Inhibitors: A Promising Approach for - American Chemical Society

Sep 4, 2015 - crucial role in the promotion of glycolysis in invasive tumor cells. Recently, hLDH5 has ...... Reshma Rani received her Master's degree...
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Recent Update on Human Lactate Dehydrogenase Enzyme 5 (hLDH5) Inhibitors: A Promising Approach for Cancer Chemotherapy Miniperspective Reshma Rani* and Vinit Kumar Department of Translational Research, National Cancer InstituteCRO, Via Franco Gallini 2, Aviano 33081, Italy ABSTRACT: Human lactate dehydrogenase (hLDH5), a glycolytic enzyme responsible for the conversion of pyruvate to lactate coupled with oxidation of NADH to NAD+, plays a crucial role in the promotion of glycolysis in invasive tumor cells. Recently, hLDH5 has been considered a vital therapeutic target for invasive cancers. Selective inhibition of hLDH5 using small molecules holds potential prospects for the treatment of cancer and associated diseases. Consequently, significant progress has been made in the discovery of selective smallmolecule hLDH5 inhibitors displaying remarkable inhibitory potencies. The purpose of this review is to discuss briefly the roles of hLDH isoforms and to compile small hLDH5 inhibitors into groups based on their chemical classes and pharmacological applications.

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INTRODUCTION Cancer Cell Metabolism and LDH. Unlike normal cells, most cancer cells rely on an enhanced rate of glycolysis that tends to ferment glucose into lactate, even under aerobic conditions. The German scientist Otto Warburg observed for the first time the metabolic switch from oxidative phosphorylation (OXPHOS) toward aerobic glycolysis (Warburg effect) and established a link between cancer and the peculiar glucose metabolism in cancer cells.1 Initially, he hypothesized that the metabolic alteration specific to cancer cells is caused by a mitochondrial defect where complete oxidation of glucose is lost; however, it was later proven that this metabolic alteration is from oncogene-directed metabolic reprogramming, not from mitochondrial dysfunction.2−4 Tumor glycolysis is highly functional and is accompanied by high glucose consumption due to a lower efficiency in energy production that ensures an adequate and rapid energy supply and biosynthetic intermediates for rapidly growing cancer cells.4−6 In essence, cancer cells are hungrier for nutrients than normal cells are; thus, tumor glycolysis provides selective advantages to tumor cells for survival and proliferation. Recently, keen research interests in tumor glycolysis are emerging because of the strong metabolic dependencies of cancer cells. Key factors, such as the enzymes and transporters involved in glycolysis, are thus considered promising targets.4,6 Among these, the lactate dehydrogenase enzyme (LDH), which plays a central role in tumor glycolysis, has attracted attention for drug development against cancer. Structurally, LDH is a tetrameric enzyme composed of two major subunits, LDHA (muscle, M) and LDHB (heart, H), that are encoded by two separate genes, ldhA and ldhB, respectively. These two subunits, LDHA and LDHB, combine into five © 2015 American Chemical Society

different possible combinations, such as A4, A3B1, A2B2, A1B3, and B4, corresponding to five isoforms or isozymes, namely, LDH5 to LDH1, respectively (Figure 1A). The

Figure 1. Schematic representation of (A) LDH isoforms and (B) metabolic pathway in normal and cancer cells.

homotetramer LDH5 (A4, LDHA) is assembled only from subunit A and is predominately found in skeletal muscle, whereas LDH1 (B4, LDHB), formed from only subunit B, primarily exists in heart muscle and other oxygenated (normoxia) tissues. The remaining three isoforms, LDH2 (A1B3), LDH3 (A2B2), and LDH4 (A3B1), are hybrid tetramers (Figure 1A).7 An additional sixth isoform, LDH-X or LDH-C4, which is involved in male fertility, was identified in human testis and sperm.8 Human LDH5 (hLDH5) plays a crucial role in the metabolic switch, where glucose is preferentially catabolized into pyruvate and finally into lactate catalyzed by hLDH5 at the end of the glycolytic process in Received: January 29, 2015 Published: September 4, 2015 487

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structural isostere of pyruvate and a well-known pyruvatecompetitive inhibitor of hLDH5 (Ki = 136 μM) and hLDH1 (Ki = 94.4 μM),21 shows a synergistic inhibitory effect on paclitaxel- and trastuzumab-resistant breast cancer cells by promoting cellular apoptosis, thus confirming the correlation of hLDH5 with resistance to chemotherapy.22,23 Overall, the knockdown of hLDH5 by shRNA or small molecules offered definite confirmation of the critical role of hLDH5 in tumor initiation, development, maintenance, and chemoresistance therapy. Probable side effects due to hLDH5 inhibition might be expected in people with hereditary deficiency of the ldh-a gene, leading to a complete lack of the LDHA subunit or patient with decreased hLDH5 activity in muscles. They showed myoglobinuria only after intense anaerobic exercise due to muscle damage, without showing any side effects under ordinary circumstances.24 Taken together, the hLDH5 enzyme is considered a suitable and safe target; hence, the selective inhibition of hLDH5 by small molecules offers a novel approach for the development of potential anticancer agents in cancer chemotherapy and for overcoming resistance to chemotherapy. The chemical space of hLDH5 inhibitors has not been explored much because very few clinical applications had been envisioned until the link between hLDH5 and the Warburg effect was established. Initially, small-molecule inhibitors of LDH were developed as antimalarial agents that target the Plasmodium falciparum LDH isoform (pf LDH), a key enzyme for the survival of the malarial parasite;25 however, they were not selective and exhibited a certain degree of hLDH5 inhibitory activity as well. A few selective hLDH5 inhibitors have been developed, but none of them have hitherto established any real clinical benefits. The key to selective action by small molecules against cancer cells can be exploited in the expansion of chemical space of new “druglike” hLDH5 inhibitors with minimal toxicity. Research groups across academia and industry are currently engaged to discover highly potent and selective new hLDH5 inhibitors.26 Recently, GlaxoSmithKline (GSK) discovered potent NADHcompetitive hLDH5 inhibitors exhibiting inhibitory activity in the nanomolar range.27 This review covers the most relevant hLDH5 inhibitors that have been investigated so far. Figure 2 gives a general overview of various chemical classes of hLDH5 inhibitors.

