On Dihydroorotate Dehydrogenases and Their Inhibitors and Uses

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On Dihydroorotate Dehydrogenases and Their Inhibitors and Uses Hélène Munier-Lehmann,†,‡ Pierre-Olivier Vidalain,§,∥ Frédéric Tangy,§,∥ and Yves L. Janin*,†,‡ †

Institut Pasteur, Unité de Chimie et Biocatalyse, Département de Biologie Structurale et Chimie, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France ‡ CNRS, UMR 3523, France § Institut Pasteur, Unité de Génomique Virale et Vaccination, Département de Virologie, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France ∥ CNRS, URA 3015, France ABSTRACT: Proper nucleosides availability is crucial for the proliferation of living entities (eukaryotic cells, parasites, bacteria, and virus). Accordingly, the uses of inhibitors of the de novo nucleosides biosynthetic pathways have been investigated in the past. In the following we have focused on dihydroorotate dehydrogenase (DHODH), the fourth enzyme in the de novo pyrimidine nucleosides biosynthetic pathway. We first described the different types of enzyme in terms of sequence, structure, and biochemistry, including the reported bioassays. In a second part, the series of inhibitors of this enzyme along with a description of their potential or actual uses were reviewed. These inhibitors are indeed used in medicine to treat autoimmune diseases such as rheumatoid arthritis or multiple sclerosis (leflunomide and teriflunomide) and have been investigated in treatments of cancer, virus, and parasite infections (i.e., malaria) as well as in crop science. Scheme 1a

I. INTRODUCTION This work aims at providing a current view on dihydroorotate dehydrogenase (DHODH) and all the series of inhibitors described so far. The first work focusing on the latter aspect of this review was published in 1999 and described the inhibitors of DHODH known at that time.1 A contemporary report is focusing on the agents interfering with dihydroorotate, succinate, and NADH oxidation of mitochondria.2 More recent surveys have reviewed the purine and pyrimidine pathways as targets for drugs in the case of Plasmodium falciparum,3,4 in autoimmune and inflammatory diseases,5 or in general.6,7 Finally, a recent review is listing many DHODH inhibitors also described here.8 As depicted in Scheme 1, the de novo uridine monophosphate biosynthetic pathway comprises six biochemical steps.9,10 In mammalian species, the first three steps are catalyzed by a trifunctional enzymatic complex comprising the carbamoyl phosphate synthetase II, the L-aspartate transcarbamylase, and the dihydroorotase.11 The next step, the oxidation of intermediate dihydroorotic acid (4) catalyzed by DHODH to give orotic acid (5), is further described below. This is followed by the orotic acid N-glycosylation to give orotidine 5′-monophosphate (6), and then a decarboxylation provides uridine 5′-monophosphate (7).

a

(i) Carbamoyl phosphate synthetase; (ii) L-aspartate transcarbamylase; (iii) L-dihydroorotase; (iv) dihydroorotate dehydrogenase; (v) orotate phosphoribosyl transferase; (vi) orotidine 5′-monophosphate decarboxylase.

present in databases, and DHODHs have been shown to vary in cofactor content, oligomeric state, subcellular localization, and membrane association. An overall sequence alignment allowed the clear distinction between two classes,12,13 which can be further divided into subclasses according to the cofactors

II. DIHYDROOROTATE DEHYDROGENASE, GENETICS, BIOCHEMISTRY, AND STRUCTURES DHODH, which is a flavin mononucleotide (FMN) (8, Figure 1) containing enzyme, catalyzes the oxidation of dihydroorotate (4) to orotate (5) and the reduction of FMN to dihydroflavin mononucleotide (FMNH2). More than 3400 sequences are © 2013 American Chemical Society

Received: December 14, 2012 Published: March 1, 2013 3148

dx.doi.org/10.1021/jm301848w | J. Med. Chem. 2013, 56, 3148−3167

Journal of Medicinal Chemistry

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using fumarate (10) as electron acceptor.14,15 In addition to a subunit (PyrDB) that is similar to class 1A DHODHs, the class 1B enzymes display an additional component (PyrK) containing an iron−sulfur cluster and a flavine adenine dinucleotide (FAD) (9). Class 1B DHODHs form heterotetramers and use NAD+ as electron acceptor.16,17 Interestingly, in some cases both types of enzymes are found in the same organisms (i.e., Lactococcus lactis, Streptococcus pneumoniae, Enterococcus faecalis). On the other hand, the archaeon Sulfolobus solfataricus has a peculiar cytosolic DHODH that is class 1S and closely related to class 1B but with a different PyrK component and a serine as the catalytic base.18 Class 2 DHODHs display a serine as the catalytic base19,20 and use coenzyme Q/ubiquinones (11)9 as the oxidant. They are membrane bound enzymes in Gram negative bacteria such as E. coli21 or integral inner mitochondrial membrane enzymes in higher eukaryotes.22,23 In the latter, the N-terminal sequence exhibits a bipartite signal of about 30 residues that mediates mitochondrial targeting and insertion into the inner membrane via a hydrophobic transmembrane sequence.23 The DHODHs from Plasmodium24−27 are also mitochondrial, contrary to DHODHs from Kinetoplastida,28 which are soluble enzymes. The 3D-structure of type 1A DHODH from L. lactis was the first to be solved in 1997 by X-ray crystallography.13,15,29 It is a homodimer, and each subunit features an α/β-barrel fold that is similar to other FMN-binding flavoproteins. Closely related structures were subsequently reported for other type 1A DHODH of T. brucei,30 T. cruzi,31,32 S. mutans,33 or L. major.34 The structure of a type 1B DHODH, also from L. lactis, was solved 3 years later.35 In this work, the catalytic subunit PyrDB and cofactor PyrK from L. lactis were cocrystallized, thus unraveling the overall structure of this heterotetrameric enzyme. The PyrDB subunit showed the characteristic α/βbarrel fold closely related to the type 1A DHODH from the same species despite only 30% sequence identity.35 The structure of human DHODH in complex with antiproliferative agents was solved in 2000.36 This provided the insights into specific features of type 2 DHODH. The membrane-association motif23 is adjacent to a pair of α-helices, α1 and α2, that are also specific of type 2 DHODH. These two helices are connected by a short loop and form the small N-terminal domain encompassing amino acids 30−68. The slot between α1 and α2 forms a hydrophobic funnel where ubiquinone is probably inserted and connects at its extremity with the FMN binding cavity. This small N-terminal domain is directly connected to the larger C-terminal domain of the human enzyme where binding sites for the substrate and the FMN are located. Again, this large domain folds into an α/β-barrel with a central structure of eight parallel β strands surrounded by eight α helices. The overall structure of this domain is highly conserved and showed strong structural similarities with DHODH from L. lactis despite only 16% sequence identity with human sequence.36 The structure determination of another type 2 DHODH, namely, from E. coli, pointed out that although both enzymes share a similar N-terminal fold, their primary sequences are very different, and this accounts for their distinct inhibition profiles.37 The α/β-barrel structure was also found for P. falciparum DHODH, although different structural features for the quinone binding site were also seen.38 As depicted in Scheme 2, the catalytic cycle leading to the oxidation of L-dihydroorotic acid (4) to orotic acid (5) takes place along the concomitant reduction of FMN (8), thus leading to the reduced FMNH2 (12). The enzyme is then able

