molecules Review DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents Maria Valeria Raimondi 1,*,† , Ornella Randazzo 1,†, Mery La Franca 1 , Giampaolo Barone 1 , Elisa Vignoni 2, Daniela Rossi 2 and Simona Collina 2,* 1 Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, via Archirafi 32, 90123 Palermo, Italy; [email protected] (O.R.); [email protected] (M.L.F.); [email protected] (G.B.) 2 Drug Sciences Department, Medicinal Chemistry and Pharmaceutical Technology Section, University of Pavia, via Taramelli 12, 27100 Pavia, Italy; [email protected] (E.V.); [email protected] (D.R.) * Correspondence: [email protected] (M.V.R.); [email protected] (S.C.); Tel.: +390-912-389-1915 (M.V.R.); +390-382-987-379 (S.C.) † These Authors contributed equally to this work. Academic Editors: Simona Collina and Mariarosaria Miloso Received: 25 February 2019; Accepted: 20 March 2019; Published: 22 March 2019 Abstract: Dihydrofolate reductase inhibitors are an important class of drugs, as evidenced by their use as antibacterial, antimalarial, antifungal, and anticancer agents. Progress in understanding the biochemical basis of mechanisms responsible for enzyme selectivity and antiproliferative effects has renewed the interest in antifolates for cancer chemotherapy and prompted the medicinal chemistry community to develop novel and selective human DHFR inhibitors, thus leading to a new generation of DHFR inhibitors. This work summarizes the mechanism of action, chemical, and anticancer profile of the DHFR inhibitors discovered in the last six years. New strategies in DHFR drug discovery are also provided, in order to thoroughly delineate the current landscape for medicinal chemists interested in furthering this study in the anticancer field. Keywords: dihydrofolate reductase (DHFR) enzyme; DHFR inhibitors as anticancer agents; heterocyclic compounds; DHFR drug discovery; hybrid compounds 1. Introduction Since the middle of the last century, the potential of the dihydrofolate reductase (DHFR) enzyme as a therapeutic target for treating infections has been evidenced [1,2]. DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate using NADPH, and it is involved in the synthesis of raw material for cell proliferation, in both prokaryotic and eukaryotic cells. DHFR inhibitors are commonly used for fighting malaria and other protozoal infections, as well as for treating fungal, bacterial, and mycobacterial infections [3]. Over the years, several compounds have been discovered and different drugs have entered the market. Among them, we have to mention pyrimethamine and proguanil as antimalarial drugs [4,5]; trimethoprim, an antibacterial drug commonly used in association with sulfonamides, like sulfamethoxazole [6,7]; and methotrexate, the first-in-class anti-cancer agent acting via DHFR inhibition [8,9]. Methotrexate inhibits DHFR with a high affinity, thus reducing the amount of tetrahydrofolates required for the synthesis of pyrimidine and purines. Consequently, RNA and DNA synthesis is stopped and the cancer cells die. From a chemical standpoint, methotrexate shows several drawbacks, such as a poor solubility and relevant toxic side effects [10–12]. DHFR inhibitors are among the most used classes of anticancer agents and finding novel agents with a Molecules 2019, 24, 1140; doi:10.3390/molecules24061140 www.mdpi.com/journal/molecules Molecules 2019, 24, 1140 2 of 19 Molecules 2019, 24, x FOR PEER REVIEW 2 of 22 promisingnovel agents pharmacological with a promising profile pharmacological still remains oneprofile of thestill majorremains challenges one of the for major medicinal challenges chemists, for asmedicinal testified bychemists, the literature as testified trend by of the the lit lasterature 20 years. trend of the last 20 years. InIn this this review, review, after after a a brief brief overview overview of of the the physiological physiological role role of of DHFR DHFR in in cells cells and and particularly particularly in cancerin cancer cells, cells, we focus we onfocus DHFR on inhibitorsDHFR inhibitors for cancer for therapy. cancer Particularly,therapy. Particularly, we highlight we compounds highlight alreadycompounds marketed already and marketed new scaffolds and new that scaffolds could be that relevant could for be anticancerrelevant for therapy. anticancer therapy. 