Methionine Aminopeptidases: Potential Therapeutic Target for Microsporidia and other Microbes

Abstract

Methionine aminopeptidases (MetAPs) have emerged as a target for medicinal chemists in the quest for novel therapeutic agents for treating cancer, obesity, and other disorders. Methionine aminopeptidase is a metalloenzyme with two structurally distinct forms in humans, MetAP-1 and MetAP-2. The MetAP2 inhibitor fumagillin, which was used as an amebicide in the 1950s, has been used for the successful treatment of microsporidiosis in humans; however, it is no longer commercially available. Despite significant efforts and investments by many pharmaceutical companies, no new MetAP inhibitors have been approved for the clinic. Several lead compounds have been designed and synthesized by researchers as potential inhibitors of MetAP and evaluated for their potential activity in a wide range of diseases. MetAP inhibitors such as fumagillin, TNP-470, beloranib, and reversible inhibitors and their analogs guide new prospects for MetAP inhibitor development in the ongoing quest for new pharmacological indications. This perspective provides insights into recent advances related to MetAP, as a potential therapeutic target in drug discovery, bioactive small molecule MetAP2 inhibitors, and data on the role of MetAP-2 as a therapeutic target for microsporidiosis.

Introduction

The unique therapeutic activity of methionine aminopeptidases (MetAPs) has been exploited for the development of new drug motifs over the past three decades (Ingber et al., 1990; Kusaka et al., 1991; Burkey et al., 2018; Cheruvallath et al., 2016; McBride et al., 2016; Heinrich et al., 2017; Goya Grocin et al., 2021). MetAP is a dinuclear metalloprotease that removes an N-terminal methionine residue from nascent proteins and facilitates post-translational modifications that are essential for the translocation, activation, regulation, and degradation of proteins in eukaryotic cells (Helgren et al., 2016; Han et al., 2018). MetAP has emerged as an influential drug target, leading to the synthesis of new compounds containing different functional groups for active drug design. MetAP plays a significant role in the survival and proliferation of both eukaryotes and prokaryotes. There are two major lineages of MetAP, namely MetAP1 and MetAP2. MetAP1 is differentiated from MetAP2 by the insertion of a polypeptide within the catalytic domain of MetAP2 (Arfin et al., 1995). The MetAP in eubacteria is closer to MetAP1 and the MetAP in archaebacteria is closer to MetAP2. In eukaryotes, both MetAP1 and MetAP2 are present, while prokaryotes only possess a single MetAP, which is consistent with the origin of eukaryotic MetAP1 and MetAP2 genes from a eubacteria and archaebacteria fusion event in eukaryotic evolution. MetAP2 is an important target due to its pivotal role in tissue repair and protein degradation (Helgren et al., 2016).

MetAP2 exhibits both enzymatic as well as non-enzymatic roles. In the case of enzymatic roles, it aids in the cleavage of initiator methionine (iMet) (Giglione et al., 2015). In addition, it is responsible for protein folding and protein degradation via N-degron pathways (Varshavsky et al., 2019; Kramer et al., 2009). MetAP2 is also involved in maintaining protein homeostasis thereby promoting protein synthesis. Moreover, MetAP2 demonstrates string aminopeptidase activity making it an efficient target for discovering novel compounds (Xiao et al., 2010; Frottin et al., 2006; Moerschell et al., 1990; Chen et al., 2002). On the other hand, MetAP2 is known to exhibit non-enzymatic roles wherein an N-terminal extension (seen in most MetAP2 genes, but absent in the microsporidia) binds to elF2 kinases thereby protecting elF2 kinases from inhibitory phosphorylation (Datta et al., 2001; Datta et al., 2007). Similarly, MetAP2 promotes translation inhibition thereby propagating protein synthesis (Figure 1) (Griffith et al., 1997).

Figure 1.

Functional Roles of MetAP2.

MetAP2 inhibitors are divided into two categories: reversible and irreversible. Interestingly, most of the inhibitors that have been studied and used at the clinical level are irreversible inhibitors containing a highly reactive epoxide ring (Figure 2; Table 1). Fumagillin and its analogs, including TNP-470, and Beloranib have been demonstrated to have promise as anticancer agents. Fumagillin has been used to treat microsporidiosis (in humans, fish, and insects) and was used as a therapy for amebiasis in the 1950s. The covalent binding of either the ovalicin or fumagillin epoxide moiety to the active site of a histidine residue in MetAP2 results in the inactivation of MetAP2. There are several reversible MetAP2 inhibitors developed in recent years which include the anthranilic acid sulfonamides (Shahlaei et al., 2010; Fassihi et al., 2012; Sheppard et al., 2006), triazole sulfonamide (Garrabrant et al., 2004), bestatin (Mauriz et al., 2010), triazole (Marino et al., 2007), and Bengamide (Xu et al., 2012) analogs (Figure 3; Table 1). Unfortunately, except for the Bengamides (White et al., 2017), the other reversible MetAP2 inhibitors have encountered limitations in clinical trials because these inhibitors are not as potent as the irreversible inhibitors. MetAP-targeted compounds have been investigated as therapeutic agents for cancer, obesity, and inflammation.

Figure 2.

Irreversible MetAP2 inhibitors

Table 1.

Examples of various MetAP2 inhibitors and their biological activities (Goya Grocin et al., 2021)

S. No	Compound	Pharmacological Activity	Type of inhibitor	Remarks	References
1		Anti-Cancer Anti-Angiogenic Anti-parasitic	First generation Irreversible	Severe Weight Loss	Ingber et al. (1990)
2		Anti-Cancer Anti-Angiogenic Immunosuppressive	First generation Irreversible	Neurotoxic Takeda	Kusaka et al. (1991) Griffith et al. (1997) O'Reilly et al. (1995) Catalano et al. (2001) Shusterman et al. (2001) Kidoikhammouan et al. (2019) Peacock et al. (1995) Sigg et al. (1968) Hartmann et al. (1978) Turk et al. (1998) Benny et al. (2008)
3		Anti-cancer (Non-Hodgkins’s lymphoma) Anti-Inflammatory Immunomodulatory	First generation Irreversible	Went into clinical trials NCT00100	Ashraf et al. (2010 & 2011)
4		Anti-Cancer	Dual metAP1 and MetAP2	Developed by Novartis. Cardiovascular toxicity in Phase 1 and varying response in patients	Towbin et al. (2003) Kinder et al. (2001) Dumez et al. (2007)
5		Anti-cancer against carcinoma, sarcoma and neuroblastoma Anti-obesity	Reversible	Developed Abbott. Acts on HUVEC	Wang et al. (2003) Huang et al. (2019) Morowitz et al. (2005)
6		Anti-Angiogenic	Reversible	Developed by GSK	Marino et al. (2007)
7		Anti-Cancer (Glioblastoma)	Reversible	Currently in Phase 1	Heinrich et al. (2019)
8		Anti-Cancer Anti-obesity	Second generation Irreversible	Halted in phase III due to thromboembolism and deep-vein thrombosis	Kim et al. (2005 & 2007) Shin et al. (2010 & 2012) Hughes et al. (2013) Proietto et al. (2018)
9		Anti-obesity. Better pharmacokinetics than beloranib	Second generation Irreversible	Halted in phase II in 2018 due to cardiovascular safety risk	Burkey et al. (2018) Malloy et al. (2018)
10		Anti-obesity	Reversible	Developed by Takeda	Cheruvallath et al. (2016) McBride et al. (2016)
11		Anti-obesity by acting on brown adipocytes	Reversible	Developed by Takeda	Huang et al. (2019) Farrell et al. (2019)
12		Treatment of Autoimmunity by effecting IgG production in B cell model	Reversible	Developed by GSK	Heinrich et al. (2019) Hirst et al. (2020)

Figure 3.