Figure 1. Structures of compounds 8−11.

involved (see Table 1). Class 1 DHODHs are cytosolic enzymes and feature a cysteine acting as the basic residue catalyzing the oxidation reaction.12 This class has been further divided into two subclasses 1A and 1B, according to the cofactors involved. The class 1A enzymes are homodimers Table 1. Examples of DHODH Types

a

species

type

Dictyostelium discoideum Enterococcus faecalis Kluyveromyces lactis Lactococcus lactis Leishmania major Saccharomyces cerevisiae Streptococcus mutans Streptococcus pneumoniae Trypanosoma brucei Trypanosoma cruzi Bacillus subtilis Clostridium oroticum Enterococcus faecalis Kluyveromyces lactis Lactococcus lactis Streptococcus pneumoniae Sulfolobus solfataricus Arabidospis thaliana Aspergillus nidulans Bos taurus (bovine) Candida albicans Drosophila melanogaster Escherichia coli Helicobacter pylori Homo sapiens Mycobacterium tuberculosis Plasmodium falciparum Rattus norvegicus (rat) Schizosaccharomyces pombe Toxoplasma gondii

I IA IA IA IA IA IA IA IA IA IB IB IB IB IB IB IS II II II II II II II II II II II II II

refsa 39 40 41 12, 42, 43 46 14, 48−50

refsb

13, 15, 29, 44, 45 34, 47 33

30 51 16 52 53 41 15, 17 18 54, 55 56 57 58 59 12, 19, 60−64 65 20, 62, 66−71 24, 27 68, 71,82, 83 14 85

30 31, 32

35

37 36, 72−79 38, 80, 81 84

References for biochemical studies. bReferences for X-ray structures. 3149



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been used only to spot the reduction activity induced by ubiquinone and dihydroorotic acid on acrylamide gels containing DHODH.66,88 Predictably, all the high-throughput assays undertaken on type 2 DHODH have used 2,6dichloroindophenol (13) to regenerate the co-oxidant, thus avoiding false positives due to eventual UV absorbance of the compounds tested.105−108

Scheme 2

III. INHIBITORS OF DIHYDROOROTATE DEHYDROGENASE AND THEIR USES III.1. Substrate-Based Inhibitors. Early work109 described weak substrate-based inhibitors of DHODH, such as the 5methylated dihydroorotic acid (15),110 the triazine 16110 (or barbituric acid111), the 5-fluoroorotic acid (17)49,112 the 5aminoorotic acid (18),112 and the azaorotic acid 19 (Figure 2).42,109 Modest activities for the 5-fluoroorotic acid (17) or the

to oxidize back FMNH2 by using a variety of co-oxidants that depend on the species considered: (FAD (9), fumaric acid (10), or coenzyme Q/ubiquinone (11)). Interestingly, in vitro, exogenous oxidants such as 2,6-dichloroindophenol/DCIP (13),60,86,87 nitroblue tetrazolium (14),66,88 and various quinones12,55,59,68,70 or potassium ferricyanide42 can be used to regenerate the coenzyme Q/ubiquinone (11) consumed by the enzyme. The mechanism of the dehydrogenation of dihydroorotic acid by DHODH has been the subject of extensive studies.12,19,42−44,62−64,89−101 It is beyond the scope of this review to provide a detailed picture of the results reported so far. Two kinds of mechanism have been envisaged: (i) the two C−H bonds of dihydroorotic acid (4) are breaking in a concerted fashion; (ii) in a stepwise mechanism, following the C−H bond break, the resulting iminium (or enol) equilibrates into orotic acid (5). The class 1 DHODHs were demonstrated to follow the concerted mechanism,12,43,52 and the class 2 compounds are using the stepwise mechanism.62,63 Different types of biochemical assays have been designed to study these reactions. The production of orotic acid has been monitored by paper chromatography,66,102 HPLC (optimal wavelength for orotic acid detection is 278.5 nm),103 or use of 13 C-marked dihydroorotic acid.104 It can also be monitored spectrophotometrically at various wavelengths corresponding to the co-oxidant/exogenous oxidants used,19,57 for instance, at 287 nm (if 2,3-dimethoxy-5-methyl-1,4-benzoquinone12 or coenzyme Q are used, 70 ), at 296−300 nm (if it is decylubiquinone,70,71), at 300 nm (if it is fumarate,19,51), and at 420 nm (if it is potassium ferricyanide42). An important shift of this frequency can be achieved when using 2,6dichloroindophenol (13), as its reduction is monitored at 600−610 nm.60,66,71,83,87 The nitroblue tetrazolium (14) has


