Dehydrogenases are oxidoreductase enzymes that play a variety of fundamental functions in the living organisms and have primary roles in pathogen survival and infection processes as well as in cancer development. We review here a sub-set of NAD-dependent dehydrogenases involved in human diseases and the recent advancements in drug development targeting pathogen-associated NAD-dependent dehydrogenases. We focus also on the molecular aspects of the inhibition process listing the structures of the most relevant molecules targeting this enzyme family. Our aim is to review the most impacting findings regarding the discovery of novel inhibitory compounds targeting the selected NAD-dependent dehydrogenases involved in cancer and infectious diseases.

Introduction

Dehydrogenases are strictly conserved enzymes in all domains of life and catalyze the oxidation of their substrate(s) through the engagement of a reducing electron acceptor, usually NAD+/NADP+, or a flavin coenzyme such as FAD or FMN. Due to the variety of functions that they perform in living organisms and in tumor cells, dehydrogenases are molecules of paramount biochemical and pharmaceutical interest.

The present minireview aims at providing a panoramic on the functions and roles of selected NAD-dependent dehydrogenases involved in diseases that affect most of the world population, i.e. tuberculosis (a global disease caused by Mycobacterium tuberculosis; MTB) which infects a quarter of the world population, killing ∼1.5 million people per year [1]), malaria (∼230 million cases worldwide, causing an estimated 405.000 deaths every year [2]) and cancer (the second leading cause of death globally, accounting for an 9.6 million deaths [3]). In this context, attention has been devoted to NAD-dependent dehydrogenases as potential and effective drug targets involved in essential metabolic pathways such as the tricarboxylic acid and the biosynthesis of the purine and pyrimidine bases. Moreover, the function and role of the large family of NAD-dependent aldehyde dehydrogenases (ALDHs) in tumor development is reported, with a focus also on the dehydrogenases involved in hypoxia, a hallmark of tumor onset. Moreover, a paragraph is dedicated to the glyceraldehyde 3-phosphate dehydrogenase, a housekeeping enzyme involved in glycolysis that has multiple roles in cancer development and is also considered a robust target for novel antimalarials.

NAD-dependent dehydrogenases in anti-tuberculosis drug development

Current antitubercular drugs target essential enzymes involved in key metabolic processes such as the cell wall synthesis, the energy metabolism, protein synthesis, phosphate transport and the metabolism of key molecules and cofactors [4]. Despite the intense ongoing research [5], the development of an effective anti-TB vaccine is still a matter of strong research focus [6], and the bacillus Calmette–Guerin (BCG) vaccine, although it has been developed two centuries ago, it is still the most used and effective vaccine nowadays, but fails in providing full protection against TB.

A hallmark of MTB infection lies in its capacity to endure in the host lungs for decades thanks also to its ability to survive severe hypoxia conditions [7]. MTB resiliency to hypoxia relies also on a modified TCA cycle (also called the ‘glyoxylate shunt’) which enables the cells to utilize fatty acids or ethanol and acetate as sole carbon sources [8]. Efforts have been invested in targeting the enzymes isocitrate lyase and malate synthase of the glyoxylate shunt [9,10], thus potentially overcoming the cross-reactivity of inhibitory molecules between the bacterial and host TCA cycles [4]. However, an alternative strategy aims at targeting those enzymes that are shared between the glyoxylate shunt and the TCA cycle such as citrate synthase, aconitase and malate dehydrogenase. The elucidation of the crystal structures of MTB NAD-dependent malate dehydrogenase (MDH) [11], of citrate synthase [11] and aconitase [12] could constitute valid frameworks for the design of specific inhibitors. However, the high sequence and structure conservation between the pathogen and human host ortholog enzymes have limited the discovery of pathogen-specific TCA inhibitors. A proposed approach for preventing cross-reactivity with the host proteins is the ‘protein interference assay’ (PIA) [13] which uses target protein mutant(s) that interfere with the oligomerization state of the wild-type protein, thus impairing target protein function and eventually affecting the pathogenicity of the parasite. The PIA approach aims at increasing drug-target selectivity while potentially minimizing their likely cross-inhibition with host and pathogen ortholog enzymes. The tetrameric MDH of MTB, in light of its structural elucidation [11], could be a possible molecular target for such approach, as has been successfully applied for MDH of Plasmodium falciparum [13].

