Characterization of Novel Diphenylamine Compounds as Ferroptosis Inhibitors.

Ferroptosis is a form of oxidative cell death that is increasingly recognized as a key mechanism not only in neurodegeneration but also in regulated cell death, causing disease in other tissues. In neurons, major hallmarks of ferroptosis involve the accumulation of lipid reactive oxygen species (ROS) and impairment of mitochondrial morphology and function. Compounds that interfere with ferroptosis could provide novel treatment options for neurodegenerative disorders and other diseases involving ferroptosis. In the present study, we developed new compounds by refining structural elements of the BH3 interacting-domain death agonist inhibitor BI-6c9, which was previously demonstrated to block ferroptosis signaling at the level of mitochondria. Here, we inserted an antioxidative diphenylamine (DPA) structure to the BI-6c9 structure. These DPA compounds were then tested in models of erastin, and Ras-selective lethal small molecule 3 induced ferroptosis in neuronal HT22 cells. The DPA compounds showed an increased protective potency against ferroptotic cell death compared with the scaffold molecule BI-6c9. Moreover, hallmarks of ferroptosis such as lipid, cytosolic, and mitochondrial ROS formation were abrogated in a concentration- and time-dependent manner. Additionally, mitochondrial parameters such as mitochondrial morphology, mitochondrial membrane potential, and mitochondrial respiration were preserved by the DPA compounds, supporting the conclusion that lipid ROS toxicity and mitochondrial impairment are closely related in ferroptosis. Our findings confirm that the DPA compounds are very effective agents in preventing ferroptotic cell death by blocking ROS production and, in particular, via mitochondrial protection. SIGNIFICANCE STATEMENT: Preventing neuronal cells from different forms of oxidative cell death was previously described as a promising strategy for treatment against several neurodegenerative diseases. This study reports novel compounds based on a diphenylamine structure that strongly protects neuronal HT22 cells from ferroptotic cell death upon erastin and Ras-selective lethal small molecule 3 induction by preventing the development of different reactive oxygen species and by protecting mitochondria from ferroptotic impairments.

Antibiotics in malaria therapy and their effect on the parasite apicoplast.

The impact of selected antibiotics on combating malaria infections was discovered in the mid of last century. Only recently, studies on their modes of action in malaria parasites have been initiated, prompted by the discovery of a prokaryotic organelle, the apicoplast. This plastid-derived structure, which originates from a secondary endosymbiotic event, possesses important metabolic as well as housekeeping functions, including fatty acid and heme biosynthesis. Due to its indispensability for parasite survival it represents a promising target for the use of antibiotics in malaria therapy. Most antibiotics cause a delayed death phenotype, which manifests in the late onset of antimalarial activity during the second replication cycle of the pathogen. This review will describe the effect of classical antibacterial agents against malaria parasites and the use of some of these compounds in clinical settings. Firstly we discuss the current knowledge about the physiological and morphological effects of antibiotics on the parasite and the apicoplast in particular, with special focus on the delayed death effect. Secondly antimalarial antibiotics are specified and their effects in vitro are compared with available in vivo data and clinical studies. Major precautions and side effects are described.

The plastid-like organelle of apicomplexan parasites as drug target.

Apicomplexan parasites infectious to humans include Plasmodium spp., Babesia spp., Toxoplasma gondii, Cryptosporidium spp., Isospora belli and Cyclospora cayetanensis. With exception of Cryptosporidium spp., these parasites possess a non-photosynthetic plastid-like organelle called apicoplast. The apicoplast possesses a small circular genome and harbours prokaryotic-type biochemical pathways. As the most important metabolic functions, the mevalonate independent 1-deoxy-D-xylulose 5-phosphate pathway of isoprenoid synthesis and the type II fatty acid synthesis system are operative inside the apicoplast. Classical antibacterial drugs such as ciprofloxacin, tetracycline, doxycycline, clindamycin and spiramycin inhibit the apicoplast-located gyrase and translation machinery, respectively, and are currently used in the clinic for the treatment of infections with apicomplexan parasites. As an inhibitor of isoprenoid synthesis, fosmidomycin was proven to be effective against acute P. falciparum malaria in clinical phase II studies. Triclosan, an inhibitor of fatty acid synthesis, was active in a malaria mouse model. In vitro antimalarial activity was shown for inhibitors of peptide deformylase and the import of apicoplast-targeted proteins. Work on various other inhibitors of apicoplast-located biochemical processes is ongoing.

2-(aminoacylamino)benzophenones: farnesyltransferase inhibition and antimalarial activity.

