Characterization and Inhibition of the 2-C-

methylerythritol 4-phosphate Pathway

Ellen Trost

Mary Baldwin University

Department of Chemistry & Physics

Spring 2021

Advisor: Dr. Maria Craig

Table of Contents

Abstract ...... 3 Introduction ...... 3 Methods Overview ...... 5 1-Deoxy-D-xylulose 5-phosphate Synthase (DXS) ...... 8 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) ...... 12 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (CMS) ...... 16 (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate synthase (HDS) ...... 21 (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate reductoisomerase (HDR) ...... 22 Conclusion ...... 24 References ...... 26 Abbreviations ...... 32

2 Abstract

The MEP pathway is a metabolic pathway that is not present in humans but is utilized by bacteria, plants, and apicomplexan protozoa to make isoprenoids, which are essential for their survival. Inhibiting the MEP pathway could deter the growth of these disease-causing microorganisms, making it an advantageous target for drug developers. This review examines the structures of the enzymes of the MEP pathway—determined via X-ray crystallography—that could be exploited by an inhibitor and evaluates several inhibitors that were tested with a variety of enzyme kinetic assays. Several studies show promising results that present clear inhibition of enzymes in the MEP pathway, but a few studies have found that inhibitors aren’t as effective in living systems for. There is also a lack of in vivo studies and trials in mammalian systems, which indicates that more research is necessary before inhibitors of the MEP pathway can unequivocally be considered drug treatments.

Introduction

The rise of antimicrobial resistance is an area of concern for scientists as new drugs are required for treatment of illnesses that we considered “cured” in the past.1 One area of interest for drug treatment is the 2-C-methylerythritol 4-phosphate pathway, which was first proposed in

1999 by Hartmut Lichtenthaler.2 A similar pathway, the mevalonate (MVA) pathway is well- established in the literature. The MVA pathway was thought to be the only pathway for the production of isoprenoids, which are the building blocks for important cellular components like cell walls and intercellular communication molecules like hormones. Lichenthaler published a literature review that compiled the growing evidence for an alternative pathway for isoprenoid synthesis and was the first to formally propose the MEP pathway.2 When Lichenthaler first published his review of the MEP pathway, only the first enzyme of the pathway, 1-Deoxy-D-

3 xylulose 5-phosphate Synthase (DXS), was established. Studies over the past two decades have characterized the rest of the enzymes and a clear pathway is now well-established in the literature (Fig. 1).

Figure 1. Enzymes and Intermediates of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Enzymes include: 1-Deoxy-D-xylulose 5-phosphate Synthase (DXS), 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (CMS), 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate synthase (HDS), (E)-4-Hydroxy-3- methyl-but-2-enyl pyrophosphate reductoisomerase (HDR).3

4 Lichtenthaler also noted that this pathway is only present in certain domains of life; bacteria, plants, and protozoans that contain a non-photosynthetic organelle called an apicoplast.2

The MEP pathway is thought to have originated from a cyanobacteria-like ancestor and is now present in some modern photosynthetic organisms through endosymbiosis. This is why MEP takes place in the chloroplasts of plants and unlike the MVA pathway, the MEP pathway utilizes the products of photosynthesis to generate isoprenoids, indicating a dependence on light for the

MEP pathway in photosynthetic organisms.2 In apicoplast-containing protozoans, such as the organism that causes , falciparum, the MEP pathway occurs in the apicoplast, also due to an endosymbiosis event,4–7 which occurs when one organism engulfs another. The apicoplast functions mainly as a compartment for vital metabolic processes including the MEP pathway.8

The presence of this pathway in disease-causing microorganisms and the absence of the pathway in humans indicates that inhibition of the MEP pathway could kill the microorganisms that cause disease but would not harm human cells. This review evaluates the current scientific literature concerning the structure and function of key enzymes in the MEP pathway as inhibition of these enzyme could be potential treatments for illnesses like bacterial infections, malaria, and tuberculosis. Several studies have characterized the enzymes of the MEP pathways and proposed viable inhibitors but researchers have yet to connect specific inhibitors with decreased proliferation of infectious diseases.