cancer cells (Figure 1B). In normal cells, glucose is metabolized into pyruvate and then CO2 and acetyl-Co-A for entry into citric acid cycle under normoxia. While under hypoxic conditions, OXPHOS remains inactive and hLDH5 catalyzes the conversion of pyruvate into lactate coupled to oxidation of the cofactor NADH to NAD+. hLDH5 has a higher affinity and higher Vmax for pyruvate reduction than does hLDH1 and thus becomes more efficient for anaerobic metabolism and pyruvate reduction such as in hypoxic tumors. Conversely, hLDH1 favors the reverse reaction and is concerned more with aerobic oxidation of pyruvate.7 Analysis in several human tumors as well as adjacent normal tissues confirms the higher concentration of LDH M subunits in the tumors than normal tissues.9 In fact, there is a marked increase in concentration of LDH M subunits in cancerous tissues (thyroid and colon) compare to H subunits.9 Therefore, hLDH5 plays a central role in the reprogramming of tumor cell metabolism, suggesting that it might be required for the maintenance and survival of tumor cells. hLDH-5 and Tumorigenesis. The overexpression of hLDH5 has been reported in highly glycolytic human cancers10−15 and is transcriptionally upregulated by transcriptional factors responsible for hypoxic adaptation.10−14 It has been identified as a direct target of hypoxia inducible factor 1 (HIF-1),10−12 c-Myc,11−14 and the HER2/neu12 oncogene and was found to be correlated with aggressive phenotypes, cell proliferation, and poor prognosis in several tumors. Very recently, Cui et al. reported that the increased expression of forkhead box protein 1 (FOXM1) upregulated hLDH5 expression by direct binding of FOXM1 with the promoter region of hLDH5, causing increased LDH activity, lactate production, and glucose utilization in pancreatic cancer cell growth.15 Collectively, several genetic studies provide proof of concept that the dysregulated expression and activation of oncogenes are directly associated with hLDH5 expression and confirmed the involvement of hLDH5 in tumorigenesis. Moreover, elevated levels of hLDH5 caused higher lactate production, which triggered increased extracellular acidosis, resulting in a low pH and facilitating tumor invasion and metastasis.16 hLDH5 is associated with the viability of tumor cells9 and the growth of transformed spheroid cell mass in hypoxic environments.17 In fact, the silencing of hLDH5 expression by shRNA displayed a reduction in the ability of tumors to proliferate under hypoxic conditions, markedly delayed tumor formation, stimulated mitochondrial respiration,12,14,18 and reduced tumor growth, even in the background of fumarate hydratase-deficient cells in a renal cancer xenograft mouse model.19 The knockdown of hLDH5 directly by lysine-5 acetylation of hLDH5 and stimulating chaperone-mediated autophagy-regulated degradation of hLDH5 confirms that lysine-5 acetylation is inversely correlated in pancreatic cancer initiation and further established the role of hLDH5 in the regulation of cell growth.20 Furthermore, the inhibition of hLDH5 by small molecules reduces ATP levels and increases oxygen consumption, thus leading to increased production of mitochondrial reactive oxygen species (ROS) and oxidative stress and resulting in cell death.14 Recently, small molecules, as potential inhibitors of hLDH5, proved to be effective in hypoxic pancreatic cancer cell lines and exhibited synergistic cytotoxic activity with gemcitabine by attacking the key mechanisms involved in the proliferation, cell-cycle control, apoptosis, and migration of pancreatic cancer cells.17 The combination of paclitaxel and trastuzumab, separately, with oxamate, a

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DIFFERENT CHEMICAL CLASSES OF SMALL MOLECULES AS INHIBITORS OF hLDH5 The X-ray crystal structure of LDH revealed that there are two domains, the larger (cofactor binding) domain formed by residues 20−162 and 248−266 and the mixed α/β substrate binding domain (smaller) comprising residues 163−247 and

Figure 2. Overview of different chemical classes of hLDH5 inhibitors. 488

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267−331. The NADH cofactor binds to LDH at one end of the central β-sheet with residues His195, Asp168, Arg171, and Thr246 in an extended conformation, and its nicotinamide group partially overlaps the substrate-binding site.28 Analysis of a ternary complex (NADH-LDH-oxamate, Figure 3A)28,29