Besides targeting the central carbon metabolism of MTB, other antimicrobial approaches aim at inhibiting essential pathways involved in MTB survival, in the supply of key nutrients and in the DNA metabolism and repair mechanisms [14, 15]. The inhibition of MTB enzymes that prevent the host lysosome–phagosome maturation process favors the MTB clearance by the host immune system [16–20], whereas phosphate deprivation impairs phosphate supply, as demonstrated by the elevated expression of enzymes of the Pho regulon in phosphate starvation conditions [21,22]. Moreover, the biosynthesis of essential molecules such as PRPP [23] and the purine and pyrimidine bases [24–26] might represent promising targets for future antitubercular strategies. In this context, the inosine-5′-monophosphate dehydrogenase (IMPDH, or GuaB2; Rv3411c), a NAD-dependent dehydrogenase whose structure was firstly reported by Makowska-Grzyska et al. [27], has been shown to be essential for the NAD+-dependent dehydrogenation and hydrolysis of inosine 5′-monophosphate to xanthosine 5′-monophosphate, and represents the rate-limiting enzyme of the de novo purine nucleotide biosynthesis [28,29]. A phenotypic screening approach revealed a promising molecule (VCC234718, Table 1) able to induce MTB mortality in macrophages and mouse lung xenografts, while retaining low toxicity on mammalian cells [25,26]. Notably, the resistant MTB strain developed a spontaneous mutation on the guaB2 gene resulting in the GuaB2-Y487C mutated variant, which conferred resistance to the active molecule. This seminal work has contributed to raise the interest in GuaB2 as a promising antitubercular drug target, and two independent studies used a structure-activity approach which culminated in the development of the GuaB2 inhibitory compound 13 (Table 1), a 5-amidophthalide derived from a library of compounds active against Staphylococcus aureus IMPDH [30], and a compound derived from an aryl benzoxazole (compound 17b) which inhibited GuaB2 with a Ki of 18 nM (Table 1) [32]. This compound also showed MTB growth inhibition properties, confirming the essentiality and the vulnerability of MTB GuaB2.