The use of amino acids as acyl substitutents at the 2-amino group of our benzophenone core structure yielded compounds with mainly good to moderate farnesyltransferase inhibitory and moderate antimalarial activity. However, these farnesyltransferase inhibitors display some degree of selectivity towards malarial parasites since there was no cytotoxic activity observed at 70-80 microM.

Novel lead structures for antimalarial farnesyltransferase inhibitors.

Through the combination of nitrophenylfurylacryloyl moiety which has been designed to occupy an aryl binding site of farnesyltransferase with several AA(X)-peptidomimetic substructures some novel farnesyltransferase inhibitors were obtained. Evaluation of their antimalarial activity and some initial modifications yielded a 4-benzophenone- and a sulfonamid-based novel lead for antimalarial farnesyltransferase inhibitors.

2-(Arylpropionylamino)- and 2-(arylacryloylamino)benzophenones: farnesyltransferase inhibition and antimalarial activity.

Structural variation of the 2-acylamino moiety of some benzophenone farnesyltransferase inhibitors led to the para-trifluoromethylphenylpropionyl derivative with relatively low farnesyltransferase inhibition but considerable antimalarial activity and no cytotoxicity.

Structure-activity relationships of novel anti-malarial agents part 8. Effect of different central aryls in biarylacryloylaminobenzophenones on antimalarial activity.

Replacement of the 2,5-disubstituted furyl residue present in the known antimalarial agents 8 by other aryl residues resulted in a more or less reduced antimalarial activity in most cases. The only exemption was the 2,4-thienylene compound 11a displaying activity with an IC50 value of 120 nM. In conclusion, the 2,5-furylene compound 8e remains to represent the most active antimalarial agent in this series of farnesyltransferase inhibitors.

Exploration of novel aryl binding sites of farnesyltransferase using molecular modeling and benzophenone-based farnesyltransferase inhibitors.

Most non-thiol CAAX-peptidomimetic farnesyltransferase inhibitors bear nitrogen-containing heterocycles in place of the terminal cysteine which are supposed to coordinate the enzyme-bound zinc. However, it has been shown that those nitrogen-containing heterocycles can be replaced by carbocyclic aromatic moieties which are unable to coordinate the zinc ion, a conclusion that resulted in the postulation of one or two hitherto unknown aryl binding sites. No indication has been given about the spatial location of these novel binding sites. Employing flexible docking of several non-thiol farnesyltransferase inhibitors known from the literature and some model compounds based on our benzophenone scaffold as well as performing GRID searches, we have identified two regions in the farnesyltransferase's active site which we suggest being the postulated aryl binding sites. One aryl binding region is located in close proximity to the zinc ion and is defined by the aromatic side chains of Tyr 300beta, Trp 303beta, Tyr 361beta, and Tyr 365beta. The second aryl binding site is defined by the side chains of Tyr 300beta, Leu 295beta, Lys 294beta, Lys 353beta, and Lys 356beta. This second aryl binding site has been used for the design of a non-thiol farnesyltransferase inhibitor (9c) with an IC(50) of 35 nM.

Synthesis, molecular modeling, and structure-activity relationship of benzophenone-based CAAX-peptidomimetic farnesyltransferase inhibitors.

Because of the involvement of farnesylated proteins in oncogenesis, inhibition of the protein-modifying enzyme farnesyltransferase is considered a major emerging strategy in cancer therapy. Here, we describe the structure-activity relationship of a novel class of CAAX-peptidomimetic farnesyltransferase inhibitors based on the benzophenone scaffold. 4'-Methyl, 4'-chloro, 4'-bromo, and 4'-nitrophenylacetic acid as substituents at the 2-amino group of the benzophenone core structure yield farnesyltransferase inhibitors active in the nanomolar range. Using diphenylacetic acid in this position further improves activity. SEAL superimposition of inhibitor 12a to the enzyme-bound conformation of a CAAX-peptide shows a markedly good resemblance of the molecular properties of the peptide. FlexX docking of 12a confirms the good fit of the molecule into the peptide binding site of farnesyltransferase. The novel benzophenone-based AAX-peptidomimetic substructure described here will be useful for the design of some novel types of farnesyltransferase inhibitors.

Structure-activity relationships of novel anti-malarial agents: 1. arylacyl and cyclohexylacyl derivatives of 5-amino-2-tolylacetylaminobenzophenone.