azaorotic acid 19 were also reported against the type 1A DHODH of T. cruzi.47 The 3-hydroxy benzoate analogues 20 and 21 were also reported to be inhibitors of the type 1A DHODH of L. lactis.45,113 as well as the salicylhydroxamic acid (22).114 The binding modes of compound 20 and 21 to L. lactis DHODH were determined by X-ray crystallography, and this further demonstrated their affinity for the dihydroorotate binding pocket (2BX7 and 2BSL).45 Many biologically active benzoquinone-bearing compounds115 such as dichloroallylawsone (23) or atovaquone (24) were investigated for a potential inhibition of DHODH.116Interestingly, quite wide variations of inhibition power were observed according to the type of DHODH studied. For instance, atovaquone (24) is a competitive inhibitor of human DHODH with regard to its ubiquinone cofactor with a Kie of 2.7 μM and an IC50 of 14.5 μM whereas for rat DHODH, the Kie is 60 nM and the IC50 is close to 700 nM.116 This actually led to the structure resolution of atovaquone (24) bound to rat DHODH which showed a strong interaction between Arg136 and its α-ketohydroxy 3150



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component along with extensive hydrophobic contacts (1UUM).84 The antimalarial drug atovaquone (24) is also a modest inhibitor of P. falciparum DHODH in vitro (Ki = 27 μM).117 But its strong effect on the pyrimidine nucleotide pool seen in vivo takes place via a selective inhibition of the parasite respiratory chain (the cytochrome bc1 complex), thus blocking the regeneration of the DHODH ubiquinone cofactor.117−121 Such weak inhibition was also recently reported for a series of quinolone inhibiting the human and Plasmodium mitochondrial electron flux.122 Interestingly, the quinone-containing derivative 25, a compound studied for its cytotoxicity, was reported to inhibit human DHODH by analysis of its effect on the COMPARE array of 60 different human tumor cell lines of the NIH.123 Antimycin A4 (26), which specifically inhibits the site of ubiquinone reduction in cytochrome bc1, turns out to also modestly inhibit human DHODH by competing with the ubiquinone cofactor.70,114 III.2. Inhibitors of Human DHODH. Leflunomide/Arava (27) is the first apparent inhibitor of DHODH124 that was approved for use in human medicine in the treatment of rheumatoid arthritis more than 10 years ago.125−127 However, this compound, which came out of a screening for pesticides, turns out to be a prodrug leading, nonenzymatically via a basecaused isoxazole ring-opening reaction taking place in plasma and intestinal mucosa, to the malonitrile amide teriflunomide/ A77-1726/Aubagio (28). It is this amide that is the inhibitor of human DHODH,67,128−133 whereas the parent leflunomide (27) is actually devoid of effect, in vitro, on DHODH.131 An excellent medical review of this compound is providing a recent picture of its potentials and risks in human medicine.134 The success of leflunomide (27) against one type of autoimmune disease has led to the use of teriflunomide (28) against multiple sclerosis.135−139 Leflunomide/teriflunomide has also been considered for the treatment of dermatological diseases such as psoriasis.134,140 Moreover, leflunomide (27) was found to have an effect on cytomegalovirus proliferation141 and it was more recently claimed142 and reported to be useful in removing the inhibition of fluid clearance caused by respiratory syncytial virus.143,144 A recent review is also suggesting a potential advantage in the use of teriflunomide (28) as an immunosuppressive agent with an antiviral effect on cytomegalovirus, since this virus remains a major cause of transplantation failure.145 However, a recent report has reported an in depth study of the reasons for an actual activation of hepatitis B virus proliferation by nucleoside biosynthesis inhibitors, including teriflunomide (28).146 The cocrystallization of teriflunomide (28) with human DHODH was achieved (1D3H).36 This inhibitor binds to the hydrophobic channel, specific to the type II enzymes, which is ended by the binding site of flavin mononucleotide (8) and thus allows the oxido−reduction reaction with ubiquinones (11). As depicted in Figure 3, aside from hydrophobic contacts with the 4-trifluoromethylphenyl moiety, the main interactions of compound 28 with human DHODH are mediated by a water molecule linking the amide oxygen and the residue Arg136 as well as a hydrogen bond between its carbonyl group and Tyr356. Although teriflunomide (28) is only a weak inhibitor of P. falciparum DHODH, its binding mode was also determined by X-ray crystallography (1TV5). It appears that this compound also binds to the (rather smaller) ubiquinone-binding hydrophobic channel of P. falciparum DHODH, although in a different fashion. Indeed, the amide side chain of teriflunomide (28) undergoes a 180° rotation, thus leading to a direct interaction between its ketone

Figure 3. Structures of compounds 27 and 28 and binding mode of 28 to human DHODH.

function and the corresponding arginine residue (Arg265). Few less efficient hydrophobic interactions with the 4-trifluoromethylphenyl moiety are also seen in this case.38 Extensive structure−activity relationship studies have been reported for analogues of teriflunomide (28). A fairly systematic variation of its substituents led to the cyclopropylbearing analogue HR325 (29, Figure 4) which had optimized


pharmacokinetic properties with regard to its plasma halflife.147 Indeed, a drastic drop of its half-life in patients (t1/2 = 3.5 h) was observed, in comparison with more than 2 weeks for teriflunomide.134 However, if this compound underwent phase II clinical trials around 1996,147 no results have been reported so far. Concerning the analogue MNA715/maritimus/FK778 (30),2,130,148,149 this compound underwent clinical trials as an 3151