Figure 3. (A) Represenative example of hLDHA-NADH-oxamate (PDB code 1I10). (B) LDH-NADH (green)-pyruvate (purple) (1.80 Å resolution, PDB code: 3D4P). (C) Structure of substrate oxamate and pyruvate. Reproduced with permission (PLoS One 2014, 9e86365 and J. Med. Chem. 2011, 54, 1599−1612).29,30

Figure 4. Gossypol and gossypol derivatives (activity in μM).

nonspecific toxicity in biological systems, can interact with several cellular components, and may interrupt many cellular functions.35 A phase I/II clinical study of R-(−)-1 for treatment in patients with non-small-cell lung cancer (in combination with erlotinib), chronic lymphocytic leukemia (with lenalidomide), and metastatic solid tumors (with paclitaxel and carboplatin) has been terminated because of unacceptable toxicity, whereas that for B-cell chronic lymphocytic leukemia (with lenalidomide) and advanced non-small-cell lung cancers (with docetaxel and cisplatin) is ongoing.36

indicated that oxamate interacts with residues Gln99, Arg105, Asn137, Arg168, His192, and Thr247 via hydrogen-bonding, whereas Leu164 and Ala237 are engaged in hydrophobic contacts. The side chain of Arg168 interacts with the carboxylate group of the ligand. The closure of a mobile loop (residues 96−107; numbering refers to hLDHA in PDB 1I10), in which the conserved Arg105 could stabilize the transition state in the hydride-transfer reaction, is indispensible for catalytic activity. A different ternary complex (NADH-LDHpyruvate, Figure 3B)30 also showed similar kinds of bonding and confirmed the importance of Arg169, Thr248, and His193 for the ligand interactions. For example, the complex depicted significant interaction of Arg169, Thr248 with carboxylate group of pyruvate as well with the carbonyl oxygen of the substrate.29,30 These cocrystal structures enabled the rational design of chemical structures possessing potential inhibitory activities. The astonishing functional and structural diversity of the various chemical classes of hLDH5 inhibitors involving natural and synthetic molecules is described in the following sections.

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GOSSYPOL DERIVATIVES Further development of compound 1 as anticancer agent failed because of the lack of selectivity and because of serious side effects; therefore, several molecules that had chemical structures closely related to compound 1 (Figure 4) were designed. A derivative of 1, 1,1′-dideoxygossylic acid (2), was designed by replacement of the two aldehyde groups with carboxyl groups and concurrent removal of two hydroxyl groups, whereas in compound 3a (gossylic nitrile 1,1′diacetate) and compound 3b (gossylic nitrile 1,1′-divalerate), nitrile groups occupied the former positions of the aldehyde groups (Figure 4).37 Compounds 2, 3a, and 3b displayed good inhibitory activity, exhibiting respective Ki values of 1.3, 2.0, and 9.1 μM against hLDH5 and 0.7, 33, and 39 μM against hLDH1. Only compounds 3a and 3b were more selective toward hLDH5 than the parent compounds 1 and 2 (Figure 4). Cyclic derivatives of 1, such as gossylic lactone (4a) and iminolactone (4b), displayed significant inhibitory activity, with respective Ki values of 0.6 and 2.5 μM against hLDH5 and 0.4 and 92 μM against hLDH1. In particular, lactone analog 4a displayed approximate similar activity toward both enzymes, while the imino analog 4b demonstrated significant selectivity toward hLDH5 over hLDH1 (Figure 4).37

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POLYPHENOLIC SCAFFOLD-BASED INHIBITORS The natural product gossypol (1) is a polyphenolic binaphthyl disesquiterpene originally isolated from the cotton seeds of gossypium that displayed a complex and broad spectrum of promising biological activities, such as antitumor, antioxidant, antiviral, and antiparasitic activities (Figure 4).31 It shows atropisomerism; thus, it exists in two enantiomeric forms, (R) and (S), via restricted rotation around the 2−2′ carbon−carbon single bond that tethers the two sterically hindered naphthalene units. For dose-dependent cytotoxic activity, the (R)-(−) form of 1 is more potent than the (S)-(+) isomer of 1, with mean IC50 values of 20 μM in melanoma, lung cancer, breast cancer, cervical cancer, and leukemia cell lines.32 Although the initial pharmacological interest in 1 arose because of its male antifertility action (caused by spermicidal activity), it also showed an ability to inhibit various LDH isoforms in an NADH-competitive manner (hLDH5 Ki = 1.9 μM, hLDH1 Ki = 1.4 μM).33,34 Though it displayed promising biological activities, there is serious concern about the toxicity of 1 because of its highly reactive chemical structure. The two aldehyde groups and catechol hydroxyl groups are highly sensitive and generate toxic metabolites of 1. They exert

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2,3-DIHYDROXYNAPHTHOIC ACID-BASED INHIBITORS With the aim of maintenance of potential activities with improved selectivity toward hLDH5, 8-deoxyhemigossylic acid (5) and its derivatives (6) (Figure 5) were discovered.38 The 4isopropyl group of compound 5 is replaced by a methyl (6a− c), n-propyl (6d−f), and isopropyl (6g−l) at the 4-position of representative compound 6, whereas the R1 group (a hydrogen, methyl, benzyl, or substituted benzyl group, Table 1) occupied 489

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POLYPHENOLIC FLAVONE-BASED INHIBITORS In addition to compound 1, some other polyphenolic scaffolds based on natural products (such as morin, 7) were found to be potential anticancer agents due to their inhibitory activity against hLDH5 (Figure 6).41 In 2013, Wang et al.42 disclosed Figure 5. 2,3-Dihydroxynaphthoic acid derivatives.