Methods Overview

The papers discussed in this review employ a diverse array of techniques and procedures. An overview of the two most pertinent methods is provided for proper context but be mindful that far more techniques were utilized than what is explicitly discussed. Before mechanisms of

5 inhibition for the enzymes in the MEP pathway could be proposed the enzymes had to be characterized so the structure could be understood with greater clarity. X-ray crystallography is frequently used to create an image of a crystallized protein like an enzyme. X-ray crystallography is quite similar to using a microscope to view a specimen except a beam of X- rays is passed through the crystal form of protein. A diffraction pattern is created by measuring the intensity of the X-rays that are scattered by the crystal. An electron density map can be computed from the angle and pattern of diffraction. With high resolution and amino acid sequence the atomic model can be constructed.9 After the structure of the enzyme has been determined, the ability of an inhibitor to decrease the activity of said enzyme can be determined using an activity assay. An activity assay, also referred to as an enzyme assay, measures the rate of a reaction using a variety of techniques. Kinetics data is commonly expressed is as a

Michaelis–Menten curve (Fig. 2), by plotting the speed of the reaction over the concentration of substrate of the enzyme. A Michaelis–Menten curve is useful for determining the Vmax, which is the maximum velocity of the reaction, this occurs when the enzyme is fully saturated with substrate. The Michael-Menten constant (KM) is the concentration of the substrate at half of the maximum velocity and is an inverse measurement of the affinity of the enzyme to its substrate.

There are a few different types of inhibitors with different mechanisms of inhibition but the most common type of inhibitor in biological systems is a competitive inhibitor, which decreases the speed of the reaction by binding to the active site of the enzyme and blocking the binding of the substrate. This type of inhibitor can be identified by a Michaelis-Menten curve with the same

Vmax as an uninhibited enzyme but an increased Km. The Vmax stays the same because at high enough concentrations the substrate will outcompete the inhibitor and the velocity will remain the same.10

6

Figure 2. Theoretical Michael-Menten saturation curves of an enzyme with no inhibitor (black), a competitive inhibitor (red), a noncompetitive inhibitor (blue), and an uncompetitive inhibitor (green).11

The studies discussed in this review utilize a variety of different assays to measure the progress of the reactions. One assay method that is used in the papers cited in this review is a spectrophotometric assay. Since NADH and NADPH, common cofactors for metabolic reactions, absorb light at a different wavelength than the oxidized forms (NAD+ and NADP+).

The speed of the reaction can directly be monitored by recording the light absorbed. Another way to determine the speed of a reaction is to measure the amounts of reactants and produces at different intervals with techniques like C-NMR, Liquid scintillation spectrometry, LC-MS/MS,

HPLC-MS, and GC-MS. Researchers also use the IC50 value, which is the concentration of inhibitor necessary to reduce the enzyme activity to half of its maximal value, as an inverse measure of an enzyme’s efficacy. The IC50 value should be less than the KM for an effective inhibitor.

7 1-Deoxy-D-xylulose 5-phosphate Synthase (DXS)

The first enzyme of the MEP pathway, DXS, was characterized in 1997 before

Lichtenthaler published his review article.12 While some of the evidence for an alternative pathway was acknowledged, it is not formally proposed as Lichtenthaler does in 1999 (Fig. 3).

DXS converts pyruvate and glyceraldehyde 3-phosphate (G3P) to 1-Deoxy-D-xylulose 5- phosphate (DXP), but it is also suggested that in E. coli, DXP plays a role in the synthesis of thiamin and pyridoxol. DXS performs an acyloin condensation where pyruvate is decarboxylated. This is also a well-established function of the thiamine diphosphate-dependent decarboxylation enzymes of the E1 domain of pyruvate dehydrogenase which uses thiamin pyrophosphate (TPP). This suggests that the enzymes may have related sequences motifs and subsequently structures. The gene that encodes DXS in E. coli was determined by comparing the known genome of E. coli with sequences similar to the E1 subunit of pyruvate dehydrogenase and other transketolases from E. coli. HPLC was used to analyze the reaction and it was found that upregulating the DXS protein increased the conversion of pyruvate and G3P to DXP, confirming the proposed function of DXS. An anion exchange column was used to purify the enzyme and the sequence of the N-terminus was found using Edman degradation. It was found that the activity of the enzyme was lost in the absence of TPP, indicating that TPP is a cofactor for the reaction.12

8

Figure 3. Steps and mechanism of DXS proposed by Lichtenthaler.2

DXS was a promising target for inhibition because it is the first enzyme in the pathway and it interacts with four different ligands, giving it four potential sites for inhibition.13 A feedback inhibition mechanism in Populus trichocarpa DXS (PtDXS) was characterized. A LC-