Reversible MetAP-2 inhibitors from different structural classes: [4; (A-832234, Anthranilic acid-sulfonainide), [5; (JNJ-4929821, Triazolesulfonamide)], [6; (A-357300, Bestatin)], [7; (Triazole)], [8; (LAF-389, Bengamide)], [9; (LAF-153, Bengamide)].

There are limited reviews in the literature related to the synthesis and activity of MetAP inhibitors (Goya Grocin et al., 2021; Han et al., 2018; Mauriz et al., 2010; Joharapurkar et al., 2014; Frottin et al., 2016; Datta, 2009), their pharmacodynamics, pharmacokinetics, safety profile, and direct and indirect mode(s) of action. In this review article, we have assembled and integrated the available data in the literature and categorized the biological activities of MetAP inhibitors, their mode of action, and the drugs that went into clinical trials. We also provide insights gleaned from the literature concerning the pharmacological potential of MetAP inhibitors as antimicrobial therapeutic agents. In addition, we briefly discuss the side effects, safety profiles, and their direct and indirect mode of action.

MetAP as a therapeutic target

Most eukaryotes possess both classes of methionine aminopeptidases, however, prokaryotes have a single class possessing either homolog of MetAP1 (eubacteria) or MetAP2 (archaebacteria).

MetAP as a target for tuberculosis

Tuberculosis (TB), infection due to Mycobacterium tuberculosis) is a major cause of mortality throughout the world. Millions of people annually become ill due to tuberculosis. The increasing drug resistance of Mycobacterium tuberculosis (Mt) to available agents has received significant attention and new anti-tuberculosis agents are a major need for managing this disease. MetAP1 is a promising target for developing anti-tuberculosis agents. Liu et al., (2010) tested a series of structurally diverse naphthoquinone derivatives against MtMetAP1a and MtMetAP1c enzymes (Figure 4) (Olaleye et al., 2010). Among the tested compounds, 2,3-dichloro-1,4-naphthoquinone (31) and 2,3-dibromo-1,4-naphthoquinone (32) were found to be the most potent against MtMetAP1a and MtMetAP1c with IC₅₀ values around 1 to 6 μM. Further, compounds (31) and (32) were evaluated in vitro against M. tuberculosis and were effective with minimum inhibitory concentration (MIC) values of 10.0 and 10.0–25 mg/mL, respectively. In addition, these two compounds were also potent against latent (non-growing) M. tuberculosis organisms in addition to those that were actively replicating. Liu et al., (2011) have extended this work and reported 7-bromo-5-chloroquinolin-8-ol (CLBQ14) as a potent and selective inhibitor of M. tuberculosis methionine aminopeptidases (MtMetAP1a and MtMetAP1c) (Figure 4) (Olaleye et al., 2011). Comparative studies of CLBQ14 with naphthoquinone demonstrated that CLBQ14 derivatives were more potent than previous compounds against replicating and latent non-growing M. tuberculosis. Moreover, the anti-mycobacterial activity of CLBQ14 was correlated with its in vitro enzymatic inhibitory activity. These results demonstrate that naphthoquinone and 7-bromo-5-chloroquinolin-8-ol-containing inhibitors are potential therapeutic agents for tuberculosis.

Figure 4.

Naphthoquinone and CLBQ14 as MetAP1 inhibitors exhibiting anti-TB activity

Subsequently, Kratky et al., (2012) developed a series of salicylanilide benzoates and pyrazine- 2-carboxylates derivatives (Figure 5). The synthesized compounds were evaluated for antimicrobial activity and exhibited MIC values in the range of 0.5-62.5 μmol/L in vitro. Moreover, these compounds exhibited moderate inhibitor activity to mycobacterial and human methionine aminopeptidases. Among all of these derivatives, compound (34) was the most potent and selective inhibitor of M. tuberculosis MetAP (41% inhibition at 10 mmol/L).

Figure 5.

Pyrazine- 2-carboxylates as potent MetAP1 inhibitors for the treatment of TB

Bengamide, a natural product, is known to possess potent anti-proliferative activity in mammalian cells, and this led to the design and synthesis of a new class of MetAP inhibitors for antibacterial and anti-tuberculosis therapeutics. Lu et al., (2011 & 2012) synthesized a series of bengamide derivatives and evaluated them for their inhibitory activity against MtMetAP1a and MtMetAP1c in different metalloforms and for their activity against both replicating and non-replicating M. tuberculosis. These compounds were also tested against human K562 cells. Compound (36) was found to be a potent growth inhibitor of M. tuberculosis as well as the most active of the series in inhibiting the growth of human cells. The X-ray structures of MtMetAP1c in complex with (35), (36), (37), and (38) revealed different binding modes at the active site and provided improved potency and selectivity of these inhibitors which will be useful for further modification of these Bengamide derivatives (Figure 6).

Figure 6.

Bengamide derivatives as promising MetAP1 inhibitors for the treatment of TB

3b. MetAP as a target for bacterial infections

Hagen and colleagues designed and synthesized a novel series of compounds containing nitroxoline, triazole, furan, and anthranilic acid sulfonamides motifs and evaluated their inhibition of Burkholderia pseudomallei (the etiologic agent of melioidosis) MetAP (BpMetAP) (Figure 7) (Wangtrakuldee et al., 2013). The inhibitory activity was evaluated against BpMetAP using an in vitro enzyme activity assay. Among all tested compounds, five compounds displayed significant growth inhibition, and compound (42) showed potent inhibition in the enzyme activity assay with IC₅₀ of 30 nM. Compound (43) also displayed potent activity in the enzyme activity assay and complete cell growth inhibition in vivo. These nitroxoline-analogs have the potential to become templates for the treatment of melioidosis.

Figure 7.

Nitroxoline derivatives MetAP1 inhibitors against melioidosis

Wang et al., (2011) reported the synthesis of salicylate-based compounds by the reaction of phenylboronic acid with corresponding brominated thiophenes through Suzuki coupling (Figure 8). A library of new compounds was synthesized and evaluated for enzyme inhibition and bacterial cell growth inhibition. The inhibitory potency of these compounds for MetAP enzymes was carried out in the presence of Co(II), Mn(II), and Fe(II). Among these compounds (44), (45), and (46) were identified as the most potential inhibitors and showed good selectivity for the Fe(II)-form of MetAP. Interestingly, these potent compounds have shown significant antibacterial activity against E. coli. Furthermore, compounds (47-50) were not selective among the metalloforms, but exhibited considerable antibacterial activity against E. coli. It is noteworthy that compounds containing hydroxyl and carboxyl groups at the adjacent position in the phenyl ring of salicylate derivatives showed good enzyme inhibition and antibacterial activity.

Figure 8.

Salicylate-based compounds as MetAP inhibitors for the treatment of E. coli infections

Zhang et al., (2013) described a novel class of 58 inhibitors containing pyridinylpyrimidine as a core structure. The inhibitory activity of compounds was determined against human MetAP1 (HsMetAP1) and human MetAP2 (HsMetAP2) (Figure 9). Among tested compounds, six compounds (51-56) were found to be the most selective and potent inhibitors of purified HsMetAP1. Based on the structure-activity relationship, the author concluded that 5-chloro-6-methyl-2-pyridin-2-ylpyrimidine was the essential motif for the inhibition of HsMetAP1. The inhibition activity against HsMetAP1 was further supported by the 5-chloro substituent on the pyridine ring. Also, the long C4 side chain played a significant role in improving HsMetAP1-selectivity. These compounds need to be evaluated against various microbial MetAPs.

Figure 9.