Table 1. R and R1 of Compounds 6a−l and Their Activity against hLDH5 and hLDH1 Ki (μM) compd

R

R1

hLDH5

hLDH1

5 6a 6b 6c 6d 6e 6f (FX11) 6g 6h 6i 6j 6k 6l

isopropyl methyl methyl methyl n-propyl n-propyl n-propyl isopropyl isopropyl isopropyl isopropyl isopropyl isopropyl

H H methyl benzyl H methyl benzyl methyl benzyl o-tolyl m-tolyl p-tolyl p-chlorobenzyl

3 34 4 0.5 1 0.1 0.05 2 0.2 3 0.2 0.03 1

92 250 190 39 49 19 1 78 7 >125 34 8 8

Figure 6. Polyphenolic flavone-based hLDH5 inhibitors.

the structure of epigallocatechin 8 from an aqueous extract of Spatholobus suberectus. It inhibited both hLDH5 and HIF-1α expression and showed significant inhibition of breast cancer growth (MCF-7, MDA-MB-231), triggered apoptosis without eliciting any toxic effect, and was considered a lead compound for further studies.42 Galloflavin (9), a tricyclic flavone-like molecule, was discovered as a nonselective inhibitor of hLDH5 exhibiting a Ki value of 5.46 μM vs pyruvate and 56.0 μM vs NADH in hLDH5 compared to hLDH1 (Ki = 15.1 μM vs pyruvate; 23.2 μM vs NADH) and was identified as a potent in vivo antitumor agent.43 In fact, compound 9 completely blocked the enzymatic activities of both LDH isoforms, showed 60% cell growth inhibition, and exerted comparable growth inhibition effects (IC50 values in the 90−150 μM range) in breast cancer cell lines (MCF-7, MDA-MB-231, and MCF-Tam cells). Indeed, the cell growth inhibition effect of compound 9 appearing in MCF-7 cell lines is caused by downregulation of ERα-mediated signals, whereas in MDA-MB-231 and MCFTam cells, it is caused by oxidative stress. The low cytotoxicity in healthy cells and the promising pharmacological effects in aggressive tumors encourage the further exploration of compound 9 (Figure 6).44

the 7-position, which is the coupling position in the formation of disesquiterpenes similar to the parent compound 1. The reference compound 5 of this series displayed more selectivity for the hLDH5 isoform (Ki of 3.0 μM) than hLDH1 (Ki = 92 μM), whereas its corresponding dimer 2 was completely nonselective for both isoforms. It appears that dimerization of 5 markedly improves the activity against only hLDH1; therefore, only half of the gossypol scaffold might be involved in the inhibition of hLDH5 and is sufficient for the inhibitory activity of hLDH5. Compounds 6a−l of this class exhibited Ki values in the low micromolar range (Table 1) against hLDH5 and showed selectivity relative to hLDH1. However, very few of these compounds were also endowed with potent (i.e., submicromolar) activity against hLDH1. The inhibition data for compounds (6a−l, Table 1) of this class are also consistent with the fact that dimerization diminishes the activity against hLDH5. Compounds 6f (FX11; R = n-propyl and R1 = benzyl) and 6k (R = isopropyl and R1 = p-tolyl) were identified as the most active hLDH5 inhibitors. In addition, compound 6f displayed modest selectivity for the inhibition of hLDH5 (in a NADH competition) and was considered a potential anticancer lead candidate from this class, although initially it was designed as an antimalarial agent.38,39 It elicited reduced ATP levels and diminished cellular lactate production, induced significant oxidative stress, and suppressed tumor progression in human lymphoma and pancreatic cancer xenografts.14 Recently, Ward et al.40 proposed that compound 6f was unable to show significant binding by biophysical assays (NMR and Bio BIAcore) as well as in biochemical assay because of denaturation of protein (LDHA). Despite the promising results, the highly reactive catechol portion of this molecule (6f) makes it unsuitable as a drug candidate for further development.

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N-HYDROXYINDOLE SCAFFOLD-BASED INHIBITORS A distinguished class of hLDH5 inhibitors with potential therapeutic applications, represented by the N-hydroxyindole (NHIs), has been discovered (Figure 7).30,45−48 NHIs, in which the central indole scaffold contains a hydroxyl group on the nitrogen atom in the 1-position and a carboxyl group in the 2position of the indole moiety, showed inhibitory activity in the competition with both the cofactor NADH and the substrate pyruvate, with Ki values in the low micromolar range against hLDH5. An extensive structure−activity relationship study revealed that the inhibitory efficiency of NHIs might be relying on the “OH-COOH” pharmacophore motif because this type of structural motif partially resembles the structure of the natural substrates of LDH, i.e., an α-keto acid (pyruvate) or αhydroxy acid (lactate). For example, the NHI derivative 10a, generated by phenyl substitution in the 5-position of the central indole scaffold, exhibited stronger activity than compound 10b where a phenyl group is present in the 6-position. Compound 10a and 10b showed Ki values of 10.4 and 19.8 μM, respectively, in the NADH competition and 15.7 and 35.4 μM, respectively, in the pyruvate competition experiments against the hLDH5 enzyme.30 The presence of two phenyl groups in the adjacent 5- and 6-positions of the NHI (compound 10c) enhances hLDH5 inhibitory activity with Ki 490