MS/MS assay, which detects the concentration of products and reactions, was used to evaluate the activity of the enzyme under various conditions. PtDXS was cloned and purified from E. coli to produce the enzyme in a high volume, so it could be more easily isolated with column chromatography. IPP and DMAPP were found to inhibit DXS activity by competitively inhibiting the binding of the cofactor TPP. The researchers hypothesized that the pyrophosphate groups of IPP, and DMAPP were what blocked TPP but when they ran the assay again with sodium pyrophosphate, they found that DXS activity was not affected. After evaluating the x-ray crystal structure of DXS (Fig. 4), they concluded that the divalent metal cation (usually Mg2+) orients the pyrophosphate groups of IPP and DMAPP but it is the hydrogen bonds and van der

Waals forces that interact with IPP and DMAPP to block TPP.14

9 A B

Figure 4. Binding of TPP (A) and IPP (B) in the active site of PtDXS. The divalent metal cation is represented by a grey sphere and coordination of the ion and hydrogen bonds are shown with yellow dashed lines and blue dashed lines represent van der Waals interactions. The orange and red group represents the pyrophosphate groups of IPP and TPP. The green structures represent the amino acids that interact with the substrate and the abbreviated names of the amino acid residues are written in white text.14

This mechanism shows clear inhibition that could be useful in the development of drug that targets the MEP pathway. Drugs are commonly developed by mimicking the structure of the substrate of the enzyme (the reactants or cofactors) to block the activity of the enzyme. This is extremely difficult with DXS because all of the substrates are important for normal human metabolism. While the pyruvate and G3P used for the MEP pathway come from the Calvin cycle of photosynthesis,15 pyruvate and G3P are important intermediates of glycolysis, which occurs in humans.16 The targeting of these structures in drug development could lead to disruption of human metabolism.13 For example, alkylacetylphosphonates (alkylAPs) have been shown to inhibit DXS by mimicking the ketoacid pyruvate and competitively binding, but they were not shown to be useful as antimicrobial agents due to their poor selectivity and potency.16 TPP is an important cofactor for enzymes like pyruvate dehydrogenase and transketolase in human cells so the targeting of TPP would also be detrimental to humans.16 David Bartee et al. synthesized an

10 inhibitor of DXS despite these constraints in 2018. They noticed that the active site of DXS had twice the volume of other enzymes that would be targeted by pyruvate inhibition (like pyruvate dehydrogenase and other transketolase). They theorized that adding steric bulk could increase specificity. DXS is also unique in that it forms a long-lived intermediate with the attachment of

TPP to pyruvate before incorporation of G3P, 2,16 which would explain the large active site.

There are two positively charged arginine residues in the G3P binding pocket that could be targeted by attaching a negatively charged carboxylic acid group to the distal end of their alkylAP backbone. A DXR-enzyme coupled assay was used to evaluate the activity of the enzyme. This method assumes that the DXR enzyme functions at optimal efficiency and is not affected by the inhibitors or substrates to model the activity of the DXS enzyme. The activity of

DXR was measured using spectrophotometry to monitor the reduction of NADPH. The addition of the negative group on the distal end was found to increase potency, binding to both the pyruvate and G3P binding sites. The most potent inhibitor, D-PheTrAP (Fig. 5), contained both a sterically bulky group and a negative distal tail. Reaction progress curves showed bisubstrate competitive binding and inhibition of DXS with increase concentration of inhibitor. These results suggest that D-PheTrAP could be an effective inhibitor of the MEP pathway and a potent anti- microbial drug.16

11 a)

c)

Figure 5. a) Structures of potential inhibitors of DXS. The most potent inhibitor, D-PheTrAP, is boxed in blue.

b) Reaction progress curves of DXS with concentrations of the inhibitor D-PheTrAP between 0M and 6.4M.

c) Theorized binding of D-PheTrAP in the active sites of the DXS enzyme. The positively charged binding

pocket is shown in blue.16

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR)

The DXR enzyme catalyzes the second reaction of the MEP pathway of isoprenoid

synthesis. DXR converts DXP to MEP and oxidizes NADPH to NADP+. In 2005 the crystal

structure of DXR was obtained with a resolution of 2.2 Å. The crystal structure suggests that the

enzyme exhibits a conformational change after binding the inhibitor fosmidomycin.5 This

conformational change upon the binding of fosmidomycin causes a loop of the enzyme to move

in front of the active site so that the substrate, DXP, cannot bind.13 This conformational change

12 also makes it difficult to model substrate binding and poses a challenge for researchers attempting to design inhibitors.17

A B

Figure 6. A) Activity of recombinant P. falciparum DXR with increasing concentration of the inhibitors fosimidomycin (open circles) and FR-900098 (closed circles). B) In vivo antimalaria activity of fosmidomycin and FR-900098. Parasitemia was determined on day 5 by Giemsa-stained blood smears. Mean values and standard deviations from three to four independent experiments, each containing four to six mice per dosage, are shown.4