Table 1.
Inhibitor compounds of selected NAD-dependent dehydrogenases involved in infection and cancer and here described
DehydrogenaseCompound nameStructural formulaReferenceIC50 (Ki) µMPDB code
M.termoresistibile
IMPDH 
VCC234718  [253.4 5J5R 
MtbIMPDH 13  [303.04  ±  0.03 NA 
MtbInhA Isoniazid  [310.75 × 10−3 (Ki1ZID 
MtbIMPDH 17b (Q151)  [320.018 NA 
MtbInhA Triclosan  [331.0 1P45 
MtbInhA Pyridomycin  [346500 4BGE 
MtbInhA GSK693  [357.0 × 10−3 5JFO (GSK625) 
MtbInhA AN12855  [360.06 5VRL 
hMDH2 LW6  [376.3 NA 
hMDH2  [383.4 NA 
hIMPDH MPA  [390.020 1JR1 
hIMPDH MMF  [400.025 NA 
hIMPDH Tiazofurin  [410.5 (KiNA 
hIMPDH VX-944  [426–10 nM (KiNA 
hALDH1/2 Disulfiram  [43ALDH1A1 = 0.15
ALDH2 = 1.45 
NA 
hALDH1/2 Daidzin  [44ALDH1A1 = 28.0
ALDH2 = 0.9 
2VLE 
hALDH1/2 DEAB  [45ALDH1A1 = 0.55
ALDH2 = 1.45 
4X0U (ALDH7A1) 
hALDH1A1 NCT-501  [460.04 NA 
hALDH1A1 NCT-505  [477 × 103 NA 
hALDH1A1 NCT-506  [477 × 103 NA 
hALDH1A1 CM026  [480.80 ± 0.26 4WP7 
hALDH1A1 CM037  [484.6 ± 0.8 4X4L 
hALDH1A1 CM053  [480.21 ± 0.04 4WPN 
hALDH1A1
hALDH1A2
hALDH1A3 
13g  [49ALDH1A1 = 0.08 ± 0.1
ALDH1A2 = 0.25 ± 0.04
ALDH1A3= 0.12 ± 0.02 
6DUM 
hALDH1A1
hALDH1A2
hALDH1A3 
CM039  [49ALDH1A1 = 0.9 ± 0.2
ALDH1A2/ALDH1A3 ≃ 20.0 
5TEI 
hALDH1A2 WIN18,446  [500.19 ± 0.05 6ALJ 
hALDH1A2 6–118  [500.91 ± 0.33 6B5G 
hALDH1A2 CM121  [500.54 ± 0.15 6B5H 
hALDH1A3 GA11  [51N.A. NA 
hALDH1A3 GA23  [51N.A. NA 
KarGAPDH Koningic acid  [520.2 NA 
PfGAPDH 2-phenoxy-1,4-naphthoquinone  [535.7 NA 
DehydrogenaseCompound nameStructural formulaReferenceIC50 (Ki) µMPDB code
M.termoresistibile
IMPDH 
VCC234718  [253.4 5J5R 
MtbIMPDH 13  [303.04  ±  0.03 NA 
MtbInhA Isoniazid  [310.75 × 10−3 (Ki1ZID 
MtbIMPDH 17b (Q151)  [320.018 NA 
MtbInhA Triclosan  [331.0 1P45 
MtbInhA Pyridomycin  [346500 4BGE 
MtbInhA GSK693  [357.0 × 10−3 5JFO (GSK625) 
MtbInhA AN12855  [360.06 5VRL 
hMDH2 LW6  [376.3 NA 
hMDH2  [383.4 NA 
hIMPDH MPA  [390.020 1JR1 
hIMPDH MMF  [400.025 NA 
hIMPDH Tiazofurin  [410.5 (KiNA 
hIMPDH VX-944  [426–10 nM (KiNA 
hALDH1/2 Disulfiram  [43ALDH1A1 = 0.15
ALDH2 = 1.45 
NA 
hALDH1/2 Daidzin  [44ALDH1A1 = 28.0
ALDH2 = 0.9 
2VLE 
hALDH1/2 DEAB  [45ALDH1A1 = 0.55
ALDH2 = 1.45 
4X0U (ALDH7A1) 
hALDH1A1 NCT-501  [460.04 NA 
hALDH1A1 NCT-505  [477 × 103 NA 
hALDH1A1 NCT-506  [477 × 103 NA 
hALDH1A1 CM026  [480.80 ± 0.26 4WP7 
hALDH1A1 CM037  [484.6 ± 0.8 4X4L 
hALDH1A1 CM053  [480.21 ± 0.04 4WPN 
hALDH1A1
hALDH1A2
hALDH1A3 
13g  [49ALDH1A1 = 0.08 ± 0.1
ALDH1A2 = 0.25 ± 0.04
ALDH1A3= 0.12 ± 0.02 
6DUM 
hALDH1A1
hALDH1A2
hALDH1A3 
CM039  [49ALDH1A1 = 0.9 ± 0.2
ALDH1A2/ALDH1A3 ≃ 20.0 
5TEI 
hALDH1A2 WIN18,446  [500.19 ± 0.05 6ALJ 
hALDH1A2 6–118  [500.91 ± 0.33 6B5G 
hALDH1A2 CM121  [500.54 ± 0.15 6B5H 
hALDH1A3 GA11  [51N.A. NA 
hALDH1A3 GA23  [51N.A. NA 
KarGAPDH Koningic acid  [520.2 NA 
PfGAPDH 2-phenoxy-1,4-naphthoquinone  [535.7 NA 

From the left column: target protein name, compound name, compound structure, reference, IC50 (Ki, in parentheses) values expressed in µM (except where noted). NA: not available.

One of the most historically used medicine against TB is isoniazid (isonicotinic acid hydrazide; INH), a first-line pro-drug which is administered throughout the early 6-months drug regimen. In vivo, INH is metabolized by the mycobacterial catalase-peroxidase enzyme KatG producing the reactive isonicotinyl acyl radical, which forms a covalent inhibitory adduct with the NAD cofactor of the enzyme InhA (Rv1484) [31,54], an enoyl acyl-carrier protein (ACP) involved in the metabolism of fatty acids and mycolic acid. Mycolic acid, a fundamental component of the cell wall of M. tuberculosis, is synthetized by two elongation systems, namely fatty acid synthase I (FAS-I), responsible for the de novo synthesis of the fatty acids with the carbon skeleton between C16 and C26, and fatty system II (FAS-II), responsible for extending the fatty acids carbon skeleton up to C56. While eukaryotic cells only use FAS-I, bacteria utilize also FAS-II for fatty acid biosynthesis. Hence FAS-II is an amenable drug target against TB.