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agent to prevent renal transplant rejection in 2004.150 Unfortunately, it appears that its development was arrested because at least two additional phase II trials were terminated.151,152 The analogue MNA279 (31)2,130,149 as well other analogues153,154 pointed out that other types of electronwithdrawing groups are possible on the phenyl position. Some functionalization of the methyl group of teriflunomide was possible because the acid 32 was only slightly less potent in vitro.155 Replacement of the amide function of teriflunomide (28) by a thioamide led to a slightly less effective inhibitor in vitro or in vivo.156 Larger substituents on the amide side of this scaffold were found to be possible.157 More recently, many biphenyl analogues of teriflunomide such as compounds 33 and 34 (or derivatives featuring a cyclopropyl instead of a methyl group) were studied for their inhibition of human and P. falciparum DHODH. However, within that series the compounds were usually far stronger inhibitors of the human enzyme (i.e., 33) and at best (i.e., 34) weak inhibitors of both.75 Extensive structural studies of the binding modes of this series with human DHODH pointed out a second type of conformation adopted by compound 33. This binding mode involves a 180° rotation of its amide bond and thus leads to a water-mediated interaction between Arg136 and the ketone moiety as well as second water-mediated interaction between Tyr147 and the cyano group (3FJ6).75 Further research led to the ester 35 with a greater potency in vivo on human DHODH than teriflunomide (28). The crystal structure resolution of compound 35 bound to human DHODH pointed out an orientation similar to the one adopted by teriflunomide (28) as well as additional hydrophobic interactions due to the additional 4-fluorophenyl ring (3U2O).78 An earlier attempt to replace the plausible cyclic structure, made by the enol hydrogen and the amide of teriflunomide (28), with a pyrazole bioisostere led to human DHODH inhibitors such as 36.158,159 However, this series has remarkably narrow structure−activity relationship and is devoid of effect in vivo. In light of the actual conformation adopted by teriflunomide (28) in the hydrophobic channel of DHODH, further studies would probably be useful to elucidate this fact. Recently, an attempt to replace the isoxazole ring with a 1,2,5-oxadiazole that led, by ring-opening, to the weak oxime-bearing inhibitor 37 pointed out the key role of the isoxazole ring.160 Quite large substituents, as seen in the structure of 38, appeared to be also possible, since this unlikely analogue displayed an effect on an arthritis animal model.161 A structure-based study led to inhibitors of human DHODH related to teriflunomide (28), devoid of a nitrile function but retaining a ketone moiety as seen in compound 39 (Figure 5) or a bioisostere such as the phenol group of salicylate 40.162 Interestingly, some structural similarities can been seen with a series of immunomodulators such as compound 41,163 not to mention the ill-fated linomide (42)164 or its backup laquinimod (43),165,166 as well as heterocyclic-bearing analogues,167 for which mechanisms of action have yet to be reported. Few trifluoromethylphenylamides such as compound 44 were claimed for their antiviral properties against human cytomegalovirus. It was actually stated that these antiviral properties were due to the inhibition of “kinases from pathogens”, although they also displayed a modest inhibition of human DHODH.106 Another amide, featuring a Michael accepting acrylate moiety, was reported for an anti-influenza effect in vitro mediated by the uncompetitive inhibition of DHODH.168 Moreover, amides such as compound 45 have been claimed for their inhibition of human DHODH and their potential use in cancer or allergy.169


A recent virtual screening followed by a biochemical assay led to an extensive list of the usual suspects (i.e., aldehydes, hydrazones, imines, polyphenols, 1,4-acceptors) as well as few weakly active amides, unrelated to 44 or 45, which could deserve further investigation.170 Brequinar (46, Figure 6), a compound initially studied for its anticancer properties,171 was shown to be one of the strongest

Figure 6. Structure of compound 46 and its binding mode to human DHODH.

inhibitor of human DHODH.102,172 Although it must be emphasized that if the IC50 for human DHODH is 10 nM, this value is 367 nM for rat DHODH.116 Unfortunately, a phase II clinical trial demonstrated only very modest anticancer effects on a number of solid tumors.173−177 This compound was also studied as a potential immunosuppressant73,178−181 to the point of a phase 1 clinical trial.182 Its use against viral diseases has also been investigated,183 including in conjunction with interferon.184 Interestingly, partially brequinar-resistant strains of dengue virus that displayed mutation on two viral-encoded proteins (a protein of the viral envelope and the nonstructural 5 polymerase domain) were also characterized.185 More recent anti-influenza screening also identified analogues of brequinar (46) and pointed out a puzzling effect on the influenza NS1mediated mRNA export block possibly caused by diminishing availability of pyrimidine nucleotides.186 The cocrystallization of brequinar (46) with human DHODH was also achieved (1D3G).36,73 This inhibitor binds to the same hydrophobic channel as teriflunomide (28). However, in this case, a direct salt interaction between the carboxylic acid and Arg136, which has chased away the resident water molecule seen for the 3152