Fosmidomycin is a known treatment of malaria, caused by the protozoan , but the reason for its potency was not established in 1999 when Lichtenthaler published his review establishing the MEP pathway.2 The DXR gene in P. falciparum was first located by comparing the known DXR genes of bacteria and blue algae to the P. falciparum genome and the expression of this gene during the proliferation stage of malaria infection was confirmed by RT-PCR. The activity of DXR was monitored using the oxidation of NADPH. The activity of DXR was found to decrease with increasing concentrations of the inhibitor fosmidomycin and its derivative, FR-900098 (Fig. 6). Fosmidomycin was found to decrease the survival of P. falciparum more effectively than the conventional drugs chloroquine and

13 pyrimethamine in all but one strain. A mouse model of malaria, where mice are infected with fatal Plasmodium vinckei (similar to P. falciparum, which infects humans), was used to test the toxicity of the drugs in a mammal system and the efficacy of the drug treatments. They found that treatments improved mouse survival and showed no adverse effects.4

While fosmidomycin has been a useful inhibitor of the MEP pathway and effective treatment for malaria, there is a strain of P. falciparum (had1) that is resistant to fosmidomycin because it produces an excess of the DXR substrate, DXP. This excess allows the substrate to outcompete the inhibitor for the active site and had1 parasites can survive6. Growing fosmidomycin resistance led researchers to develop new inhibitors that target the DXR enzyme.

Bisubstrate inhibitors were synthesized to bind to active sites of DXP and NADPH with fosmidomycin as a backbone for the design. Of the compounds synthesized, the most potent inhibitor had an IC50 value of 17.8 μM, which is neither comparable to the IC50 value of fosmidomycin (0.31 μM), nor in an acceptable range for drug developers (less than 10 μM).

Since there is a significant deviation from the positive control one can conclude that their synthesized inhibitor is not an effective inhibitor.

Previous studies showed that fosmidomycin is an effective treatment for malaria since it inhibits DXR by competitively binding and blocking the substrate DXP and uncompetitively blocking cofactor NADPH. Fosmidomycin analogues were synthesized (Fig. 7) to optimize inhibition through a series of synthesis steps but the analogue with N-Formyl (12a) and N-

Formyl the with POM prodrug (18a) (Fig 7A) were found to be the most effective inhibitors for

DXR and P. falciparum. The cLogP value for 18a indicated that 18a was better than 12a at permeating the plasma membrane and then converted to 12a in the cell. HepG2 cells treated with both 12a and 18a showed no toxic effects on human cells. Growing P. falciparum on a growth

14 medium supplied with IPP restored the parasite growth, indicating that 12a and 18a inhibit the

MEP pathway specifically. The synthesized 12a and 18a analogues were more effective in

deterring the growth of had1 mutants than fosmidomycin. The increased inhibition is probably

due to the bisubstrate binding of their synthesized inhibitor, which demonstrates both

competitive inhibition with the reactant DXP and uncompetitive inhibitor with the cofactor

NADPH. An in vivo model, using a P. berghei mouse model of malaria, found that 12a and 18a

were effective in treating malaria and showed no adverse effects on the mice.6

A

B

Figure 7. A) Most potent DXR inhibitors 12a and 18a. The R=H for both 12a and 18a. B) Growth of P.

falciparum with different concentrations of 1a (fosmidomycin) and the two synthesized inhibitors 12a and 18a.

B) The had1 mutation results in higher levels of the DXR substrate DXP, and mutants are resistant to DXR

inhibition as indicated by a shift in the IC50 curve (had1; open shapes, black line) when compared to WT P.

falciparum (3D7; closed shapes, gray line). Sensitivity was restored if a WT copy of had1 was supplied in the

mutant strain (had1 + HAD1-GFP; closed shapes, black line). Please note that the scales of the x-axes are not

the same.6

15 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (CMS)

The bacterium that causes tuberculosis (TB), Mycobacterium tuberculosis, is concerning because it has shown resistance to conventional and first-line drugs. The role of the MEP pathway in M. tuberculosis is being studied because eubacteria synthesize IPP and DMAPP through the MEP pathway exclusively so an inhibitor of one or more of the enzymes in this pathway could be used as a treatment method for tuberculosis. The third enzyme of the MEP pathway, CMS, converts MEP and CTP to CDP-ME (4-diphosphocytidyl-2C-methyl-D- erythritol and pyrophosphate. CMS has been shown to be vital for the pathway and the survival of M. tuberculosis.18