Essential for the synthesis of fatty acids involving the FAS-II system in MTB is InhA which catalyzes the NADH-dependent reduction of the double bond at position two of the nascent fatty acid [54,55]. Due to its essentiality in mycolic acid biosynthesis, InhA has been the target of structural investigations [56] and of the elucidation of the molecular determinants of its inhibition. In this aspect, the broad-spectrum antimicrobial Triclosan inhibits InhA with an IC50 of 1 μM and has cidal activity against MTB [33]. Interestingly, the crystal structure of InhA in complex with Triclosan (PDB: 1P45 [57]) revealed a unique 2 : 1 Triclosan : InhA stoichiometry with two molecules of inhibitor stacked over the NAD cofactor, and only one inhibitor molecule interacting with the cofactor (Figure 1). Notably, the inhibitor molecules most distant from the NAD cofactor fill the void of a hydrophobic pocket which, according to structural data (PDB: 1BVR [58]), allows the accommodation of the nascent fatty acids during their biosynthesis. These data suggest a distinctive binding region of Triclosan that could be exploited for the design of novel InhA inhibitors.

Binding promiscuity of InhA.

Figure 1.
Binding promiscuity of InhA.

In the figure is reported the surface of MTB InhA (white) together with NAD (in red), the C16 fatty acid analog ((A), in grey; protein surface, NAD and fatty acid analog are taken from PDB 1BVR), the two Triclosan molecules ((B), in cyan; PDB: 1P45) and Pyridomycin ((C), in magenta; PDB: 4BGE). The fatty acid analog occupies a hydrophobic cavity which is also exploited by the two Triclosan molecules for binding. Pyridomicin also binds the hydrophobic pocket but extends itself toward the NAD binding site, hampering cofactor binding (in (C) the NAD molecule, although absent from the 4BGE model, is reported for explanatory purposes).

Figure 1.
Binding promiscuity of InhA.

In the figure is reported the surface of MTB InhA (white) together with NAD (in red), the C16 fatty acid analog ((A), in grey; protein surface, NAD and fatty acid analog are taken from PDB 1BVR), the two Triclosan molecules ((B), in cyan; PDB: 1P45) and Pyridomycin ((C), in magenta; PDB: 4BGE). The fatty acid analog occupies a hydrophobic cavity which is also exploited by the two Triclosan molecules for binding. Pyridomicin also binds the hydrophobic pocket but extends itself toward the NAD binding site, hampering cofactor binding (in (C) the NAD molecule, although absent from the 4BGE model, is reported for explanatory purposes).

The binding promiscuity of InhA has been spontaneously exploited by Pyridomycin, a natural compound produced by the bacterium Dactylosporangium fulvum with antibacterial properties. In their work, Hartkoorn et al. [34] showed that Pyridomycin is a competitive inhibitor of InhA by binding to its NADH-binding site and showing a Ki of 6.5 mM. The same authors reported also the crystal structure of InhA in complex with Pyridomycin which showed that it binds to the NADH-binding pocket and extends itself toward the substrate-binding pocket (PDB ID: 4BGE).

The efforts of pharmaceutical companies in searching for novel antitubercular scaffolds lead to the identification of a family of thiazole compounds with nanomolar inhibitory potencies against InhA which culminated in the compound GSK693 (Table 1) which showed in vivo efficacy similar to isoniazid, with the advantage of not being a pro-drug [35]. More recently, a HTS approach identified the compound AN12855, a diazaborine, as an InhA inhibitor with a binding mode independent of the cofactor and able to bridge the cofactor NAD+ and substrate-binding site, as already observed for Pyridomycin (Figure 1). Moreover, AN12855 showed efficacy in chronic and acute murine models of TB infection, indicating a novel cofactor-independent InhA inhibitor and a promising candidate to be tested in clinical trials [36].

Antimalarial drugs targeting NAD-dependent dehydrogenases

Together with TB, malaria is one of the major global infectious diseases, and due to its concurrent geographical overlap with TB, an interplay has been postulated between the two maladies [59].

The central carbon metabolism of the malarial vector P. falciparum has been the object of intense research for antimalarial drug development, also due to the fact that several evidences suggest the presence of a functional TCA cycle in P. falciparum [60]. Hence the TCA cycle could be considered as a potential target for the research of antimalarial remedies. However, the cross-reactivity of potential inhibitors targeting the TCA cycle of P. falciparum with those of the host could potentially impair their specificity.