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Figure 7. N-Hydroxyindole (NHI) scaffold-based hLDH5 inhibitors.

values of 5.4 μM vs NADH and 7.4 μM vs pyruvate.30 Incorporation of a p-chlorophenyl (11a) and a biphenyl (11b) group in the 6-position of the NHIs displayed similar activity (Ki = 5.3 μM, 11a; 4.5 μM, 11b) in competition with NADH, whereas in competition with pyruvate, compound 11a demonstrated a markedly negative effect on the Ki values (Ki = 56 μM).30,45 This is the general observation, where aromatic substitution in the 6-position of the central scaffold usually favors the hLDH5 activity. This is further confirmed by the sulfonamide-bearing compound 12 (N-methyl-N-pchlorophenylsulfonamide) and compound 13 (isoindolinylsulfonamide) that exhibited Ki of 6.6 and 7.7 μM vs NADH and 5.6 and 8.8 μM vs pyruvate, respectively.46 Furthermore, the additional insertion of an electron-withdrawing group (trifluoromethyl) in the 4-position of 10b resulted in compound 14a, which demonstrated more potency than 10b and showed Ki values of 8.9 μM in competition with NADH and 4.7 μM in competition with pyruvate, whereas its methyl ester derivative 14b displayed a slightly lower Ki value of 5.1 μM in the NADH competition assay. Specific binding of 14a to hLDH5 was further supported by 2D NMR analysis depicting Kd value of ∼9 μM (in NADH competition); however, BIAcore studies showed nonspecific binding.40 Moreover, both NHI derivatives 14a and 14b showed a synergistic cytotoxic effect in combination with gemcitabine under hypoxic conditions and also inhibited spheroid growth, cell migration, and invasion in pancreatic cancer cells.17 Recently, compounds 15a and 15b of this class were discovered by glucose conjugation of the -OH group present in the 1-position of 14a and 14b. Although the glucose derivatives 15a and 15b displayed weaker inhibitory activity against hLDH5, exhibiting Ki values of 19.5 and 37.8 μM, respectively, in the NADH competition assay, they exhibited more efficacy than their N-OH analogs in cell-based assays.47 Both compounds 15a and 15b efficiently reduced dose-dependent lactate production in the HeLa cancer cell line and compromised cell proliferation in several cancer cell lines. Compound 15b showed better cell permeability and reduced lactate production and antiproliferative activity.47,48 Molecular modeling studies of this class depicted that these derivatives bind in similar fashion as pyruvate bind in substrate binding pocket (Figure 3B) The carboxylic group showed interactions with Arg169 (R) via salt bridge formation and with Thr248 (T) by hydrogen bond formation, whereas the N-OH group was involved in direct and water-mediated hydrogen bonding through Thr248 (T) and His193 (H). The aromatic (phenyl) group in the 6-position of the NHIs was located in a lipophilic pocket at the entrance of the binding cavity of the enzyme and

captures a part of the cofactor binding site.30 The 1H, 15N NMR pattern of LDHA backbone amide induced by NHI derivative (14a) exhibited its alternate binding to the adenine subpocket of the cofactor rather than the nicotinamide/ substrate region.40 In short, the SAR of the NHIs exposed that the OH-COOH pharmacophore motif, aromatic substitution in the 6-position, and an additional electronwithdrawing group in the NHIs favor the potency toward hLDH5, and O-glucoconjugation improves the efficacy of these molecules. Importantly, NHIs are highly selective toward hLDH5 over the hLDH1 isoform.

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CHIMERIC (BIFUNCTIONAL)-TYPE INHIBITORS Chimeric hLDH5 inhibitors are defined as bifunctional molecules in which two portions, one a substrate mimic and the other a cofactor mimic, are conjugated by a linker to cover the whole binding pocket of the enzyme. The first chimeric LDH inhibitor glycolic acid−NADH conjugate (16) was synthesized in 2010 by conjugating a glycolic acid to the reduced nicotinamide ring of NADH in which the α-hydroxy acid portion was intended to mimic the pyruvate substrate (Figure 8).49 Interestingly, compound 16 exhibited strong

Figure 8. Glycolic acid−NADH conjugate.

NADH-competitive LDH inhibitory activity on LDH (from bovine heart and rabbit muscle) in the nanomolar range and also demonstrated both in vitro and in vivo cardioprotective effects, promoting cell survival in cultured mouse cardiomyocytes exposed to hypoxia−reoxygenation stress. Unfortunately, it displayed limited clinical applications as a cardioprotective agent because of its low cell permeability, which would reduce the inhibitor’s efficiency;49 nevertheless, it provides a future direction for the development of chimeric molecules as hLDH5 inhibitors. Later, a new generation of chimeric molecules, 17a and 17b, were designed to target hLDH5 using a fragmentbased click chemistry approach in which the bis(indolyl)maleimide moiety (an adenosine mimetic NADH-like frag491

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DIHYDROPYRIMIDINE- AND PYRAZINE-BASED INHIBITORS In 2013, Genentech (CA, USA) disclosed dihydropyrimidine (20)52 and pyrazine (21)53 scaffold-based hLDH5 inhibitors by following a high-throughput screening approach (Figure 10).

ment) was conjugated through a linker comprising alkyl or arylalkyl chains of different length and by means of a 1,2,3triazole ring, with the other fragment, such as a carboxylate group, which mimics the natural substrate pyruvate (Figure 9).50 Interestingly, unlike sodium oxamate (IC50 value of 130.6

Figure 10. Dihydropyrimidine and pyrazine-based hLDH5 inhibitors. Figure 9. Chimeric molecule-based hLDH5 inhibitors.