2.2. Physiological Physiological RoleRole andand StructureStructure of DHFR FolicFolic acidacid (FA)(FA) isis aa water-solublewater-soluble vitaminvitamin important for biological systems. systems. It It is is not not biologically biologically activeactive perper se,se, butbut itit isis thethe precursorprecursor ofof thethe activeactive form known as tetrahydrofolate (THF), (THF), which which is is essentialessential forfor thethe dede novonovo synthesissynthesis ofof purines,purines, aminoamino acids, and thymidylate thymidylate (TMP) (TMP) [13]. [13]. It It has has been been demonstrateddemonstrated that that its its absence absence causes causes the inhibitionthe inhibiti ofon cell of growth cell growth and proliferation and proliferation [14]. The [14]. synthetic The pathwaysynthetic that pathway allows that the allows transformation the transformati of FA inon THF of FA is reportedin THF is inreported Figure1 in. Figure 1. Figure 1. Reduction of of FA FA in in THF. THF. TheThe synthesissynthesis ofof folatesfolates inin bothboth eukaryoticeukaryotic and prokaryotic cells cells is is strictly strictly dependent dependent on on the the activitiesactivities ofof twotwo enzymes:enzymes: DHFRDHFR and dihydrofolatedihydrofolate synthase (DHFS), (DHFS), whose whose inhibition inhibition leads leads to to cell cell death.death. FromFrom a medicinala medicinal chemistry chemistry perspective, perspective, the ubiquitousthe ubiquitous enzyme enzyme DHFR DHFR is of particularis of particular interest sinceinterest it is since essential it is foressential folate metabolismfor folate metabolism and purine and and purine thymidylate and thymidylate synthesis in synthesis cell proliferation. in cell Poorproliferation. DHFR activity Poor causesDHFR tetrahydrofolate activity causesdeficiency tetrahydrofolate and cell deficiency death [15]. Thisand mechanismcell death [15]. is reported This inmechanism Figure2. is reported in Figure 2. FromFrom aa structuralstructural standpoint,standpoint, DHFR is a relative relativelyly small water-soluble water-soluble protein protein with with a a molecular molecular weightweight of of 18.000–25.000 18.000–25.000 Da. Da. Over Over the years,the years, DHFR DHFR has been has extensivelybeen extensively studied studied and several and attemptsseveral haveattempts been have made been to elucidate made to theelucidate structure the of struct DHFRure isoforms. of DHFR Toisoforms. date, the To Protein date, the Data Protein Bank (PDB)Data hasBank collected (PDB) overhas onecollected hundred over structures one hundred obtained st fromructures both obtained eukaryotic from and prokaryoticboth eukaryotic organisms and (humans,prokaryoticEscherichia organisms coli ,(humans,Lactobacillus Escherichia casei, Pneumocystis coli, Lactobacillus carinii casei, Micobacterium, Pneumocystis tuberculosis carinii, Micobacterium, etc.), alone or intuberculosis complex, with etc.), different alone or ligandsin complex [16]. with Briefly, different DHFR ligands consists [16]. of eightBriefly, sheets, DHFR which consists form of aeight rigid skeleton:sheets, which seven form sheets a rigid run parallelskeleton: and seven the sheets other ru runsn parallel antiparallel. and the All other the enzymeruns antiparallel. isoforms All contain the atenzyme least four isoformsα-helices contain intersecting at least in four the longα-helices loops intersecting of the sheets. in Furthermore, the long loops one of loop the forms sheets. the bindingFurthermore, site for one the loop substrate, forms whilethe binding another site two for form the substrate, the binding while site another of the coenzyme two form NADPH. the binding It is interestingsite of the coenzyme to note that NADPH. DHFR It has is interesting no disulfide to note bridges that andDHFR it doeshas no not disulfide need to bridges be coordinated and it does by metalnot need ions to exercisebe coordinated its biochemical by metal activity ions to [8 exer]. Ancise important its biochemical structural activity element [8]. of An the important enzyme is thestructural presence element of the of “Met20” the enzyme or “loop is the 1” presence consisting of the of "Met20" residues or 9-24 "loop [17 1"–21 cons]. Itisting helps of to residues stabilize 9-24 the nicotinamide[17–21]. It helps ring to of stabilize NADPH the to promote nicotinamide the passage ring of of NADPH hydride fromto promote NADPH the to passage dihydrofolate of hydride and it isfrom able NADPH to open, closeto dihydrofolate or occlude, theand active it is able site to of open the enzyme, close or [22 occlude,,23]. The the amino active acid site residue of the enzyme Asp27 is [22,23]. The amino acid residue Asp27 is also crucial, because it helps the protonation of the also crucial, because it helps the protonation of the substrate and keeps it in a conformation favorable substrate and keeps it in a conformation favorable to hydride transfer [24]. The structure of the to hydride transfer [24]. The structure of the human DHFR in complex with NADPH and with the human DHFR in complex with NADPH and with