The NAD-dependent MDH of P. falciparum catalyzes the conversion of malate in oxaloacetate with the reduction of NAD+ to NADH and has been also the target for new approaches for antimalarial drug discoveries and development. In a seminal paper, Lunev et al. [13] disrupted the interfaces of MDH oligomers by mutating key amino acids which, once mutated, could impair protein dimerization. Moreover, they studied the possibility of incorporation of the mutated monomer in the P. falciparum wild-type enzyme and demonstrated that the dimerization-impairing mutant could be incorporated in the native one, hence competing with the wild-type monomer for dimerization, and eventually interfering with its catalysis. Such innovative approach could be generally exploited for the development of novel biological drugs interfering with protein–protein interactions thus circumventing the problematics associated with drug selectivity due to the structure and sequence conservation between host and pathogen ortholog enzymes.

Targeting NAD-dependent dehydrogenase in cancer

Hypoxia, besides having a prominent role in MTB infection mechanism and survival, has been also recognized as a peculiar trait of certain tumors [61], and the expression and activity of enzymes of the TCA cycle have been also linked to the expression of many mitochondrial enzymes, related to the TCA cycle, which control the cellular process involved in hypoxia-related tumor formation and progression [62–65]. An important element in eliciting an hypoxia-induced programming for cell survival is the hypoxia-induced factor-1 (HIF-1) which has been correlated with poor cancer prognosis and chemoresistance in various types of cancer [66]. The NAD-dependent MDH isoform 2 (MDH2) induces the expression of HIF-1 under hypoxia condition and has been recently recognized as an attractive target for cancer treatment [67]. The research of HIF-1 inhibitors led to the discovery of LW6 (Table 1), an aryloxyacetylamino benzoic acid derivative, which acts as a novel HIF-1 inhibitor [68]. Later, the development and synthesis of LW6-derived chemical probes led to the identification of mitochondrial MDH2 as the target protein [37]. With the aim of finding compounds targeting MDH2, a virtual screening approach identified a benzohydrazide-based chemical (Compound 7, Table 1), which competitively inhibited MDH2 activity [38], thus rationalizing the evidence of its anti-cancer activity and encouraging the development of MDH2-based anti-cancer drugs.

As previously reported for MTB, IMPDH is also essential for the survival and proliferation of tumor cells, since IMPDH is involved in the de novo biosynthesis of purine nucleotides which are particularly relevant in diseases and conditions which involve proliferating cells (such as cancer, infection and organ transplants rejection) where a rapid generation of ATP and GTP is required [69]. Humans code for two isoforms of IMPDH (hIMPDH), namely hIMPDH1 and hIMPDH2; however, the type 1 isoform is constitutively expressed in both normal and neoplastic cells and covers ‘housekeeping’ functionalities, while the type 2 is preferentially up-regulated in tumor cells [70].

The first hIMPDH inhibitor to be identified was mycophenolic acid (MPA) which is referred as the very first antibiotic to be discovered [71,72]. MPA showed antiviral, antifungal and antibacterial properties [73] and also anti-tumoral efficacies in model cell lines such as leukemia, lymphoma, prostatic cancer and in murine models [74–77]. MPA inhibits hIMPDH with an IC50 of 0.020 µM and its hIMPDH-complexed structure has been described [39]. Moreover, it has been recognized as an anti-tumor agent in various cancer cell lines and mouse models [78–80]. However, the severe side effects provoked by MPA hampered the approval as an anti-cancer drug.

Historically, the inhibitory effect of MPA over hIMPDH activity has been recognized in the 1970s [81] and contemporarily it was also recognized as an immunosuppressant [82,83] which eventually lead to the development of the ester derivative mycophenolate mofetil (MMF) with an inhibition potency comparable to that of MPA (IC50 = 0.025 µM) [40], but with improved bioavailability [84]. Moreover, the role of IMPDH in hematological malignancies are evidenced by the high expression of IMPDH compared with non-malignant leukocytes, and it is proved that IMPDH inhibition leads to depletion of guanine nucleotides with the consequence of reducing cancer cells proliferation [85].