The representative molecule of the dihydropyrimidine series, 20a, showed hLDH5 enzymatic inhibitory activity with an IC50 of 8.8 μM; however, it demonstrated no selectivity toward both isoforms, exhibiting a similar activity on the hLDH1 isoform (IC50 = 11.1 μM).52 With the aim of improvements in potency and selectivity toward hLDH5, several analogs of 20a were generated by either replacing or modifying the sulfonamide substituent present on the anilide ring and by various substitutions on the peripheral phenyl ring. The inhibitory activity profile of this series depicted that the p-sulfonamide group in the anilide ring and the cyano group in the dihydropyrimidine scaffold play critical roles in the hLDH5 inhibitory activity. In light of these facts, compounds 20b−e were prepared by substitution in the peripheral phenyl ring directly attached to the central dihydropyrimidine scaffold, where the aliphatic methylene moiety is substituted by a methyl or ethyl group. Compounds 20b (R1 = CH3, IC50 of 0.48 μM) and 20c (R1 = C2H5, IC50 of 0.65 μM) showed strong improvements in inhibitory activity that were markedly sustained, even by further insertion of additional halogens on the phenyl ring in the ortho position with respect to the pchloro group in compounds 20d (R = Cl, IC50 of 0.75 μM) and 20e (R = F, IC50 of 0.65 μM).52 Dissociation constants of 20a− e measured by SPR showed IC50 values of 6.1, 2.2, 3.0, 5.1, and 2.9 μM, which were in accordance with inhibitory activity determined by biochemical assays. However, compounds 20b− e exhibited inhibitory potency in high nanomolar range (0.48− 0.75 μM) toward hLDH5 isoform but displayed moderate selectivity against hLDH1 (IC50 = 2−4 μM) along with poor cell membrane permeability and high protein binding affinity. Unfortunately, the effect of the chirality of 20b−e on the inhibitory activity was not disclosed. In addition, the most active pyrazine derivative 21 exhibited an IC50 value of 0.50 μM against hLDH5 and demonstrated favorable aqueous kinetic solubility and cell permeability.53 Compound 21 displayed 4fold selectivity toward hLDH5 over hLDH1 and 10-fold selectivity over hLDHC.53 Compound 21 inhibited lactate production in MCF7 cells (40% reduction) at 50 μM (highest compound concentration), and the minimal cellular activity could be related to insufficient biochemical potency or high plasma−protein binding.53 In fact, the improved potency and selectivity of these compounds illustrated that these molecules bind in the active site of the protein in the presence of NADH and interact with the catalytic residues involved in substrate catalysis.

± 1.2 μM), compounds 17a and 17b demonstrated moderate IC50 values of 14.8 ± 1.2 and 35.9 ± 1.2 μM, respectively, against hLDH5 with good selectivity over hLDH1 (inactive even at 50 or 100 μg/mL).50 The activity of 17a was 9-fold higher than that of sodium oxamate (reference compound) against hLDH5, which led to this molecule being considered for further development. Computational analysis revealed that compound 17a overplayed well with NADH and presented favorable interaction with the protein (binding energy = −10 kcal mol−1) displaying hydrogen bonding between the protein and carboxylic group and bis(indolyl)maleimide moiety. The carboxyl moiety present at the terminal position favors the strong polar interactions in the substrate and cofactor binding sites of the enzyme.50 In addition, by adopting a fragmentbased lead generation strategy, diacid malonate scaffold-based inhibitors of hLDH5, such as 18a−c,40 were identified by AstraZeneca UK in which two fragments benzothiazole and malonate were tethered by a linker at the opposite end of molecule (Figure 9). The most active compound 18a displayed an IC50 value of 0.27 μM (enzyme inhibition assay) and a Kd value of 0.008 μM (BIAcore binding affinity assay). Its analogue, compound 18b (X = NH, exhibiting an IC50 value of 0.29 μM, Kd ≈ 0.06 μM) showed potency similar to 18a.40 Further, with insertion of a methyl group instead of s-propyl group (R) in the benzothiazole fragment, resulting compound 18c showed a modest improvement in enzyme inhibitory activity (IC50 = 1.9 μM) with similar binding affinity (Kd ≈ 0.069 μM) to that of compound 18b. Furthermore, ARIAD Pharmaceuticals (MA, USA) reported novel chimeric hLDH5 inhibitors in which fragments that bound to the substrate and cofactor sites were tethered through flexible polyhydroxyl linkers.51 The resulting compounds 19a and 19b differ only by the structure of the linker containing an ether-type oxygen atom (X = O, 19a) or an amine-type nitrogen atom (X = NH, 19b). The ether analog 19a exhibited a strong inhibitory activity (IC50 = 0.44 μM), but the amine analog 19b proved to be the more potent molecule (IC50 = 0.12 μM), approximately 4-fold more effective than 19a.51 Crystallographic studies determined that the carboxylic acid moieties of 18 and 19 interact with the basic side chain of the hLDH5 residue Arg168 in a manner similar to oxamate-derived substrate inhibitors and carboxylic acid functional groups that can ionize at physiological pH.51 492