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was investigated for its effect on an animal model of arthritis.188 Its seven-membered ring appears to be optimal in comparison with a previously reported tetracyclic derivative featuring a sixmembered ring instead.189 More recently, a patent claiming antiviral compounds, very likely inhibitors of human DHODH, further illustrated the variations possible on the first phenyl ring of brequinar (46). This ring was replaced by a variety of heterocycles, often by a pyrazole as for compound 50 or an indole, as previously seen in compound 48. Interestingly, the furan equivalent to such series was reported for their antimicrobial activity.190 Moreover, the naphthyridine analogue 51 was also claimed in this patent.191 If the replacement of the quinoline by a pyridine ring led to a compound devoid of effect on DHODH,104 this was not valid for benzene-containing series of inhibitors of human DHODH such as AMX02 (52)72 or compound 53.74 The structure of compound 52 bound to human DHODH pointed out an expected interaction between Arg136 and the carboxylic acid as well as a hydrogen bond between Tyr356 and the amide moiety (2B0M).72 The structure of compound 53 bound to human DHODH pointed out an interaction between Tyr356 and its methoxy group as well as the interaction between Arg136 and the carboxylic acid (2PRL).74 Comparatively fewer hydrophobic interactions for these two inhibitors probably explain their affinity decrease in comparison with brequinar (46). Many benzoic acid derivatives, such as compound 54, were claimed for their inhibition of DHODH and their use as “antiinflammatory agents, autoimmune disease remedies, immunosuppressants, anticancer drugs, fungicides, and virucides”.192 Even the ester 55, somehow structurally related to brequinar, turned out to inhibit the growth of P. falciparum and was found to be a modest inhibitor of human or P. falciparum DHODH.108 In a research program actually aiming at avoiding such inhibition for a series of P-selectin antagonists, the orthohydroxylated acids 56 and 57 were reported to be strong inhibitors of human DHODH.193 Somehow related to this aspect, the hydroxybearing naphthyridine derivative 58, a good inhibitor of phosphodiesterase 10A with a potential as an antipsychotic or antidiabetic drug, was also found to inhibit human DHODH.194 The cytotoxic redoxal (59, Figure 8) which features two anthranilate groups was also found to be a human DHODH inhibitor by analysis of the response pattern on the NIH COMPARE panel of 60 different human cancer cell lines.123,195 From the activity of this compound, along with the structure of 53, extensive research led to many series of strong human DHODH inhibitors. The human DHODH inhibitor LAS186323 (structure not disclosed), currently in phase I clinical trial,196 is probably related to the 2-phenylpyrimidine analogue 60 or the nicotinic acid derivative 61, as both are representative examples of the series claimed by the research group.197−201 These compounds have the potential to be used against autoimmune diseases (alone or in conjunction with methotrexate) or against proliferative skin diseases.197−201 A series of more rigid bicyclic derivatives such as the benzimidazole 62 were also claimed for their inhibition on human DHODH as well as for their cytotoxic effect.202 Fragments “extracted” from the structures of known inhibitors of DHODH have provided insights into the design of inhibitors. This approach led to the triphenyl anthranilate 63.77 An X-ray derived structure pointed out that, as for brequinar (46), the carboxylic acid group of compound 63 interacts tightly with the Arg136 and Gln47 whereas, contrary to brequinar (46), the amide moiety is able to interact with the

interaction with teriflunomide (28), provides the main polar interaction. Further hydrogen bonding with Glu47 is also seen. The Tyr356 residue is not in interaction with brequinar (46), but nine hydrophobic contacts with the enzyme are existing. These hydrophobic contacts include the interaction between Phe62 residue and the 2-fluorophenyl component of brequinar (46). Interestingly, in rat DHODH, this residue is replaced by a valine which explains the significant loss,71 from an IC50 of 6 nM to 127 nM, of brequinar activity in this case (1D3G for the 2′-defluoro analogue of 46 and 1UUO).36,84 Prior to 1999, the structure−activity relationship for brequinar derivatives has been well reviewed.1,104 These studies pointed out the importance of the quinoline ring (a pyridine analogue was devoid of effect), the requirement for a group as bulky as a biphenyl on carbon 2, and the necessity of a carboxylic acid moiety on carbon 4. The fluorine atoms were found to be optimal as well, although other electron-attracting groups could replace the fluorine on carbon 6.104,184 The meta substituted phenyl ether analogue 47 (Figure 7) was also found to be an effective inhibitor as well as bicyclic derivatives such as indole 48.187 The more rigid DHODH inhibitor KF20444 (49)

Figure 7. Structures of compounds 47−58. 3153



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hydroxyl moiety of Tyr356 (2WV8).77 Other acids such as the cyclopropyl or the ethyl bearing inhibitors 64 and 65 were found by virtual screening, although the structures actually selected by the computer model had been the corresponding amides that only contained trace amounts of acids 64 and 65.76 Finally, out of all these anthranilate derivatives,77,203 the human DHODH inhibitors ABR-222417 (66) and ABR-2224050 (67) have been assayed fairly successfully in an in vivo model of graft rejection.204 Concerning the binding mode of compound 64, a hydrogen bond is seen between the pyridine nitrogen and Thr63, the carboxylic acid is bonded to the Arg136 residue, and one of its oxygen atom forms a water-mediated bond with Gln47. Moreover, the carbonyl oxygen of the ethylamide is hydrogen-bonded to Tyr356 (3KVJ).76 Interestingly, the cyclopropyl moiety of compound 64 appears to provide optimal hydrophobic interactions in comparison with the ethyl analogue 65, since a decrease in affinity (qualified as dramatic) for human DHODH was mentioned.76 Another series of carboxylic acid bearing human DHODH inhibitors, such as the amide 4SC-101/SC12267/vidofludimus (68, Figure 9), have been studied in depth. This compound was reported for its in vivo effect against a murine model of the systemic lupus erythematosus73,205−208 as well as in a model of inhibition of graft rejection.209 Concerning human trials for this inhibitor, the company Web site mentions “a positive exploratory study” against inflammatory bowel disease,210 but another trial, against rheumatoid arthritis, “did not meet its primary end point”.211 The structures of an array of analogues of compound 68 bound to human DHODH were determined.73 As for brequinar (46), these inhibitors occupy the ubiquinone binding site. However, contrary to brequinar (46), the carboxylic acid function of compound 69 was found to adopt a rotated conformation as it is in the interaction, via a hydrogen bond, with Tyr356 and with Tyr147 via a water-

mediated interaction (2BXV). Even more unexpected, slight structural modifications led to analogues with a dual mode of binding for their carboxylic functions, as “brequinar-like” and “non brequinar-like” orientations could be seen in the structures obtained.73 Replacement of the acid function by the bioisosteric hydroxyfurazan moiety as depicted for compound 70 also led to good rat DHODH inhibitors.212 An inhibition of human DHODH for the urea derivative 71 and the hydrazone 72 was also found in the course of the virtual screening which led to compounds 64 and 65. The X-rayderived structures of compound 71 or 72 bound to human DHODH confirmed the interaction between their carboxylic moiety with Arg136 (3KVK and 3KVM).76 A series of phthalimide derivatives, structurally related to compound 68, such as 73 have been claimed recently for their strong inhibition of human DHODH.213 Somehow unrelated to these inhibitors, Michael accepting benzylidene thiazolidines214 or the amides 74 and 75 belong to a series of compounds claimed for their inhibition of antibodies production via their action on human DHODH.215 The biphenylpyridine 76 is an example of another series of very strong inhibitors of human DHODH.216 Dicoumarol (77, Figure 10), a compound with many reported biological effects, was demonstrated to act on DHODH because the addition of orotic acid (5), but not dihydroorotic acid (4), in the culture medium alleviates its effect on HL-60 cell growth.217 Following this discovery, few additional 4-hydroxycoumarins were also demonstrated to modestly inhibit human DHODH, the 4,5-dihydroxycoumarins being the strongest inhibitors.77 The inhibition of DHODH as a mechanism of action for the cytotoxic azacarboline NSC 665564 (78) was also identified by its effect profile on the 60 different human cancer cell lines of the NIH COMPARE research program.218 In view of GSK 983 (79), a compound reported for a broad-spectrum antiviral effect in cellulo,219−222 a 3154