Pyridinylpyrimidines compounds as potent HsMetAP1 inhibitors

Helgren et al., (2017) described a series of compounds containing furoic acid, 1,2,4-triazole, and quinolinol motifs as their core structure. This series of compounds was screened for their in vitro enzymatic activity assay against Rickettsia prowazekii MetAP (RpMetAP). Based on this in vitro and silico study, the most potent compounds were tested for inhibition of R. prowazekii growth in a pulmonary vascular endothelial cell (EC) culture infection model system. These studies revealed that 1,2,4-triazole and quinolinol-based compounds had significant inhibitory activity at reasonable concentrations. The activity of the most potent compound (57) was monitored by fluorescence staining and microscopy and this provided a visual representation of its anti-rickettsial activity in the presence of mammalian host cells (Figure 10). Helgren et al., (2018) recently extended their work, by examining furoic acid, biaryl, and 1,2,4-triazole-containing compounds for their inhibitory activity against RpMetAP. All tested compounds were found to inhibit RpMetAP enzymatic activity with IC₅₀ values of less than 10 mM. Among them, compounds (58) and (59) showed potential inhibitory activity with IC₅₀ values of 0.27 and 0.40 mM, respectively. Further, a docking study performed against RpMetAP, using the open-source docking software AutoDock, determined the potential binding mechanism and relative activities of these hit compounds. Triazole-based compounds should prove to be a potent antimicrobial agent for the treatment of Rickettsia.

Figure 10.

1,2,4-Triazole and furoic acid as antimicrobial agents

Rose et al., (2015) utilized high-throughput screening to identify lead candidates from a large compound collection. From the screening campaign, aepinone amide derivatives were found to have moderate biochemical potency against E. coli, H. influenza, and S. pneumonia (Figure 11). Among tested compounds, (60) was found to be most active with IC₅₀ values of 0.8, 1.1, and 0.3 mM against E. coli, H. influenza, and S. pneumonia, respectively. The X-ray crystal structure of compound (60) demonstrated several strong polar interactions between MetAP and the compound backbone. Additionally, these novel MetAP inhibitors had many non-specific hydrophobic interactions with protein, and no direct interaction was observed between these compounds and the divalent metal cofactor.

Figure 11.

Azepinone amide exhibits antibacterial activity by inhibiting MetAP

Multidrug-resistant bacteria have emerged as a serious healthcare threat in recent years. New antibiotics with novel mechanism(s) of action are highly desirable to overcome drug resistance. In this regard, MetAP is an essential enzyme for cell survival in several organisms, including bacteria. Chai et al., (2010) described MetAP identifies itself as a viable target in the quest for innovative antibiotics. Numerous strong inhibitors of the refined enzyme, yet, were unable to demonstrate appreciable antibacterial action. Which divalent metal MetAP employs in bacterial cells as its natural cofactor is unknown. A cell-based assay utilizing overexpressed MetAP in permeabilized Escherichia coli cells to track the decomposition of a fluorogenic precursor was developed. The assay was validated using a series of MetAP inhibitors. The biological targets for which this cell-based assay is appropriate have a native function that is not well defined, which increases the probability of finding inhibitors capable of inhibiting the target.

Chai et al., (2011) described a series of seven compounds with MetAP activity and evaluated them for growth inhibition of a methicillin-susceptible Staphylococcus aureus ATCC 43300 strain (MSSA) and a methicillin-resistant S. aureus ATCC BAA1680 strain (MRSA) using E. coli AS19 strain as a guide for the selection of the compounds to be tested (Figure 12). All seven compounds showed considerable activity against the tested strains, compound (61) showed the most promising antibacterial activity with an IC₅₀ value of 2.8 mM or MIC at 1.0 mg/ml for the MSSA strain and an IC₅₀ of 1.0 mM or MIC at 0.7 mg/ml for the MRSA strain. Similarly, compound (62) also displayed potent inhibition activity against MRSA, MSSA, and E. coli. Bacterial MetAP enzymes, including Acinetobacter baumannii MetAPs (AbMetAPx and AbMetAPy), have also been demonstrated to be potential drug targets (Yuan et al., 2011).

Figure 12.

Thiophene as an antibacterial agent against MRSA by inhibiting MetAP1

MetAP as a potential target for leishmaniasis

Bhat et al., (2020) designed and synthesized a library of 2-(8-hydroxyquinolin-2-yl)-3-phenylthiazolidin-4-one derivatives from quinoline-carbaldehyde (Figure 13). The synthesized compounds were screened in an inhibition assay to find novel inhibitors for leishmanial methionine aminopeptidase 1 (LdMetAP1) by using molecular, biochemical, biophysical, and computational approaches. Among these tested compounds, compounds (63) and (64) had potential inhibitory activity against LdMetAP1 with IC₅₀ values of 1.31 and 1.10 μM, respectively. In biochemical studies, both compounds were found less effective and less toxic against purified HsMetAP1. Furthermore, structural studies demonstrated a key difference in the binding modes of (63) and (64) to LdMetAP1 and HsMetAP1 providing a structural basis for differences in inhibition.

Figure 13.

Hydroxyquinoline derivatives

MetAP as a potential target for malaria

Chen et al., (2006) described the participants of the methionine aminopeptidase class have been studied as possible antimalarial candidates. One of the two MetAP proteins in the P. falciparum genome, methionine aminopeptidase 1b (PfMetAP1b), was utilized to analyze a database of 175,000 compounds for inhibitors. A class of structural inhibitors with a 2-(2pyridinyl)-pyrimidine core was identified as potential therapeutic agents. Through structure-activity investigation, XC11, a strong PfMetAP1b inhibitor, was discovered. Its IC₅₀ was 112 nM. primary human fibroblasts did not show significant cytotoxicity on exposure to XC11, and this compound was found to be very selective for PfMetAP1b. XC11 was effective in mice malaria models and in vitro, it was inhibitory for P. falciparum 3D7, which is chloroquine (CQ)-sensitive, and P. falciparum Dd2, which is multidrug-resistant. These findings indicate that XC11 is a lead molecule for the creation of innovative malaria drugs and that PfMetAP1b is a potential target. Poreba et al., (2012) reported two malaria aminopeptidases, PfM1AAP and PfM17LAP, may be targeted for novel anti-malarial drugs. To determine the unique specificity of the substrate and kinetic properties of these enzymes, a new library of fluorogenic precursors constructed from natural and synthetic amino acids was developed. The PfM1AAP enzyme's aminopeptidase fingerprint closely matches the earlier published information for the three mammalian orthologs of this enzyme (rat, pig, and human), indicating that the enzyme's affinities for substrate detection have not undergone significant mutations during evolution. This implies that creating inhibitors that stop the malaria enzyme's function without affecting the host enzyme will be extremely difficult. However, the unique characteristics of each malaria aminopeptidase in terms of how it binds substrates in the S1 pocket suggest that it should be possible to design inhibitors that either selectively or collectively inhibit each of the enzymes for use in combination therapy. Data by (Valmourougane et al., 2011) and (Harbut et al., 2011), who created PfM1AAP- and PfM17LAP-specific inhibitors utilizing the fundamental bestatin scaffold, lend support to this theory, even though their studies did not demonstrate improved parasite death over bestatin. These findings can serve as a foundation for the creation of inhibitors and the future selection of particular substrates for this class of proteases, which may assist in clarifying the distinct functions that each plays in the evolution of malaria.