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DIHYDROPYRONE- AND CYCLOHEX-2-ENONE-BASED INHIBITORS Recently, the dihydropyrone derivative 2254 (Genetech, Inc.) depicted significant inhibitory potency (IC50 value of 30 nM) against hLDH5 with high solubility at pH = 7.4 and with high plasma−protein binding and lipophilicity. One carbonyl group of dihydropyrone core displayed interaction with His192 via hydrogen bond and was stabilized by formation of hydrogen bond with Asp165. Overall, the thioether and remaining carbonyl moiety of dihydropyrone core engaged with Arg168 in a bidentate hydrogen bond formation (Figure 11). Very

Figure 12. Quinoline scaffold-based hLDH5 inhibitors.

aromatic ring conjugated through the NH group, and finally, the 7-position is substituted by various aromatic or heteroaromatic rings (Figure 12). For example, compound 26 demonstrated excellent inhibitory activity against hLDH5, displaying a pIC50 value of 7.2 (IC50 = 63.1 nM).60 Recently, the sulfonamide-bearing quinoline derivatives 27a and 27b were disclosed as selective hLDH5 inhibitors; in fact, they were the most potent hLDH5 inhibitors discovered so far, exhibiting IC50 values of 2.6 (27a) and 16 nM (27b) and providing 16fold and 13-fold selectivity, respectively, for hLDH5 over hLDH1 (IC50 of 43 and 220 nM, respectively) (Figure 12).27,61 The most active compound 27a reduced glucose consumption and inhibited cell proliferation and cell survival in the highly glycolytic Snu398 and HEPG2 human hepatocellular carcinoma cells. In A549 cells, it displayed an increased oxygen consumption rate and ROS activity at a 10 μM concentration, altered metabolic intermediates concentrations, and reduced the rate of formation of tumorspheres. These molecules were endowed with promising hLDH5 inhibitory activities, although they showed poor pharmacokinetic properties that caused limitations for the further use of these molecules in in vivo experiments.61 Molecules of this series penetrated the cellular membrane possibly because of high protein binding of the compounds or an active transport mechanism in hepatocellular carcinoma cells. The crystal structures of compounds bound to LDH5 demonstrate their binding in the NADH pocket only; thus, these are noncompetitive versus pyruvate. In summary, different classes of hLDH-5 inhibitors, including the natural product gossypol, its derivatives, FX-11, the gallic acid derivative galloflavin, NHI inhibitors, and novel small molecules, such as chimeric (bifunctional) molecules, quinolinesulfonamide inhibitors, and others developed by fragmentbased and high-throughput screening approaches, were discovered so far. Among them, some compounds are attractive candidates due to their facile synthesis, selective toxicity toward cancerous cells, and in vitro and cell culture efficacy. In addition, various other small molecules were also disclosed earlier that exhibited only modest LDH-inhibitory activity while demonstrating strong activity against pf LDH.

Figure 11. Dihydropyrones and cyclohex-2-enone derivatives.

recently, by following a high-throughput screening approach, the cyclohex-2-enone derivatives 23a55 and 23b56 were disclosed, showing marked selectivity toward hLDH5 (IC50 value of 0.87 μM (23a) and 6 nM (23b)) over LDH1 (IC50 value of 6.9 μM) (Figure 11). The dissociation constant (KD) of compound 23a (IC50 value of 1.8 μM) against hLDH5 measured by surface plasmon resonance (SPR) was in close agreement with biochemical values.55 Essentially, the ionized enol moiety of 23 mimics the carboxylate moiety of oxamate and interacts with the catalytic Arg168, while the ketone moiety is involved in hydrogen bonding with the side chain of His192 and Asp137. Oral administration of compound 23a in rats also displays a good pharmacokinetic profile but was unable to reduce lactate production in cancer cell lines.55 Indeed, compound 23b is ∼100 times more active than 23a, though inactive in cells. Similar to 23a, compound 23b bound in the substrate binding pocket showed significant enhancement in biochemical potency against hLDH5. This increased activity might be due to the addition interaction shown by the carbonyl and the phenyl group of the ester moiety that seems in close proximity with the protein via formation of hydrogen bond and partial π−cation interactions, respectively.

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QUINOLINE SCAFFOLD-BASED INHIBITORS In 1972, Baker and Bramhall reported quinoline-based (24) and quinolone-based (25) LDH inhibitors in which the central quinoline or quinolone scaffold is substituted by a hydroxyl group in the 4-position and by a carboxyl group in either the 2position or 3-position (Figure 12).57−59 These molecules from both series (24, 25) demonstrated marked selectivity toward LDH over other dehydrogenase enzymes, exhibited inhibitory activity in the submicromolar range, and demonstrated that a carboxyl group in the 3-position provides better LDH activity than the 2-carboxyl counterparts; thus, the OH-COOH (in adjacent positions) motif supports inhibitory activity on the LDH enzyme.57−59 Since then, quinoline has been considered a potential scaffold in the search for new LDH inhibitors. In 2012, GSK reported potent hLDH5 inhibitors60 in which the central quinoline scaffold is substituted by an amide or sulfonamide group in the 3-position, and the 4-position of the quinoline is occupied by a carboxyl moiety bearing an