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the series, only compound 86 showed a modest effect on P. falciparum growth in vivo.107 From of a high-throughput screening on P. falciparum DHODH, the amide 87 demonstrated a modest effect on the parasite growth.233 This was followed by a medicinal chemistry program, and far more promising amides such as 88−90, endowed with an effective antimalarial effect in vivo, were obtained.81 Extensive preclinical studies pointed out an hERG channel inhibition issue (IC50 < 2.8 μM) for compound 90.81 Further work led to the design of the nitrile-bearing derivative Genz-669178 (89) with an hERG channel inhibition of 53 μM. This, along with an oral efficacy on mouse models of P. berghei or P. falciparum infections, led to the selection of compound 89 for further development.234 From another high-throughput screening, an efficient series of triazolopyrimidines inhibiting P. falciparum DHODH was recently reported. From the initial hit 91 (Figure 12),107,235 extensive medicinal chemistry led to the strong inhibitors 92 and 93 which are the most advanced of this series toward a clinical trial against malaria.235−239 In general, the trifluoromethyl group of compound 92 or the noteworthy SF5 group of DSM 265 (93) greatly improved the metabolic stability of


cellular DHODH inhibition does not look so improbable. However, so far, no study supports this hypothesis solely based on structural similarities. Somehow related to compounds 78 and 79, the tetracyclic derivative 80 is an example of a series of inhibitors of human DHODH claimed in the past.223 The broad spectrum antiviral 81 was found to exert its effect via the inhibition of the host cell DHODH.224 Similarly, following a phenotypic screening, the lipophilic broad-spectrum antiviral pyrazole−isoxazole (82) was demonstrated to act on the host cell DHODH.225,226 Interestingly, this DHODH inhibitor is sharing structural features with R803 (83), a substance reported for an effect on hepatitis C virus proliferation, for which a mechanism of action has yet to be reported.227 III.3. Inhibitors of P. falciparum DHODH. The elimination of P. berghei parasites in mice using the anticancer drug 5-fluoroorotic acid (17) or 5-aminoorotic acid (18) could have provided a proof of concept regarding the use of DHODH inhibitors in the treatment of malaria.112 However, concerning 5-fluoroorotic acid (17), as this compound is converted into 5fluorouracil, a good inhibitor of the parasite thymidylate synthase, the antimalarial effect seen was probably due to the inhibition of this enzyme rather than DHODH.228,229 In any case, another study pointed out that inhibitors of the mammalian DHODH were often weak inhibitors of P. falciparum DHODH. This study also concluded that the design of parasite-specific inhibitors could lead to antimalarial drugs, especially since a pyrimidine nucleoside salvage pathway is not present in this parasite.24 The stabilized enamine derivative 84 (Figure 11) is representative of a series of inhibitors of P. falciparum DHODH with some structural points in common with teriflunomide (28).230 Related (and smaller) analogues were studied for their effect against human DHODH.231 Few ortho carboxyanilides such as compound 85 were designed by the same group and studied for their effect on P. falciparum DHODH.232 A high-throughput screening on P. falciparum DHODH led to an array of amides as potent in vitro inhibitors also selective over the mammalian orthologue. However, out of

Figure 12. Structures of compounds 91−99. 3155



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the arylamine component of this series.237,238 On the basis of this aspect as well as a good in vivo efficacy on the humanized SCID mouse model, compound 93 has been selected for further development.238 An intrinsic dipole occurrence (the tautomeric pyrimidine-2,4(1H,3H)-diimine form) was reported to be the key to the mode of binding of this type of inhibitor to P. falciparum DHODH.80 Extensive rescaffolding of the core heterocycle led to compounds such as 94−97. Their biological activity further confirmed this dipole occurrence as compounds 94 and 95 retain effects on P. falciparum DHODH, whereas 96 is a 100 times less active than 91 and compound 97 is inactive.240 Of note is a recent report describing the triazolo[1,5-a]pyrimidine 98 (depicted with its pyridone tautomeric form) as a dual inhibitor of human and P. falciparum DHODH. Interestingly, variation of the sulfuryl group greatly altered the selectivity of this inhibition and compound 99 turns out to be a very strong and selective inhibitor of human DHODH.79 The ensuing X-ray based crystal structure study pointed out a 3 Å shift of the ubiquinone binding pocket of the human DHODH, which is caused by the 2-chloro substituent on the benzyl moiety of compound 99. As such shift does not seem to be possible for the P. falciparum DHODH, a fairly strong selectivity of inhibition is thus observed (3ZWT).79 The interaction mode of P. falciparum DHODH with compound 9081 or compound 9180 was determined by X-ray crystallography (3O8A and 3I65). Both compounds are, as teriflunomide (28), bound to the ubiquinone binding tunnel. As depicted in Figure 13, in the case of compound 90, the Arg265 and His185 residues are in interaction, via hydrogen bonds, with the oxygen and nitrogen of the amide moiety. As seen for the cyclopropyl group of compound 64 bound to human DHODH, the cyclopropyl substituent of compound 90 is very close from the FMN in its binding site, and two crucial hydrophobic interactions are thus secured. Additional hydro-