Arico-Muendel et al., (2009) discussed the efficacy of fumagillin analogs against trypanosomes, amoebas, and malaria parasites, together with an enhanced pharmacokinetic profile. They also demonstrated that these inhibitors were effective in a murine malaria model. They investigated the possibility of preserving TNP-470 function in compact, nonlabile carbamate derivatives with a variety of functional groups. Most of the chemicals in this group have potent inhibitory effects on all three parasite types, comparable to fumagillin and TNP-470. The compounds that exhibit the most favorable action across the panel are carboxylate 66 and hydrazide 67, suggesting that either basic or acidic functions can be tolerated (Figure 14). A second series of compounds had spacers made of amino acyl residues that were conformationally restricted to lengthen the carbamate group. This extension had little impact on their effect on P. falciparum (D2 strain) and T. brucei, but it significantly reduced their effectiveness against Entamoeba histolytica, by a factor of 10–100. In addition, compounds 68 and 69 (PPI-2788 and PPI-2791) were examined for efficacy in mice infected with P. berghei. PBS-treated controls did not live more than 7 days after infection, while those treated with these inhibitors were alive at 12 days. These findings demonstrate that these prototype drugs are capable of achieving notable in vivo efficacy.

Figure 14.

2-(2-pyridinyl)-pyrimidine core (65) for the potent PfMetAP1b inhibitor and fumagillin analog (66-69) for MetAP2 inhibitors on in vitro growth of Entamoeba histolytica, Plasmodium falciparum, Trypanosoma brucei

Novel MetAP2 derivatives for the therapy of microsporidia.

Microsporidia: Biology

For almost a century, researchers have been intrigued by the peculiar eukaryotic, obligatory intracellular parasites known as microsporidia. Microsporidia are highly specialized intracellular pathogens that have developed a very complex and distinctive infection apparatus, the polar tube, and have also through reductive evolution become dependent on their host cell. Microsporidia have streamlined metabolic processes, have expansion of transporters consistent with their obligate intracellular lifestyle, and reduced gene numbers and gene complexity. In the midst of the 19th century, while pebrine, often known as "pepper disease," was devouring silkworms across southern Europe as well as posing a danger to the European textile sector, the first known microsporidian was identified. Nageli identified the pebrine agent as a tiny parasite in 1857 (Kawarabata, 2003) and gave it the name Nosema bombycis. Whereas taxonomy at that moment failed to sufficiently capture the richness of microbial life, and Schizomycetes were a mishmash of bacteria and yeasts, Nageli believed Nosema was probably part of the schizomycete fungus. Following additional research, Balbiani, (1882) classified Nosema as a distinct group in 1882 and named it Microsporidia, which remains in use today. Microsporidia for many years were considered “primitive protozoa”, however more recent phylogenetic data suggests that microsporidia are related to the Cryptomycota (Han et al., 1882).

Microsporidia are a remarkably varied group of parasites. Currently, about 220 genera have been described representing over 1500 species of microsporidia (Becnel et al., 2014; Sprague et al., 1992). Microsporidia have been found in both vertebrates and invertebrates. In addition, there have also been reports of microsporidia infections in other protists, including ciliates and gregarine apicomplexan (Vivier, 1975). The range and abundance of microsporidia that are currently recognized suggest that there are likely considerably more microsporidia that exist than are currently identified and that these pathogenic organisms are probably found in all vertebrates and invertebrate species. Microsporidia have been used as biological weapons against insect pests and also cause damage to fish, apiculture, and certain key crustaceans that are utilized in aquaculture (Becnel et al., 2014; Shaw et al., 1999).

Encephalitozoon cuniculi was initially discovered in rabbits in 1922 and is the very first microsporidian disease officially reported in mammals (Wright et al., 1922). It is currently known that this species commonly infects a wide variety of mammals. The first documented instance of a human microsporidian disease was in 1959 (Matsubayashi et al., 1959). However, before the mid-1970s, when the prevalence of immunocompromised people increased, as a consequence of the HIV epidemic, microsporidiosis was rarely seen in humans (Weber et al., 1994). As a consequence of the HIV epidemic, Enterocytozoon bieneusi was identified in 1985 as a gastrointestinal parasite that caused "wasting syndrome," a potentially fatal form of diarrhea (Desportes et al., 1985; Sandfort et al., 1994). It is now appreciated that E. bieneusi can also sporadically infect immunocompetent individuals, leading to a self-limited intestinal illness (Franzen et al., 2001; Vavra et al., 1999; Weber et al., 1999). To date, thirteen species of microsporidia have been identified in human infections including organisms in the genera: Nosema, Vittaforma, Pleistophora, Trachipleistophora, Enterocytozoon (i.e., E. bieneusi), Encephalitozoon (i.e., E. cuniculi, E. hellem and E. intestinalis), Septata (reclassified as Encephalitozoon), Brachiola (reclassified as Anncaliia, i.e., A. algerae), Tubulinosema, and Microsporidium. These various infections have been associated with disease in almost all organ systems including gastrointestinal disorders, keratoconjunctivitis, pneumonia, nephritis, urinary tract inflammation, prostatitis, liver disease, encephalitis, myositis, and pancreatitis (Weber et al., 1994; Franzen et al., 2001; Weber et al., 1999; Wittner et al., 1999).

The spore is a central feature of this organism's life cycle (Fig. 15). Spores vary in size, from as small as 1 μm in Enterocytozoon bieneusi to as large as 40 μm in Bacillidium filiferum, they can be spherical, oval, rod-shaped, or crescent-shaped, with the majority being oval. Spore morphology is generally extremely consistent for a given microsporidia; however, some microsporidia may form distinct spores at different times of their reproductive cycles (Vavra et al., 1999). Typically spores have an exospore and endospore layer. The exospore layer is typically homogenous and composed of a solid, granulofibrous, protein-containing substance (Bigliardi et al., 1996; Vavra et al., 1986). At the top portion of the spore, the endospore layer is thinner than elsewhere on the spore. The endospore layer consists of proteins and alpha-chitin. The sporoplasm and nucleus are found inside the spore wall (Vavra et al., 1999). The sporoplasm has a cytoplasm packed with ribosomes, one nucleus, or two nuclei grouped as a diplokaryon (two tightly appressed nuclei). In addition, the spore contains a posterior vacuole, a polar tube (called a polar filament while in the spore), and the polaroplast. The polaroplast is a membrane arrangement that resides in the spore's anterior region (consisting of the vesicular and lamellar polaroplast). The polar filament, also known as the polar tube, is the most noticeable organelle in the spore and is essential for the transmission of these pathogens. The protein filament has a width of 0.1 to 0.2 μm and an average length of 50 to 500 μm (Vavra et al., 1999; Keohane et al., 1999). The anchoring disk, an umbrella-shaped framework, is where the polar tube is anchored at the highest point of the spore and reaches across the anterior of the spore. The polar tube is linear for roughly one-third to one-half of its total width and then is spirally twisted around the sporoplasm (Wang et al., 2018; Esvaran et al., 2018). The posterior vacuole is where the polar tube ends (Lom et al., 1963; Weidner, 1972). The polar tube and the posterior vacuole appear to have some sort of physical connection, although it is unknown if the polar tube truly enters its vacuole or merely interacts with it because the point where they meet hasn't been defined by ultrastructural studies (Vavra et al., 1999; Keohane et al., 1999; Vinckier et al., 1993).

Figure 15.

Microsporidia life cycle

Microsporidia undergo merogony followed by the development of mature spores (Fadhilah et al., 2023). The spore is capable of withstanding environmental exposure and is the transmissible, i.e., infectious, stage of the life cycle (A). When it encounters appropriate environmental conditions it germinates extruding the polar tube which makes contact with its host eukaryotic cell (B). The polar tube (C) delivers the sporoplasm into the host cell. The sporoplasm then begins to reproduce inside the cell, which is characterized by merogony (binary or multiple fission) resulting in the formation of meronts (D). This stage of development takes place in different places inside the cell depending on the species of microsporidia. For example, Enterocytozoon and Nosema replicate within the host cell cytoplasm and are not surrounded by a vacuole, whereas Encephalitozoon replicates within a parasitophorous vacuole, (E). Meronts then undergo sporogony forming sporonts and then mature spores. Eventually, the spores exit the host cell, usually due to rupture of the host cell, and are free to infect new host cells (F).