Tiazofurin represents the first of a new class of IMPDH inhibitor. Tiazofurin is a pro-drug which requires its transformation into the metabolite thiazole-4-carboxamide adenine dinucleotide (TAD) which acts as a NAD mimic and interacts with the NAD binding domain of hIMPDH1 and hIMPDH2 in a noncompetitive fashion (Ki = 0.5 µM) [41,85]. Tiazofurin anti-tumoral activity was observed in colon, lung, ovarian and renal cancer cell lines [86–88] and is most effective in myeloid malignancies, with encouraging results in chronic myeloid leukemia [89]. However, a major drawback of the effectiveness of Tiazofurin as an anti-cancer agent resides in its similarity to NAD which determines its toxicity and the severe complications in patients treated with Tiazofurin for more than 15 days [90]. Other distinct MPA analogs have been developed which were structurally distinct from MPA and the nucleoside analogs [91]. Of these, compound VX-944, developed by Vertex pharmaceuticals using a structure-based drug design approach, strongly inhibited hIMPDH1 and hIMPH2 (Ki = 6 and 10 nM, respectively) [92] and showed broad anti-cancer properties in vitro [42,93]. Unlike Tiazofurin, VX-944 does not require intracellular activation, hence circumventing one of the mechanisms of resistance to Tiazofurin.

Another important family of NAD+-dependent dehydrogenase of pharmaceutical interest in antineoplastic therapy are the members of the ALDHs superfamily (EC 1.2.1.3) consisting of 19 putatively functional isoenzymes that share about 40% of sequence identity. All these isoenzymes have prominent roles in the biosynthesis of important molecules such as the retinoic acid, the folic acid and betaine and in the metabolism of the γ-aminobutyric acid (GABA), an important neurotransmitter. ALDHs are also responsible for the detoxification of endogenous and exogenous compounds, especially in the detoxification of aldehydes, by converting toxic aldehydes into their respective carboxylic acid [94]. This activity is typically related to the action of different ALDH isozymes that can undergo overexpression under stress conditions. Hence, ALDHs interfere with the chemotherapies, and has been proposed that relapse in cancers could be due to the high activity of ALDHs in cancer patients [95–98]. ALDHs are constitutively expressed in different mammalian tissues, with the highest concentration levels in the liver due to their detoxifying role, followed by kidney, uterus, prostate, retina and brain [99]. Moreover, many ALDHs isozymes possess regulatory and metabolic roles in cancer; in particular, cancer stem cells (CSCs) have been observed to have elevated level of ALDHs expression, especially of the ALDH1A isoform [51,100].