phobic interactions are also seen with the benzimidazole ring system. In the case of compound 91, the Arg265 and His185 residues are interacting, via hydrogen bonds, with two nitrogens of the triazolopyrimidine which adopts the depicted tautomeric pyrimidine-2,4(1H,3H)-diimine form. An additional watermediated interaction is also seen between Tyr258 and the third nitrogen of the triazole ring system. Few hydrophobic interactions are also seen with the β-naphthyl moiety which adopts a very different conformation than the benzimidazole group of compound 90 (not properly depicted by this 2D projection). This difference of orientation, which is the consequence of a different architecture of this part of the DHODH binding pocket, is allowing the edge to face interaction between Phe188 and the naphthyl ring. The X-ray derived structure of compound 93 bound to P. falciparum DHODH shows the same key interactions with the heterocyclic part of compound 91 (3SFK).238 Moreover, as for the cyclopropyl group of compound 90, the difluoroethyl group of compound 93 is providing the additional hydrophobic interactions that make this compound one of the strongest of the series. III.4. Inhibitors of Bacterial, Coccidian, or Fungi DHODH. Hydantoins such as 100 (Figure 14) were reported

Figure 14. Structures of compounds 101 and 102.

to be inhibitors of Clostridium oroticum DHODH.241 Along with additional work,242,243 a recent report on a related series is probably a good reason to consider today such an inhibition with caution.244 Following a biochemical screening, a series of pyrazoles featuring three amide functions, such as compound 101, were found to selectively inhibit the ubiquinone binding site of H. pylori DHODH.65 Interestingly, the isomeric pyrazole (with regard to the nitrogen-bearing 4-methoxyphenyl group) was also found to be active (although 50 times less) on H. pylori growth.245 The same screening led to the identification of a series of bacterial DHODH inhibitors such as the thiadiazolidinedione 102 which is only a weak inhibitor of bacterial growth and probably a reactive chemical entity.246 Series of such compounds, claimed for their antibacterial effect,247,248 have also been reported as inhibitors of S. aureus alanine racemase249 and more recently for nanomolar affinity for retinoblastoma protein pRb.250 Such a lack of selectivity is all too often the hallmark of frequent hitters,251,252 promiscuous253−256 as well as nuisance compounds257 or PAINS258 that tend to plague the results of too many reported biochemicalbased screenings. The triazinetrione-bearing antiparasite toltrazuril (103, Figure 15), which is used as a coccidiostat/growth promotant in farms, was demonstrated to be a modest inhibitor of rat DHODH.2,259 However, a primary effect on the parasite

Figure 13. Binding mode to P. falciparum DHODH of compounds 90 and 91. 3156



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when assessing the biological effects on any animal model. If the historical inhibitors were found because of their cytotoxicity on cancer cell lines, quite a few series arose from biochemical screenings or more recently from phenotypic assays for antiviral compounds.186,191,225,226 In vitro, this class of inhibitors is more cytostatic than overly cytotoxic on cancer cell lines but they display a strong and selective effect on lymphocyte proliferation.268 This effect may well be at the source of their action on the immune system and thus on uncontrolled immune responses phenotypes. However, the question of whether such immunosuppresion is due to the depletion of the pyrimidine nucleotide pool of lymphocyte T or to more complex intracellular/intercellular responses (i.e., interferon or interleukin-based)210,269−273 remains a very current research area outside of the scope of this review. The established use of leflunomide (27)/teriflunomide (28) against the autoimmune diseases rheumatoid arthritis and now multiple sclerosis139,274 has triggered a second wave of investigations in the domain. The issue of target identity and selectivity is recurrently the focus of concern for some DHODH inhibitors.275 These appeared in light of either in-depth cell biochemistry studies or puzzling cases of closely related analogues devoid of an effect on this enzyme but still displaying in assays a phenotypic pattern somehow related to DHODH inhibition.106,158,159,276−280 Interestingly, at least for two compounds, some of the phenotypic effect of DHODH inhibitors on mice (brequinar (46)181 and leflunomide (27)278,279,281) could not be alleviated by the coadministration of uridine. A more recent work has also reported a troubling causative link between aryl hydrocarbon receptor expression and the effect of leflunomide (27), but not teriflunomide (28), on melanoma cells proliferation.282 All this can also be considered in light of LFM-A13 (110, Figure 16), a not so selective kinase


respiratory side chain along with secondary effects on pyrimidine biosynthesis has been suggested.260 Although toltrazuril (103) was not specifically investigated, the inhibition of Toxoplasma gondi DHODH by some of the earlier human DHODH inhibitors (i.e., the orotic acid analogues 15 and 18) has been reported.261 In any case, the tricarbonyl pattern of 103 is also found in a recent patent, claiming compounds such as the Michael acceptor 104 for their inhibition of bacterial DHODH.262 The mechanism of action of the lipophilic antifungal LY214352 (105), which was found by analysis of resistant strains of Aspergillus nidulans, turned out to be the inhibition of its DHODH.56 Interestingly, this compound is structurally related to a series of mildewicide such as quinoxyfen (106),263,264 although DHODH may not be the primary biochemical target of compound 106.265 An extensive biochemical screening using the cloned oomycete Pythium aphanidermatum DHODH was also undertaken and led to few hits.105 The ensuing structure−activity relationship study yielded a series of strong and also very lipophilic oomycete DHODH inhibitors such as compounds 107 and 108. Unfortunately, these inhibitors were not stable enough under sunlight to be further developed as fungicides.266 Finally, quinazolinones such as 109 have been recently claimed as inhibitors of fungal DHODH with antifungal properties.267

Figure 16. Structure of compound 110.