Microsporidiosis; Epidemiology, Disease and Diagnosis

Microsporidiosis occurs worldwide in vertebrates and invertebrates. Spores of Nosema sp. (and probably Anncaliia algerae) have been discovered in ditch water, and Enterocytozoon bieneusi and Vittaforma corneae (syn. Nosema corneum) have been found in surface waters. There are cases of donor-derived transmission of microsporidiosis (Encephalitozoon cuniculi) after organ transplantation. In the agricultural sector, microsporidia have been used to control harmful insects, however, infections occurring in beneficial insects, such Nosema apis and N. ceranae in bees, can affect crop production. Honeybees infected with N. ceranae can have a mortality rate as high as 94% (Higes et al., 2007). Additional examples of microsporidiosis in agriculture and aquaculture, include Loma salmonae in salmon and Thelohania and Enterocytozoon species in shrimp.

Human microsporidiosis has been described from all continents (except Antarctica). In humans, the clinical symptoms of microsporidiosis are contingent upon the causative species and mode of exposure. Diarrhea linked to Enterocytozoon bieneusi in immunocompromised patients is the most commonly reported microsporidian infection in humans. As can be seen in Table 2 microsporidia can infect numerous organ systems in humans. To date 17 species of microsporidia have been described as causing infections in humans, with Enterocytozoon bieneusi and Encephalitozoon intestinalis being reported most commonly (Lobo et al., 2012) Enterocytozoon bieneusi was originally identified as an AIDS-defining opportunistic infection that occurred in patients with CD4+ T cells counts under 100 per mm3 (the majority having CD4+ counts less than 50 per mm3) (Morpeth et al., 2006). Because of antiretroviral medication and improved hygiene, the incidence of microsporidiosis in those living with HIV has steadily declined (Didier et al., 2011). Reported prevalence rates for microsporidiosis with diarrhea in HIV-infected patients, before 1998 and the widespread use of combination antiretroviral therapy, demonstrated a prevalence that varied between 2% and 70%, depending on the symptoms of the population studied and the diagnostic techniques utilized. When combined these studies provide a rate of 15% (Zhang et al., 2005). A more recent meta-analysis of 131 studies of gastrointestinal infection in HIV-infected individuals found a pooled prevalence of 11.8% (CI: 10.1%-13.4%) for microsporidiosis using a random effects model (Didier et al., 2011). In immune-competent humans, such as travelers and children, stool surveys have found microsporidia in from 2 to 27% of samples surveyed (Zhang et al., 2005). Microsporidia infection is an important cause of morbidity in people with HIV infection.

Table 2.

Microsporidiosis in humans

Microsporidia	Infections Reported
Anncaliia algerae	Eye, muscle, skin
Anncaliia connori	Systemic
Anncaliia vesicularum	Muscle
Encephalitozoon cuniculi	Systemic, Liver, Peritoneum, Brain, Urethra, Prostate, Kidney, Sinuses, Eye, Bladder, small intestine, Skin
Encephalitozoon hellem	Eyes, Sinuses, Lungs, Kidney, Urethra, Bladder, small intestine
Encephalitozoon intestinalis	Small intestine, gall bladder and biliary tree, kidney, eye
Enterocytozoon bieneusi	Small intestine, gall bladder and biliary tree, bronchioles, nasal mucosa
Endoreticulatus spp.	Muscle
Microsporidium spp.	Eye
Nosema ocularum	Eye
Pleistophora ronneafiei	Muscle
Trachipleistophora anthropopthera	Systemic, brain, eye
Trachipleistophora hominis	Eye, muscle, sinuses
Tubulinosema acridophagus	Systemic, muscle
Vittaforma corneae	Eye, bladder

Due to their tiny size microsporidia are challenging to identify. Microscopically observing stool samples is the basis for the majority of diagnosis of this infection, although it cannot identify microsporidia down to the specific species level. The Chromotrope 2R method, or its variants, is the labeling method most commonly employed. Calcofluor white and similar agents that stain chitin (which is found in the spore wall) are also helpful for quickly identifying spores in fecal smears. The spores of Enterocytozoon bieneusi range in size from 0.8 to 1.4 μm, while those of Anncaliia algerae, Encephalitozoon spp., Vittaforma corneae, and Nosema spp. measure 1.5 to 4 μm. Speciation of microsporidia has traditionally been done by electron microscopy and this remains the standard method; however, molecular methods (usually rRNA gene sequence data) can be used for speciation and diagnosis. To this end, PCR is commonly used to identify the Encephalitozoonidae and Enterocytozoon biensusi (Ghosh et al., 2009).

Microsporidiosis: Treatment

Albendazole and fumagillin are commonly used medications to treat microsporidiosis in humans as well as animals (Figure 16). Organic mercury-containing substances, e.g., Nosemack, have also been utilized in bees for microsporidiosis, but compared to fumagillin, they are less effective and have higher toxicity. Fumagilin, a drug produced by Aspergillus fumigatus that was used for its amoebicidal activity in the 1950s, was found to reduce Nosema apis growth in honeybees. It has subsequently been demonstrated to have broad activity against many microsporidia, including Enterocytozoon bieneusi and the Encephalitozoonidae. Albendazole, an antihelmintic and antifungal drug, binds to tubulin preventing its polymerization (Didier, 2005). It has varied efficacy towards Enterocytozoon bieneusi, however, it is highly effective for the treatment of the Encephalitozoonidae that cause microsporidiosis in humans and other mammals (Didier et al., 2009). The amino acid sequence of the Enterocytozoon bieneusi and Vittaforma corneae tubulins have amino acids that have been associated with resistance to albendazole, in contrast, the primary amino acid sequence of the tubulins from Encephalitozoon hellem, Encephalitozoon cuniculi, and Encephalitozoon intestinalis are consistent with the reported sensitivity of these microsporidia to albendazole (Tremoulet et al., 2004). Restoring the immune system with antiretroviral therapy has been associated with the resolution of the symptoms of microsporidiosis in immunocompromised HIV-positive patients (Didier Elizabeth et al., 2006).¹¹⁵ In addition, HIV protease inhibitors have been reported to have activity in vitro against Encephalitozoon intestinalis (Menotti et al., 2005). Itraconazole and similar antifungal medications have been used for microsporidiosis, usually in combination with other medications such as albendazole (Didier Elizabeth et al., 2006).

Figure 16.

Structures of Fumagilin, Albendazole, and Ovalicin commonly used medications to treat microsporidiosis in humans as well as animals.

In a review by Han et al., (2018) triosephosphate isomerase (TIM), tubulin, methionine aminopeptidase 2 (MetAP2), topoisomerase IV, chitin synthases, and polyamines were identified as targets that had been investigated for the therapy of microsporidiosis. Vittaforma cornea has been found to have a topoisomerase IV gene, which suggests that fluoroquinolones may be useful for the therapy. In vitro and animal experiments using polyamine analogs have also demonstrated the potential anti-microsporidia efficacy of these compounds.

