A study on the inhibition of dihydrofolate reductase (DHFR) from Escherichia coli by gold( i ) phosphane compounds. X-ray crystal structures of (4,5-dichloro-1H-imidazolate-1-yl)-triphenylphosphane-gold( i ) and (4,5-dicyano-1H-imidazolate-1-yl)-triphenylphosphane-gold( i )by Rossana Galassi, Camille Simon Oumarou, Alfredo Burini, Alessandro Dolmella, Daniela Micozzi, Silvia Vincenzetti, Stefania Pucciarelli

Dalton Trans.

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Year
2015
DOI
10.1039/C4DT01542H
Subject
Inorganic Chemistry

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Cite this: Dalton Trans., 2015, 44, 3043

Received 26th May 2014,

Accepted 3rd December 2014

DOI: 10.1039/c4dt01542h www.rsc.org/dalton

A study on the inhibition of dihydrofolate reductase (DHFR) from Escherichia coli by gold(I) phosphane compounds. X-ray crystal structures of (4,5-dichloro-1H-imidazolate-1-yl)triphenylphosphane-gold(I) and (4,5-dicyano-1Himidazolate-1-yl)-triphenylphosphane-gold(I)†

Rossana Galassi,*a Camille Simon Oumarou,a Alfredo Burini,a Alessandro Dolmella,b

Daniela Micozzi,c Silvia Vincenzettic and Stefania Pucciarellic

An unprecedented study on the inhibitory activities of a class of phosphane gold(I) complexes on E. coli dihydrofolate reductase (DHFR) is reported. The gold(I) complexes considered in this work consist of azolate or chloride ligands and phosphane as co-ligands. The ligands have been functionalized with polar groups (–COOH, –COO−, NO2, Cl, CN) to obtain better solubility in polar media. Neutral, anionic and cationic gold(I) complexes have been tested as DHFR inhibitors by means of a continuous direct spectrophotometric method. X-ray structural characterizations were performed on ((triphenylphosphine)-gold(I)-(4,5dicyanoimidazolyl-1H-1yl) and on the analog (triphenylphosphine)-gold(I)-(4,5-dichloroimidazolyl-1H1yl). The inhibition constants obtained from the enzyme tests range from 20 μM to 63 nM (auranofin) and are conducive to promoting these compounds as potential DHFR inhibitors.

Introduction

Dihydrofolate reductase (DHFR), a ubiquitous enzyme in all eukaryotic and prokaryotic cells, is of pivotal importance in biochemistry and medicinal chemistry. DHFR catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), using NADPH as a coenzyme. THF is required for the de novo synthesis of purines, amino acids and thymidylate (TMP).

Therefore, the inhibition of DHFR activity in the absence of salvage mechanisms hampers the supply of TMP for DNA biosynthesis, and eventually leads to cell death. The ability to preferentially kill cells that replicate at a high rate is the basis of anticancer chemotherapy. Thus DHFR has long been recognized as a drug targeted at treating cancer, and many DHFR inhibitors have been designed as antineoplastic drugs such as methotrexate (MTX), representing the first class of antimetabolites to be introduced to the oncology clinic for the chemotherapeutic treatment of childhood acute lymphoblastic leukemia (ALL), nearly 60 years ago.1 Although most of the patients respond effectively to anti-folate treatment, many of them relapse due to the development of resistance. Even if DHFR inhibitor methotrexate (MTX) remains a mainstay in single and combination cancer chemotherapy, there is a continuous search for new chemical entities with the potential to target this enzyme.2 In fact blocking DHFR enzymatic activity is a key element not only in the treatment of many diseases in addition to cancer, like bacterial and protozoal infections and also opportunistic infections associated with AIDS (Pneumocystis carinii pneumonia, PCP), but also in various autoimmune diseases, e.g., rheumatoid arthritis.3 The ability to inhibit enzymes is a well-known characteristic of gold(I) drugs; a number of putative mechanisms of action based on the interaction of gold(I) compounds with enzymes have been proposed during the “golden years” of anti-arthritis treatments research.4 Recently, some studies addressed other enzymes including cytochrome C and lysozyme,5 cysteine protease,6 thioredoxin 1 and 27 and zinc finger protein PARP 1.8

Although gold compounds have been well studied as anticancer drugs and many of them have been found very powerful,9 cancer therapy based on gold drugs, from what we know, has never been seriously considered, mainly because †CCDC 997733 and 997734. For crystallographic data in CIF or other electronic format see DOI: 10.1039/c4dt01542h aSchool of Science and Technology, Chemistry Division, University of Camerino, Via

S. Agostino, 1, 62032 Camerino, Italy. E-mail: rossana.galassi@unicam.it;

Tel: 0039 0737 402243 bDepartment of Pharmaceutical and Pharmacological Sciences, Università di Padova,

Via Marzolo 5, 35131 Padova, Italy cSchool of Biosciences and Biotechnology, University of Camerino, Via Gentile III da

Varano, 1, 62032 Camerino, Italy

This journal is © The Royal Society of Chemistry 2015 Dalton Trans., 2015, 44, 3043–3056 | 3043

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View Journal | View Issue the mechanism of action is not completely clear. However, as comparative cytotoxicity studies have shown,10a the presence of the metal center has its own meaning: the presence of the metal activates biological effects on the organic ligand, which otherwise would be non-existent. Among all possible enzymes, seleno-enzymes such as thioredoxin reductase (TrxR) have been extensively considered as the most likely molecular target.7,11 Some azolate gold(I) phosphane complexes synthesized by some of us have been tested on TrxR and on the structural analog disulfide glutathione reductase (GR), showing the strongest inhibition on the former.10 These results promoted further enzymatic studies on this class of gold(I) compounds both on the structure–activity relationship and on the mechanism of action. For this latter aspect, and to evaluate the potential antimicrobial activity, a new crucial enzyme named dihydrofolate reductase (DHFR) was considered for the first time. The study was first done on a class of compounds whose anticancer activity on many cancer cell lines was previously ascertained, followed by a class of azolate gold(I) phosphane compounds related to the former but with enhanced polarity and hydrophilicity.

Results

Syntheses

In Table 1 the investigated compounds are listed and named.

Azolate gold(I) phosphane compounds 1, 2, 11 and 12 corresponding to the general formula AzAuL (1, Az = 3,5-bis-trifluoromethyl-1H-pyrazolate-1-yl and L = PPh3, triphenylphosphane; 2,