inhibitor283−285 with a structure related to teriflunomide (28), although allegedly devoid of effect on DHODH,280,286,287 which has been considered for the treatment of acute lymphoblastic leukemia.280 In any case, the sometimes severe side effects of leflunomide (27)/teriflunomide (28), such as diarrhea, abnormal liver tests, nausea, and hair loss,139 may not all be due to DHODH inhibition by 28. Accordingly, would another (more potent) human DHODH inhibitor, with a fully different chemotype, display the same clinical benefit and side effects profile? The answers to such questions are probably what will be used for assessing the interest of providing in the future a second DHODH inhibitor to the human pharmacopoeia. Other encouraging aspects for this class of inhibitors are the demonstration, on models of melanoma, of the interest of associating a DHODH inhibitor and an anticancer drug288 or the recent patent claiming the use of leflunomide (27), teriflunomide (28), or 29 for the treatment of central nervous system trauma such as spinal cord injury.289 Concerning the selective inhibitors of P. falciparum DHODH, the coming years will see if the current inhibitors fulfill their promises in the

IV. CONCLUSION The search and development of inhibitors of DHODH is a risky one. It is indeed strewn with compounds that failed to pass the stage of clinical trials (i.e., brequinar (46), 29, and 30). If no data are available for the failures of 29 and 30, brequinar (46) displayed disappointing results in clinical trials aimed at demonstrating an antitumor effect. It may just be that the tumor cell pyrimidine salvage pathway could compensate for the pyrimidine nucleoside pool depletion, thus avoiding the expected anticancer effect. Concerning current research on DHODH inhibitors, the difference of inhibition power of, for instance, atovaquone (24) or brequinar (46) toward human or rat DHODH116 is also a crucial issue that must be addressed 3157



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treatment of malaria. In any case, the great potential of this target against malaria will certainly lead to renewed efforts to obtain additional series. The relevance of this target is very likely due to the fact that this parasite is lacking a pyrimidine salvage pathway. In the case of the protozoan parasite L. donovani, extensive genetic-based research pointed out that the de novo and the salvage pathways had to be affected to secure a loss of infectivity.290 A similar study on the de novo pyrimidine biosynthesis pathway of Trypanosoma cruzi may be more promising, as no pyrimidine salvage pathway seems to exist for this parasite.291 Recent work on antibacterial or antifungal compounds could also lead to series of interest. Finally, it is quite possible that inhibitors of plant DHODH are known/ used but the actual elucidation of their mechanism of action remains to be achieved/reported.

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Bisagni at the Institut Curie, France. He joined, for a 2-yearlong postdoctoral position, Dr. David S. Grierson at the ICSN, Gif/Yvette, France. He then enjoyed a postdoctoral year in Prof. Povl KrogsgaardLarsen’s research laboratory at the Danish School of Pharmacy in Copenhagen. Following 6 years at the Institut Curie as a junior CNRS scientist, he went on sabbatical for a year at the research facilities of Vitry/Seine Aventis, France, before joining the Institut Pasteur, France, in 2004. Across all these years, he has worked on various medicinal-chemistry-driven syntheses of heterocyclic derivatives concerning oncology, virology, neurobiology, and now infectious diseases.

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ACKNOWLEDGMENTS The Institut CarnotPasteur Maladies Infectieuses (Programme STING) is acknowledged for its financial support. Dr. Emile Bisagni is also acknowledged for his support and interest.

AUTHOR INFORMATION

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Corresponding Author

*Phone: 33 (0)1 40 61 39 92. E-mail: [email protected].

ABBREVIATION USED DHODH, dihydroorotate dehydrogenase; FAD, flavine adenine dinucleotide; FMN, flavin mononucleotide; FMNH2, dihydroflavin mononucleotide; hERG, human ether-a-go-go-related gene; NADH, dihydronicotinamide adenine dinucleotide; NIH, National Institutes of Health; PAINS, pan assay interference compounds

Notes

The authors declare no competing financial interest. Biographies Hélène Munier-Lehmann obtained her Ph.D. in Biochemistry in 1992 from the University of Paris VII, France, under the guidance of Dr. Octavian Barzu at the Institut Pasteur, France. In 1993, she went to Germany for a 3-year postdoctoral training at EMBL in the Cell Biology Program in the group of Dr Bernard Hoflack. In 1996 she obtained a permanent position at INSERM as a Research Associate and went back to France at the Institut Pasteur. For 15 years, she has been studying enzymes involved in the metabolism of nucleosides. Since 2007, she has been managing a screening platform on chemical libraries in order to obtain research tools or chemical starting points for therapeutic applications.

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REFERENCES

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Pierre-Olivier Vidalain is a research scientist from CNRS, France, and a project manager at Institut Pasteur, France, in the Viral Genomics and Vaccination Unit (G2V) headed by Dr. Frédéric Tangy. He did is Ph.D. on measles virus, studying cellular mechanisms responsible for immunosuppression induced by this important human pathogen (2002). After postdoctoral training in proteomics at the Dana-Farber Cancer Institute in Dr. Marc Vidal’s laboratory (Boston, MA, 2002− 2005), he developed at the Institut Pasteur a yeast two-hybrid platform dedicated to the high-throughput mapping of virus−host protein− protein interactions. This led him to identify and characterize several novel interactions between viral virulence factors and host signaling components. Since 2008, he has been developing phenotypic assays to screen chemical libraries in order to identify broad-spectrum antiviral molecules. Frédéric Tangy is the head of the Viral Genomics and Vaccination Research Unit at the Institut Pasteur, France. After a Ph.D. in 1980 and a Dr.Sc. in 1984 at Paris VI University, France, he made a career in virology. He is currently director of the Institut Pasteur international vaccinology course and vice president of the Institut scientific council. In the past 10 years, he has developed two research programs: (1) the generation of polyvalent viral attenuated vaccines derived from measles vaccine which led to preclinical and clinical development in the domain of HIV, dengue, and malaria; (2) the study of the interactions between viral and host proteins using modern biochemical tools. This has led the identification of new drug targets and determinants of pathogenicity/attenuation. Yves L. Janin obtained his Ph.D. in Organic Chemistry in 1993 from the University of Paris VI, France, under the guidance of Dr. Emile 3158



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