Fumagalin, an antibiotic and antiangiogenic drug produced by Aspergillus fumigatus, has activity against most microsporidia including Enterocytozoon bieneusi and all of the Encephalitozoonidae. Systemic administration of fumagillin has resulted in the resolution of diarrhea due to Enterocytozoon bieneusi. The major toxicity of systemic fumagillin has been the development of thrombocytopenia. Topical application of fumagillin, as eye drops, has been successful in the treatment of microsporidian keratoconjunctivitis (Champion et al., 2010). Microsporidia keratitis has also been successfully treated with topical fluoroquinolones (ciprofloxacin 0.3%, moxifloxacin 0.5%, gatifloxacin 0.5%, levofloxacin 0.5%, and norfloxacin 0.3%) either alone or in association with topical fumagillin and/or systemic albendazole (Tremoulet et al., 2004).¹¹⁴

MetAP2 as a target for therapy of microsporidia

TNP-470, fumagillin, and ovalicin bind irreversibly to a common bifunctional protein identified by mass spectrometry as methionine aminopeptidase type 2 (MetAP2). Yeast deficient in MetAP1 (Δmap1) that only have MetAP2 are killed by fumagillin, TNP-470, or ovalicin, but yeast deficient in MetAP2 (Δmap2) that contain only MetAP1 is not inhibited by these drugs (Griffith et al., 1997). Deletion of both MetAP1 and MetAP2 is lethal. This confirms that fumagillin selectively targets MetAP2 and not MetAP1. Removal of the terminal methionine of a protein is often critical for the function and post-translational modification of a protein. Direct genetic evidence that EcMetAP2 is a target of fumagillin is provided by the evidence that yeast dependent on EcMetAP2 are killed by fumagillin (Upadhya et al., 2006). Microsporidia lack MetAP1, based on the genome data available at MicrosporidiadB.org; therefore, inhibition of MetAP2 by fumagillin results in cell death, analogous to the situation in Δmap1 yeast. Overall, the available data supports the concept that the cellular target for fumagillin and its analogs is the microsporidian MetAP2 homologue and that MetAP2 is an essential enzyme for these organisms.

Esvaran et al., (2018) reported the MetAP2 of Nosema bombycis, the etiologic agent of microsporidiosis (pebrine) in the silkworm Bombyx mori binds to fumagillin. The MetAP2 gene (cDNA of 1077 bp) from N. bombycis was isolated, cloned, and characterized in this study. NbMetAP2 was expressed equally throughout the silkworm embryonic stages. Fumagillin-B inhibited the growth of N. bombycis in the silkworm. Pandrea et al., (2005) described the identification of methionine aminopeptidases from genotypes within the Encephalitozoonidae. Using genomic DNA extracted from Encephalitozoon hellem, Encephalitozoon intestinalis, and Encephalitozoon cuniculi genotypes I–III, they amplified the methionine aminopeptidase genes from these Encephalitozoonidae. Analysis of the inferred amino acid sequences revealed that the microsporidian sequences were consistent with MetAP2 instead of MetAP1. These microsporidian MetAP2 sequences demonstrated the presence of the five residues (Asp, Asp, His, Glu, and His), which are thought to be essential for catalysis along with organizing the cation co-factor (e.g. cobalt). There were, however, some differences between the Encephalitozoonidae MetAP2s and the yeast and human MetAP2s including a lack of the NH2-terminal polylysine tract.

Zhang et al., (2005) amplified the MetAP2 gene from the human pathogenic microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Brachiola algerae, and Enterocytozoon bieneusi using a PCR homology cloning approach. The Encephalitozoon cuniculi MetAP2 (EcMetAP2) gene was cloned into a baculovirus vector. Utilizing chromatographic methods, recombinant EcMetAP2 was generated in baculovirus, isolated, and subsequently this was used to determine the ability of TNP-470 and to inhibit the activity of recombinant EcMetAP2 (Alvarado et al., 2009) The IC₅₀ values of fumagillin and TNP470 for EcMetAP2 were 11.1 nM and 10.6 nM, respectively, while recombinant HuMetAP2 demonstrated a significantly higher IC50 value for both fumagillin (188.5 nM) and TNP470 (95.0 nM) (Figure 17).

Figure 17.

Inhibition of EcMAP2 and Human MetAP2 (HsMetAP2) by Fumagillin (A) and TNP470 (B). MetAP2 enzyme (0.5μg) was incubated with or without Fumagillin or TNP470 in an enzyme dilution buffer containing CoC12 and the substrate Met-Ala-Ser as previously published.

Subsequently, Alvarado et al., (2009) used recombinant EcMetAP2 for the production of crystals for X-ray diffraction studies to determine the structure of EcMetAP2 in its apo, fumagillin, and TNP-470 bound states at resolutions of 2.2, 2.5, and 2.9 Å. Using these data sets, they also developed an in-silico model of Enterocytozoon bieneusi MetAP2. The EcMetAP2 structure was used to generate a homology model of E. bieneusi MetAP2 (EbMetAP2). EcMetAP2 exhibits a classic ‘pita-bread’ fold present in other methionine aminopeptidases from E. coli, S. aureus, M. tuberculosis, T. maritima, P. furiosus, and H. sapiens. The ‘pita-bread’ fold of EcMetAP2 is composed of a central beta-sheet with anti-parallel beta-strands β3, β5, β6, β9, β10, β15 and flanking alpha-helices α1, α2, α3, and α4. A dinuclear metal center and the active site are located on the concave face of the central beta sheet. Unlike the type-1 MetAP which possesses a ‘pita-bread’ fold with pseudo-two fold symmetry, this symmetry is broken in EcMetAP2 by the insertion of a sixty-one residue subdomain (β12, α5, α6, α7, β13) found in type-2 MetAP's and a two-stranded anti-parallel β-sheet (β7, β8) which is present only in the eukaryotic MetAP2 family. X-ray structures for MetAP2 of Pyrococcus furiosus, Homo sapiens, and E. cuniculi all differ at the N-terminus of the catalytic domain. Within the S1 hydrophobic methionine-binding subsite, which has been used as a region for the design of MetAP2 inhibitors (Poreba et al., 2012), E. cuniculi, and Enterocytozoon bieneusi possess V263 and A245, respectively, while methionine is present in the same position in the HuMetAP2 (M384). These changes to smaller amino acid residues in the S1 subsite of the microsporidian enzymes illustrate one of the opportunities for the generation of fumagillin analogs with increased specificity to microsporidia MetAP2. This illustrates how the X-ray crystal structures of HuMetAP2 and EcMetAP2 can be utilized for in silico screening of MetAP2 inhibitors.

Upadhya et al., (2006) used microsporidian MetAP2 genes to develop Saccharomyces cerevisiae strains that lacked S. cerevisiae MetAP1 (ScMEtAP1) and ScMetAP2 but expressed EcMetAP2 on episomal uracil selectable tetracycline-regulated plasmid (i.e., an EcMEtAP2ΔScMetAPiΔScMetAP2 yeast strain). Additionally, they were able to use 5-fluoroorotic acid-mediated plasmid shuffle in the EcMetAP2 yeast strain to make a yeast strain dependent on human MetAP2 (i.e., a HuMEtAP2ΔScMetAP1ΔScMetAP2 yeast strain). This system enables the production of an S. cerevisiae strain dependent on any MetAP2 gene that is cloned into the plasmid shuffle vector. These yeast strains can be used for screening compounds for efficacy against various microsporidian MetAP2s.