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CONCLUDING REMARKS AND FUTURE PROSPECTS Development of new therapies that kill tumor cells without disturbing healthy tissues is still a challenging task in cancer research. In this regard, selective advantages of the metabolic dependencies of tumor cells can be exploited for cancer treatment because either directly or indirectly metabolism is a 493

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Biographies

vital need in everything a cell does. The discovery of hLDH5 as a vital hallmark in cancer and its association with transcription factors make it an attractive anticancer target for drug development. Significant progress has been made in understanding the biology and function of hLDH5 in various human cancers. Recent scientific advances proved that hLDH5 occupies an optimal checkpoint at the “pyruvate bifurcation point” in the glycolytic pathway where pyruvate is directed toward lactate fermentation in cancer cells and differentiates normal cells toward oxidative phosphorylation. Consequently, therapeutic inhibition of hLDH5 is highly preferable to the other glycolytic enzyme in order to obtain a selective starvation of cancer cells. Therefore, hLDH5 inhibition should only affect tumor tissues, with minimal interference in normal cells undergoing normal metabolism. For these reasons, development of cancer drugs from hLDH5 inhibitors would be a quite successful approach and may constitute safe agents that interfere with tumor growth and invasiveness. However, at some point, hLDH5 has been considered a difficult target due to the highly polar nature and small size of its natural substrate (pyruvate), along with the relatively large and open active site. Despite advances in high-throughput screening, structure- and ligand-based molecular modeling, fragment-based and hit-tolead screening approaches, very few hLDH5 inhibitors have progressed to the preclinical stage and into clinical trials. Overall, the selective and potent hLDH5 inhibitors discovered so far exhibit common structural features, such as the presence of a carboxylate group, and hydroxyl and carboxyl carbons in close proximity to the similar structural features of the original substrates, lactate (α-hydroxy acid) and pyruvate (α-keto acid). However, some organic molecules identified so far depicted selective hLDH5 inhibitory activity in nanomolar range but present poor pharmacokinetic profile and poor penetration inside cells. These factors preclude these molecules for further development as a therapeutic agent. Taken collectively, biological results of these various chemical classes of hLDH5 inhibitors monitored by biochemical, biophysical, or both suggested that some of above-reported promising molecules were validated human lactate dehydrogenase inhibitors worthy of additional investigation. Therefore, discovery of newer analogues with improvement in selectivity, inhibitory activity, delivery, and good pharmacokinetic profile of hLDH5 inhibitors probably enhances viability of hLDH5 targeting as a vital approach for cancer treatment. Molecules active against hLDH5 have the potential of generating completely new medical tools to be used either alone or in combination therapies against currently untreatable cancers. The top challenges include the development of more potent and selective hLDH5 inhibitors; therefore, progressive increase in newly reported hLDH5 inhibitors with better “drug-like” properties hopefully might reach the clinic soon. Therefore, drugs belonging to several classes of compounds that inhibit the glycolytic process via interruption of hLDH5, when used either as monotherapies or in combination with standard chemotherapeutic regimens, may provide a potential breakthrough against cancer and can fill a gap that is still inadequate on the metastatic front.

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Reshma Rani received her Master’s degree in Organic Chemistry in 2003 and completed her Ph.D. in Bioorganic Chemistry in 2010 under the supervision of Prof. S. M. Sondhi at the Indian Institute of Technology, Roorkee, India. In 2011, she received a postdoctoral fellowship from Osaka University Japan with Prof. Kazuhiko Nakatani in the area of medicinal chemistry. In 2012, she won a Marie Curie International Incoming Fellowship for 2 years with Prof. Filippo Minutolo at the University of Pisa, Italy, in the area of design and discovery of LDH inhibitors. During the past 10 years, she has been working in the same area, focusing on cancer and inflammatory diseases. Currently, she is working as a visiting researcher at the CRONational Cancer Institute, Aviano Italy. Vinit Kumar received his Ph.D. in 2010 from the Indian Institute of Technology, Roorkee, India. In 2011, he joined the Frontier Institute for Biomolecular Engineering Research, Japan, as a postdoctoral research fellow and worked in the area of structural and functional properties of RNA. In September 2012, he moved to the National Cancer Institute, Aviano, Italy, and there, he focuses on the development of new technologies for cancer theranostics.

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ACKNOWLEDGMENTS Authors are thankful to Prof. Filippo Minutolo (University of Pisa, Italy) and Prof. Giuseppe Toffoli and Dr. Flavio Rizzolio (CRONational Cancer Institute, Aviano Italy). V.K. is thankful to Italian Ministry of Education MIUR (FIRB prot. RBAP11ETKA) for funding.

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ABBREVIATIONS USED LDH, lactate dehydrogenase; hLDH5, human lactate dehydrogenase; NADH, nicotinamide adenine dinucleotide; OXPHOS, oxidative phosphorylation; ATP, adenosine triphosphate; ROS, reactive oxygen species; HIF, hypoxia inducible factor; HER2, human epidermal growth factor; FOXM1, forhead box protein 1

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +39 0434 659816. Notes

The authors declare no competing financial interest. 494

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