ALDHs represent an important drug target, and several efforts have been employed for the structure-based engineering of molecule targeting ALDHs. The ALDH enzymes share a wide range of common physiological functions and substrates. Nevertheless, ALDHs isozymes have distinct substrate specificities [101], which would potentially facilitate the design of selective inhibitors. Many ALDH isozymes have been co-crystallized both in the apo form and in complex with their substrates, a process that helped out in the characterization of the active site structures [102–104]. ALDH2 and ALDH1A1 play an important role in the alcohol metabolism; indeed they are responsible for the oxidation of acetaldehyde to acetic acid, hence contributing to the detoxification of the by-product of ethanol consumption. The prominent role played by ALDH enzymes in alcohol metabolism led to the discovery of different inhibitors, such as Disulfiram and Daidzin [44,105] (Table 1), which eventually causes the accumulation of acetaldehyde following ethanol intake, leading the patient to experience a series of unpleasant symptoms which recapitulates the effects of heavy alcohol consumption [106]. Both inhibitors act on the NAD+-dependent aldehyde dehydrogenases ALDH1A1 and ALDH2, conserving the same mechanism of full competitive inhibition, with different affinity and potency [43,107], and causes acetaldehyde accumulation leading the individuals to suffer alcohol-intoxication related discomforts such as low blood pressure, tachycardia, facial flushing, nausea and vertigo [106]. These symptoms, collectively called disulfiram ethanol reaction [106], discourage the individual from indulging in alcohol intake. In particular, Disulfiram inhibits ALDH1A1 more potently than ALDH2 [106], probably due to the fact that in ALDH1A1 the hydrophobic access tunnel to the catalytic site is able to accommodate the bulky molecule disulfiram more effectively [43,107]. Several ALDHs inhibitors have been developed that reversibly and irreversibly target the ALDHs enzymes. One of the most studied ALDHs inhibitor, DEAB (N,N-diethylaminobenzaldehyde, Table 1), often used in research, inhibits six ALDH isoforms and it is a substrate for at least five more isoforms [45,48]. The scarce selectivity displayed by the DEAB is also characteristic of the covalent inhibitor disulfiram, whose metabolites inhibit also other ALDH isoforms [102]. A HTS approach discovered the noncovalent ALDH1A1 inhibitor NCT-501 [46] (Table 1), which lead to the development of the derivative compound NCT-505 and NCT-506 (Table 1), with increased solubility and selectivity toward this isoform [47]. A recent work reports a new inhibitor class of ALDH1A1 represented by compounds CM026, CM037 and CM053 (Table 1) showing high potency and selectivity towards ALDH1A2 and ALDH1A3. Notably, these three compounds inhibit the protein activity with different mechanisms: CM026 is a noncompetitive inhibitor for acetaldehyde and a competitive for NAD+, while CM053 is a noncompetitive inhibitor for NAD+ and CM037 is a competitive inhibitor for acetaldehyde [103]. Moreover, compound 13 g (Table 1) inhibits ALDH1A1 with an IC50 in the low nanomolar range and showing the same mechanism of inhibition of CM039, but without selectivity towards the ALDH1As isoforms [49]. Recently, three different inhibitors of ALDH1A2 have been biochemically and structurally characterized. The compound WIN18,446 covalently binds the enzyme to the highly conserved catalytic cysteine inducing a significant conformational change as observed in other cysteine-based enzymatic systems [108], resulting, in this case, in a lower affinity for NAD+. WIN18,446 is a non-selective potent inhibitor of the ALDH1A subfamily, and it is also able to inhibit ALDH2 [109]. Unfortunately, it also sequestrates zinc and shows teratogenic properties, hence it can be considered an imperfect scaffold in the development of active molecules to understanding the role of these enzymes. The other two inhibitory molecules 6–118 and CM121 (Table 1) are reversible competitive inhibitors and the structure revealed that both compounds bind in the active site, near the catalytic cysteine which bind to the active site in proximity to the catalytic cysteine without changing NAD+ affinity [50]. Moreover, two novel yeast ALDH inhibitors, GA11 and GA23 (Table 1), derivatives of the natural product Daidzin and able to penetrate the blood–brain barrier, inhibited the ALDH1A3 enzymatic activity with IC50 values in the micromolar range, and were able to specifically reduce the growth of MES83 glioma neurospheres [51 and Authors' unpublished data]. Despite the number of efforts in the research and development of compounds with selectivity for the ALDH isoforms, no selective inhibitors of the ALDH1A3 isoform have been discovered. The recent crystal structure of the ALDH1A3 isoform [110] provides molecular details of the retinoic acid binding pocket, and the structure could constitute the basis for the development of ALDH1A3 specific inhibitors (Figure 2A). Analysis of the ALDH1A3 structure revealed two different conformations for NAD+ and retinoic acid representing two snapshots along the catalytic cycle. As detailed in Figure 2B, the isoprenic moiety of retinoic acid points either toward the active site cysteine, mimicking retinal binding (Rea_D), or moves away adopting the product release conformation (Rea_C); at the same time, the nicotinamide ring of NAD+ moves in a concerted manner, either close to the active site cysteine (NAD_D) or toward the protein surface (NAD_C).

The overall structure of human ALDH1A3 (PDB_CODE 5FHZ).

Figure 2.
The overall structure of human ALDH1A3 (PDB_CODE 5FHZ).

(A) Ribbon representation of the human ALDH1A3 tetramer with chains A, B, C and D colored in blue, green, cyan and magenta, respectively. (B) The two different and related conformations adopted by retinoic acid (Rea) and NAD+. Upward, a zoom-in showing the Rea, yellow stick, in the two conformations as observed in monomers D and C, after optimal superposition; the catalytic Cysteine C314 is depicted as stick. Below, a zoom-in showing the NAD+, grey stick, in the two conformations as observed in monomers D and C, after optimal superposition.

Figure 2.
The overall structure of human ALDH1A3 (PDB_CODE 5FHZ).

(A) Ribbon representation of the human ALDH1A3 tetramer with chains A, B, C and D colored in blue, green, cyan and magenta, respectively. (B) The two different and related conformations adopted by retinoic acid (Rea) and NAD+. Upward, a zoom-in showing the Rea, yellow stick, in the two conformations as observed in monomers D and C, after optimal superposition; the catalytic Cysteine C314 is depicted as stick. Below, a zoom-in showing the NAD+, grey stick, in the two conformations as observed in monomers D and C, after optimal superposition.