Methionine aminopeptidase 2 (MetAP2) has been the object of research aimed at elucidating the mode of its activity and discovering less toxic, more potent fumagillin-related medications. From a structure-activity relationship (SAR) point of view, as well as from the literature on MetAP2 inhibition by fumagillin and its analogs, it is clear that the C4 position, C6 position, and the spiro epoxide of fumagillin play important roles in MetAP2 inhibition. To improve the pharmacokinetic and safety profile of MetAP2 inhibitors (i.e., fumagillin/TNP-470/ovalicin analogs) one group modified the structure of the substituent at C6 on the cyclohexane ring, which is a polyolefinic acid in fumagillin and a reactive chloroacetylcarbamate in TNP-470. Crystallographic studies demonstrated that the primary interactions between fumagillin and MetAP2 involved the reactive spiro epoxide moiety as well as the prenyl-containing substituent at C4, whereas the ester chain at C6 extends through a narrow hydrophobic channel to the protein exterior. We designed and synthesized novel function-oriented small molecule libraries based on a Limited Rational Design (LRD) approach. We approached the generation of new compounds based on the existing inhibitors (fumagilin and TNP470); incorporating insights gained from the crystal structure of EcMetAP2 and our in-silico models of EbMetAP2. Computational protein structure modeling programs were utilized to study the MetAP2 active site and fit small molecules into this active site to model protein-drug complexes facilitating the development of new compounds. We developed several compound libraries that are based on modifications of the structure at fumagillin at the spiro epoxide, 4, and 6 positions to improve the activity, specificity, and reduce toxicity of our derivatives. We performed computer-assisted binding and docking studies using the ICM platform (an internal coordinate force field, Molsoft-ICM www.molsoft.com) to allow global optimization and analysis of small molecule geometries by performing free geometry optimization in Cartesian space using the MMFF94 force field including fully automated atom type assignments. One of our strategies was to add different functional groups at the spiro-epoxide position to prevent ring opening by other nucleophile substrates, as the epoxide is very prone to ring opening. In several of the compounds, we replaced acid groups with acid bioisosteres (boronic acid) as boron has a vacant orbital and inter-converts with ease between the neutral sp² and the anionic sp³ hybridization states, which generates a new stable interaction between the boron atom and donor molecules. Small molecules containing boron have demonstrated improved biological activity in pharmaceutical design (Das et al., 2023). To this end, one of our lead compounds BL6, a boron-containing compound (Figure 19), had excellent activity in vitro and in vivo against microsporidia (Figure 20).

Figure 19.

Structure of BL-6: a new MetAP2 inhibitor

Figure 20. Evaluation of novel MetAP2 inhibitors.

A. Cell culture assay demonstrating inhibition of the growth of Encephalitozoon cuniculi and Vittaforma corneae in host cells treated with BL6. B. Evaluation of BL6, D61, D62, and D6) using EcMetAP2 dependent yeast. There was no activity against wild-type yeast or yeast dependent on HuMetAP2

As illustrated in Figure 21, the Docking of the BL6 ligand was performed using a series of progressively refined protocols available in the GOLD Protocols. 2D and 3D images were derived using PyMOL, Discovery Studio (BIOVIA), and Protein-Ligand Interaction Profiler (PILP). Initially, the virtual screening protocol was used to dock BL6 to both the entire surface of MetAP2, as well as only at the ligand binding site. Next, a rigid receptor protocol was used to refine the docked conformations at the binding site. Finally, an induced fit protocol was used to allow protein flexibility at the cleft binding site to further refine the binding affinity. numerous molecular interactions occur between BL-6 and the amino acid residues lining the binding cavity, as well as with the di-metal co-factor CO²⁺ of HuMetAP2, in addition to forming a hydrogen bond with Asn204 (3.11 A°). Other interactions include pi-pi stacking with His208, a Pi-Anion bond with Glu241, several hydrophobic bonds with the co-factor, and various alkyl interactions, all contributing to binding affinity. In the case of EcMetAP2, BL-6 exhibits more robust hydrogen bonding with His562 (3.12A°, 3.01A°) and Asp560 (3.39A°), along with hydrophobic interactions with other amino acids and the Co-Factor FE (III) ion. Additionally, it forms various other interactions such as Pi-Anion and Pi-Pi interactions, leading to enhanced binding affinity.

Figure 21. Docking Studies of BL6 with METAP2:

A) 3D representation of HuMetAP2 (PDB ID:1B6A) with BL-6. And Ligand interaction 2D mapping of HuMetAP2/BL-6. B) 3D representation of EcMetAP2 (PDB ID:3FMQ) with BL-6 ligand interaction 2D mapping of Protozoal MetAP2/BL-6.

Usage of fumagillin and analogs

Mycotoxins include fumagalin. Encapsulated on genomic eight, it was first identified from A. fumigatus in 1949 and is located inside a supercluster. This mycotoxin attaches to and consistently eliminates the enzyme methionine aminopeptidase (MetAP) type 2. Since MetAPs play a crucial role in the hydrolyzation of the initial methionine (iMet) found in the N-terminal of newly generated proteins, any disruption caused by MetAP2 inhibition may affect several proteins, some of which are involved in the proper upkeep of cellular safety (Guruceaga et al., 2019). The many effects of fumagillin have their basis in this action. This toxin exhibited a dual effect of acting as an antibiotic by blocking the development of Entamoeba histolytica and acting similarly when interacting with macrophages (Novohradska et al., 2017). Casadevall et al., (2019) postulated that fungal infection sensitivity may be predicated on defense mechanisms created to ward off ameboid predators, among other findings. Furthermore, as fumagillin is the only chemical medication that effectively treats nosemiasis generated by the parasitic fungi found in the Microsporidia phylum on Apis spp., it offers pharmaceutical promise for the treatment of microsporidiosis (Molina et al., 2002). It is typically applied for managing pests in bee hives. Fumagillin is unable to be utilized extensively and must be used cautiously because of its toxicity. Thus, in certain uses, less hazardous substitutes for fumagillin are being created. However, fumagillin exhibits anti-angiogenic activity (Sin et al., 1997),¹³⁰ most likely as a result of its inhibitory effect over the MetAP2 enzyme; as a result, it has significant pharmacological promise and may be utilized for treating cancer (Mauriz et al., 2010). Furthermore, because of its antiangiogenic qualities, this toxin damages lung epithelial cells, which makes room for fungal invasion. It also inhibits neutrophil function, causing erythrocyte cell death (Fallon et al., 2010; Zbidah et al., 2013; Guruceaga et al., 2018).

Kanno et al., (2015) discovered that fumagillin, a strong vascular inhibitory agent, in primary effusion lymphoma cell lines, promoted Kaposi sarcoma-associated Herpesvirus (KSHV) multiplication. 1-10 μM fumagillin enhanced the replication transcriptional activator (RTA) transcript and protein product at 24 and 48 hours, respectively. Western blot research showed that other KSHV-encoded lytic proteins were also stimulated to generate RTA at a concentration of 10 μM fumagillin. The expression profiles of KSHV stimulated by fumagillin were identical to those produced by 12-O-tetradecanoylphorbol-13-acetate (TPA), although the levels of every gene were lesser compared to those induced by TPA, according to a real-time PCR array used for detecting KSHV gene expression. At last, in real-time in fumagillin-stimulated cells, PCR showed an increase in the viral DNA amount copied per cell. Initial effusion lymphoma cell lines, which demonstrate KSHV replication. Besides TPA, 10 μM fumagillin caused primary effusion lymphoma cell lines to stop growing. These findings imply that a substance with significant impacts on cell proliferation and KSHV resurrection is an angiogenic inhibitor of primary effusion lymphoma cells. Blanchet et al., (2014) reported similar to fumagillin, ligerin is a naturally occurring chlorinated merosesquiterpenoid that has been shown to have both in vivo anticancer action in a mouse model and specific antiproliferative effect towards osteosarcoma cell lines. The purpose of the semisynthesis of ligerin analogs aimed to investigate the impact of both the tweaking of the C6 chain and the replacement of a halogenated component for the C3 spiroepoxide. The outcomes demonstrated that all analogs had antiproliferative properties towards osteosarcoma cell lines in vitro and that the biological activity of chlorohydrin molecules was on par with or greater than that of their spiro epoxy models. The parent molecule outperformed other semisynthetic analogs in terms of effectiveness towards SaOS2 and MG63 human osteosarcoma cells, and it was fourfold less hazardous against HFF2 human fibroblasts. This made it the greatest choice for additional research.

