentration-(25-100 microM) and time-dependent (0.25-4 h) loss in basal and nystatin stimulated oxygen consumption and tubule viability. Investigations using isolated renal cortical mitochondria (RCM) showed that NDPM was a potent inhibitor of mitochondrial function. Isolated RCM respiring on pyruvate/malate and exposed to NDPM exhibited a concentration (25-100 microM) dependent decrease in state 3 and state 4 respiration. Inhibition of mitochondrial state 3 respiration by NDPM was mediated through site 1 of the respiratory chain. NDPM did not inhibit cytochrome c-cytochrome oxidase or the electron transport chain. These results indicated that NDPS, its known metabolites, and NDPMA were not directly toxic to rat RPT. However, the postulated metabolite NDPM, was a potent tubule cytotoxicant that inhibited mitochondrial function in isolated RCM and RPT and may produce cell death through this mechanism.