This structure and the plethora of existing ALDHs structures will help the study of specific inhibitors for this superfamily and will help the scientific community in the elucidation of the role of this enzyme class in several diseases.

Targeting glyceraldehyde 3-phosphate dehydrogenase

The enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH; E.C: 1.2.1.12) is a key enzyme in the glycolysis pathway and converts the d-glyceraldehyde 3-phosphate in d-glycerate 1,3-biphosphate via a reversible NAD+/inorganic phosphate-dependent reaction. For many years, GAPDH has been regarded purely as a housekeeping glycolytic enzyme, mainly present in the cytoplasm. However, a variety of studies have suggested that GAPDH is a multifunctional protein involved in a variety of cellular processes like apoptosis [111–114], DNA repair [115], and cytoskeletal dynamics [116], and the multifunctional properties of GAPDH are directly linked with different oligomerization states of this enzyme, post-translational modifications, and subcellular localizations [117]. Moreover, GAPDH interacts with cytoskeleton proteins such as tubulin and actin, facilitating microtubule bundling and actin polymerization [116,118], and could be also observed not only in the cytoplasm but also in other subcellular compartments vesicles, mitochondria and in the nucleus [117].

Experimental evidences showed that altered levels of GAPDH are strictly connected with cancer and neurodegenerative diseases, and overexpression of GAPDH has been observed in different solid tumors like colon-rectal carcinoma [119], pancreatic cancer [120] and lung cancer [121], indicating that higher GAPDH levels may be associated with cell proliferation, tumor progression and chemoresistance.

Despite its key role in glycolysis and the multifaceted role in many different diseases, to date no structures of human GAPDH in complex with its inhibitor(s) exists. This is surprising since the Koningic acid, a potent GAPDH inhibitor (IC50 = 200 nM [122]) that binds its catalytic cysteine [123], has been discovered more than three decades ago [124].

The absence of structural reports of GAPDH in complex with Koningic acid has contributed to limiting the study of novel specific GAPDH inhibitors. The development of potent and specific inhibitors of P. falciparum GAPDH could impair the glycolysis of the parasite, potentially hindering its role in the Warburg effect in vivo [52]. Recently, however, Bruno et al. [53] reported the inhibitory effects of derivatives of the covalent inhibitor 2-phenoxy-naphthoquinone, with potencies in the micromolar range towards T. brucei rhodesiense and P. falciparum GAPDH, respectively. The 2-phenoxy-1,4-naphthoquinone and its analogs showed also antitrypanosomal and antiplasmodial activities in vivo at submicromolar concentrations. Hence the 2-phenoxy-1,4-naphthoquinone scaffold can be considered as an important class for the identification of GAPDH inhibitors with increased potency and selectivity against P. falciparum.

Conclusions and perspectives

  • The NAD-dependent dehydrogenase family comprises a wide number of enzymes that cover primary roles in many organisms and are essential in numerous pathogenic processes.

  • Efforts are continuously devolved in the development of inhibitory compounds against NAD-dependent dehydrogenase; however, major difficulties arise regarding their specificity, especially for the ALDH family, where the large number of isozymes represent a multiplying difficulty factor for drug discovery.

  • We have herein summarized a selection of, in our judgment, the most impacting findings regarding the discovery of novel inhibitors targeting a restricted sub-group of NAD-dependent dehydrogenases involved in major infectious diseases worldwide as well as in cancer. NAD-dependent dehydrogenases has been identified as robust drug targets a long time ago and still remain an important area of investigation in drug discovery; up-to-date experimental approaches promises to keep the pace of discovery in this area by possibly tackling the major issue of selectivity of drug action.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

D.M.F. kindly acknowledges the grant ‘Roche per la Ricerca 2017 for funding.

Abbreviations

     
  • ALDHs

    aldehyde dehydrogenase

  •  
  • FAS-I

    fatty acid synthase I

  •  
  • FAS-II

    fatty system II

  •  
  • HIF-1

    hypoxia-induced factor-1

  •  
  • IMPDH

    inosine-5′-monophosphate dehydrogenase

  •  
  • MDH

    malate dehydrogenase

  •  
  • MDH2

    malate dehydrogenase isoform 2

  •  
  • MPA

    mycophenolic acid

  •  
  • PIA

    protein interference assay

  •  
  • TAD

    thiazole-4-carboxamide adenine dinucleotide

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