Esser et al., (2016) described a viable lipase-labile prodrug of the unstable anti-angiogenesis mycotoxin fumagillin has been developed and added to integrin-targeted lipid encapsulated nanoparticles (αvβ3-Fum-PD NP). An evaluation was conducted on multimodal anti-angiogenic therapy, which involved the combination of αvβ3-Fum-PD NP and zoledronic acid (ZA), a long-lasting osteoclast inhibitor that may have anti-angiogenic effects. αvβ3-Fum-PD NP decreased (P b 0.05) the viability of endothelial cells in vitro, but did not affect the survival of macrophages. At large dosages, ZA reduced (P b 0.05) macrophage vitality yet did not affect endothelial cell growth. When ZA was added to rabbit Vx2 tumors, 3DMR neovascular imaging revealed no change, but ZA and αvβ3-Fum-PDNP reduced angiogenesis (P b 0.05). Immunohistochemistry demonstrated improved microvascular diminution (P b 0.05) using dual therapy and diminished microvascularity (P b 0.05) with αvβ3-Fum-PD NP and ZA. While αvβ3-Fum-PD-NPs decreased both measurements, ZA did not diminish the number of tumor macrophages or the growth of cancer cells in vivo. If there is a rise in macrophage ZA uptake, then dual therapy with ZA and αvβ3-Fum-PD-NP might provide improved neo-adjuvant usefulness. Asula et al., (2019) investigated to assess fumagillin bicyclohexylamine's effectiveness in treating corneal neovascularization in rats that was caused by cauterization with silver nitrate. The group's vascular endothelial growth factor (VEGF-C) levels did not significantly differ from one another (P = 0.994). There was no apparent distinction across the two categories when the angiogenic microvessel density for the peripheral corneal zone was assessed (P = 0.113). On the other hand, compared to Group 3, median vascular activity in Groups 1 and 2 was considerably larger for the central and mid-peripheral corneal zones (P = 0.015, P = 0.003).

An et al., (2018) described The current approaches to obesity are associated with adverse consequences and an absence of long-term benefits. By inhibiting methionine aminopeptidase-2, fumagalillin reduces appetite but does so more effectively than pair feeding when it comes to weight loss. In this instance, we demonstrate that giving rats a diet rich in fat enriched with fumagillin (HF/FG) decreases the hyperactive feeding seen in the HF/PF pair-fed controls and modifies circadian gene regulation in comparison to the HF/PF group. Rats that are HF/FG show multiple indicators of decreased use of energy, but HF/PF rats do not. Additionally, HF/FG rats show elevated energy harvesting by the gut bacteria, altered gut hormones associated with food intake, and calorie leaking in the urine. Fumagillin's impacts on the consumption of energy, but not feeding actions, have been shown in gnotobiotic mouse experiments. These consequences could be controlled by the gut flora. Fumagillin, in summary, activates behavioral and physiological pathways that cause weight reduction that are different from those triggered by merely limiting calories. Li et al., (2014) reported Three oxygenases derived from the fumagillin biosynthesis pathway have been identified and characterized. These include an ABM family monooxygenase for antioxidant cleavage of the polyketide molecules, a hydroxylating nonheme-iron-dependent dioxygenase, and a multifunctional cytochrome P450 monooxygenase. In particular, it is demonstrated that the P450 monooxygenase stimulates the two epoxidations, bicyclic ring opening, and consecutive hydroxylations that produce the sesquiterpenoid core structure. Additionally, we identified a shortened polyketide synthase that contains a ketoreductase activity that regulates the arrangement of modified precursors at position C-5.

Morgen et al., (2016) created a new family of MetAP2 inhibitors that can be substantially adaptable and were intended to resemble the molecular framework of fumagillin while having better pharmacological characteristics. These compounds were shown in biochemical enzymatic experiments toward three MetAP isoforms to be strong and selectively inhibiting MetAP2. Compared with their diastereomeric and enantiomeric isomers, inhibitors that shared fumagillin's comparative and unrestricted stereoconfiguration exhibited noticeably greater efficacy. The inhibitors covalently alter His231 in the MetAP2 active site by ring-opening a spiroepoxide, according to X-ray crystallographic research. Though similarly situated inactive isomers had no impact on the proliferation of either cell type, biochemically active compounds reduced the growth of endothelial cells and a MetAP2-sensitive cancer cell line. Modified N-terminal metabolism of the protein 14-3-3-γ was linked with these consequences. Ultimately, it was shown that certain compounds had better permanence in mouse plasma and microsomes when compared to beloranib, a fumagillin analog that was studied in clinical trials.

We have summarized the table on the efficacy of MetAP inhibitors for the treatment of microsporidian infections (Table 3).

Table 3.

Treatment of microsporidian infections in humans and animals using MetAP inhibitors

Drug	Species of Microsporidia	The host	References
Fumagillin	Nosema apis	Honey bees	Huang et al. (2013)
	Octosporea bayeri	crustacean	Zbinden et al. (2005)
	E. bieneusi	Human	Molina et al. (2002)
	Loma spp.	Chinook salmon	Higgins et al. (1998)
	N. salmonis	salmonid fishes	Higgins et al. (1998)
	Cystosporogenes sp	spruce budworms	Van Frankenhuyzen et al. (2004)
	N. bombycis	Silkworm	Esvaran et al. (2018)
TNP-470	Nucleospora salmonis,and Heterosporis finki	salmonid fishes	Van Frankenhuyzen et al. (2004)
	Encephalitozoon intestinalis, Vittaforma corneae, and Encephalitozoon hellem	Human (intestine, Cornea, and respiratory tract respectively)	Kent et al. (2014)
Fumagillin + Albendazole2	Encephalitozoon cuniculi	Rabbit	Kotkova et al. (2013)
Fumagillin, TNP-470, ovalicin, NSC 9665, NSC 141539	Vittaforma corneae	athymic mice	Didier et al. (2006)
Fumagillin, TNP-470, ovalicin, 9168, 9665, 56407, 141538, 676019, 174554	Encephalitozoon intestinalis	In-Vitro (RK-13 host cells)	Didier et al. (2006)

Conclusion

This review article describes current advances and perspectives on MetAP as a target in drug design and development and provides a bird's eye view for medicinal chemists for the rational design of novel MetAP inhibitors. There is increasing interest in MetAP as a therapeutic target and some compounds have been designed and explored as MetAP inhibitors. These various inhibitors have exhibited significant anticancer, antiprotozoal, and antimicrobial activities besides their role in anti-obesity, autoimmune disorders, and related complications. The MetAP inhibitors have significant promise as therapeutic agents for various infectious diseases. Despite, however, these efforts and investments by pharmaceutical companies, no MetAP inhibitor has yet been approved for the clinic. Inadequate data about the mode of action and inconsistency among clinical use and drug development endeavors have slowed down the advancement of MetAP2 inhibitors. Fumagillin, a MetAP2 inhibitor, has demonstrated efficacy in clinical trials for the treatment of Enterocytozoon bieneusi infection (microsporidiosis) in humans. However, fumagillin is no longer commercially available, despite its efficacy for this infection. With an improved understanding of substrate structure, and mode of action of existing MetAP inhibitors, it is expected that new therapeutic MetAP2 inhibitors could be moved into clinical use.

Figure 18.

Heterologous expression of EcMetAP2 and human MetAP2 (HuMetAP2) in yeast. Tenfold serial dilutions of the wild type and isogenic yeast strains containing vector alone or vectors containing either EcMetAP2 or HuMetAP2 were spotted onto YPD medium containing fumagillin (5 nM), ovalicin (1 nM) demonstrating inhibition by these compounds¹⁴⁶. ΔScMetAP1 is Δmap1 and ΔScMetAP2 is Δmap2.