We have identified compound 1 as a lead structure of a novel class of anti-malarial agents. Here, we report on our continuing studies towards the establishment of structure-activity relationships by varying the terminal phenyl moiety and the alkyl linker of the acyl substituent at the 5-amino function of the benzophenone core structure. Most of the derivatives of our lead structure 1 essentially display the same anti-plasmodial activity as the lead.

Synthesis of potential aldose reductase inhibitors based on minimal pharmacophore requirements.

A series of 17 compounds were synthesized based on the premise that the minimal pharmacophore for aldose reductase inhibition requires the presence of both an aryl group and polar group connected by a linking structure. Three groups of compounds were synthesized, the first possessing an aniline-4-(2'-6'-methylbenzothiazole) or 2-aminobenzothiazole group as the aryl group, the second possessing a 2-naphthyl as the aryl group and the third possessing either a 4-(2-phenylthiazole) or 2-(5-2'-nitrophenylfuran) as the aryl group. In all three of these groups the carboxylate or its methyl ester are linked to the aryl group through various lengths of methylene carbons and amide or cinnamide groups. Optimal activity was observed when the carboxylic group was separated from the aryl group by a linking structure of five atoms in length. Both a double bond and an amide moiety are well tolerated in the linking structure.

Discovery of a novel lead structure for anti-malarials.

From a library of 61 compounds available from former studies 2,5-bis-acylaminobenzophenone 7p was identified as a lead structure for a novel class of anti-malaria agents active against multi-resistant Plasmodium falciparum strain Dd2. Some structural modifications of this initial lead demonstrated the potential for further improvement of the anti-plasmodial activity of this novel class of anti-malarials.

Diaryl ester prodrugs of FR900098 with improved in vivo antimalarial activity.

The fosmidomycin derivative FR900098 represents an inhibitor of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase with potent antimalarial activity. Prodrugs of FR900098 with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield phosphodiaryl esters. One diaryl ester prodrug demonstrated efficacy in mice infected with the rodent malaria parasite Plasmodium vinckei comparable to i.p. drug administration.

Non-thiol farnesyltransferase inhibitors: structure-activity relationships of aralkylsubstituted benzophenones.

We describe a novel class of benzophenone-based farnesyltransferase inhibitors exploiting a novel aryl binding region in the farnesyltransferase's active site. The present study was mainly focussed on structural modifications of the trimethylene spacer of the 4-phenyl butyroyl residue of our lead structure (IC50 = 530 nM). These modifications turned out to have little effect on activity as had the replacement of the terminal aryl by cyclohexyl (IC50 = 440 nM vs. IC50 = 530 nM).

Structure activity relationship of antiproliferative N-acyl-aspartic acid dimethyl ester. 2. Variation of the aspartyl moiety.

Structural variations of the L-aspartic acid substructure of (S)-N-3-phenoxycinnamoylaspartic acid dimethyl ester which shows a selective antiproliferative activity against THP-1 tumor cells, demonstrated that the L-aspartic acid moiety is absolutely mandatory for antiproliferative activity as well as for selectivity.

Structure-activity relationships of novel anti-malarial agents. Part 2: cinnamic acid derivatives.

We have described compound 1 as a lead structure for a novel class of anti-malarial agents. Replacement of the 3-phenylpropionyl moiety of the lead structure 1 by a 4-propoxycinnamic acid residue resulted in a significant improvement in antimalarial activity. Compound 3q represents an important step in the development of lead structure 1 into an anti-malarial drug candidate.

Non-peptidic, non-prenylic inhibitors of the prenyl protein-specific protease Rce1.

Several compounds designed as bisubstrate analogues of protein farnesyltransferase inhibited the prenyl protein-specific protease Rce1, qualifying them as lead structures for a novel class of non-peptidic, non-prenylic inhibitors of this protease.

Non-peptidic, non-prenylic bisubstrate farnesyltransferase inhibitors. Part 3: structural requirements of the central moiety for farnesyltransferase inhibitory activity.

Recently, we have described non-peptidic, non-prenylic bisubstrate analogues as a novel type of farnesyltransferase inhibitor composed of a farnesyl-mimetic, a linker and an AAX-peptidomimetic substructure. With this study, we showed that the amide function connecting the farnesyl-mimetic and the linking substructures of our inhibitors is crucial for their activity. We suggest that the amide is bound to the essential zinc ion in the farnesyltransferases active center. We identified succinic and glutaric acid, respectively, in addition to the initially used 1-alanyl moiety as suitable linking structures. Glycine can also be used in this function provided the distance between the alpha-amide group and the center of the peptidomimetic substructure is enlarged by introduction of an additional methylene unit into the peptidomimetic substructure.