Figure 8. (A and B) Two different views of the structure of AthCMS with cylinders representing α-helices and arrows representing β-sheets. The different subunits are shown in blue and green. Binding of cytidine monophosphate is shown and labeled. (C and D) structural views of AthCMS with the structure of EcCMS overlayed in grey.19

16 The CMS enzyme of Arabidopsis thaliana (AthCMS) contains a plastid targeting sequence at the N-terminus, and a catalytic domain that starts around 79 residues. Previous studies have established that AthCMS is less effective that EcCMS (from E. coli). Structural analysis of these two proteins confirmed the catalytic residues of the CMS enzyme that determine the structure and function. CMS forms a dimer, assisted by a β-arm that could not be modelled in AthCMS due to missing residues in that region. AthCMS is lacking an α-helix in front of β11 due to disordered residues that are lacking secondary structure. The binding site for

CTP was conserved between AthCMS and EcCMS (Fig. 8). Two lysine residues were shown to stabilize the pentavalent transition state and aspartic acid, arginine, and two threonine residues facilitate the binding of MEP. 13C NMR spectroscopy was used to analyze the activity of the enzyme by stopping the reaction using EDTA. The activity of the enzyme was found to decrease significantly when the concentration of CMP increased. While the individual elements of the secondary structure are similar, minute changes impacted the rotation of the subunits of the dimer, causing very large changes in quaternary structure.19

Table 1. M. tuberculosis CMS kinetic parameters.18

To more easily isolate the enzyme, the gene for CMS, Rv3582c, in the M. tuberculosis genome was identified based on its similarity to the CMS gene in E. coli. The gene was then amplified with PCR and cloned into E. coli. Liquid scintillation spectrometry was used to monitor PPi release as an assay for CMS activity. CMS was purified using a nickel-affinity

17 column and its activity was monitored with increasing pH, and the optimal pH for activity was found to be 8.0. The activity was shown to be dependent on a divalent metal cation and utilized

CTP over any other nucleotide 5-triphosphates. They showed that CMS has a slightly higher affinity CTP than MEP (Table 1) so an inhibitor that resembles CTP and can competitively bind could be a useful treatment option, but CTP is used in other processes within the human body.

An inhibitor that prevents the binding of MEP might be a better treatment option (Table 1).18

An inhibitor of CMS was found using high-throughput screening (HTS). HTS is a method that quickly and effectively evaluates a large library of inhibitors for drug discovery using a robot to administer and analyze the library of inhibitors.20 A library of 3200 compounds was screened to determine if any of them inhibited CMS. Domiphen bromide (DMB) showed significant inhibition of CMS verified by the PPi activity assay and with HPLC-MS. The release of PPi was monitored to evaluate the activity of CMS with various concentrations of substrate.

The efficacy of DMB as a tuberculosis treatment was tested using M. smegmatis, which is similar to M. tuberculosis but overexpresses the CMS enzyme. Overexpressing and downregulating the M. smegmatis enzyme confirmed that DMB inhibited CMS and the growth of M. smegmatis by preventing cell wall biosynthesis.21

A paper published more recently22 found a large amount of false positives in their high- throughput screen of inhibitors for both DXR and CMS. Recent resistance to fosmidomycin and the potential of an allosteric site for inhibition pushed researchers to find an inhibitor that wasn’t a derivative of fosmidomycin and could possibly inhibit more than one enzyme of the pathway.

HTS was used to screen 1280 commercially available compounds and 150 natural products. No potent inhibitors were determined. Since the previous researchers21 screened over twice the

18 amount of potential inhibitors it is possible that the 2018 study didn’t test DMB, or perhaps it wasn’t considered because it is only effective in inhibiting CMS.

2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS)

The fifth enzyme of the MEP pathway, MCS, catalyzes the cyclization reaction that converts diphosphocytidyl-2-methylerythritol 2-phosphate into 2-methylerythritol 2,4-cyclodiphosphate.

The X-ray crystal structure of MCS from Bacillus subtilis revealed six monomers, arranged as two trimers. The structure and the sequence of BsMCS (MCS from Bacillus subtilis) are similar

EcMCS (MCS from E. coli) and are even more conserved in the active sites. The crystal structures show a hydrophobic cavity between two subunits as well as three other active sites that could be targeted by inhibitors.23

An enzyme with sequence similarities to CMS and MCS was found in the genome of

Campylobacter jejuni (a disease-causing bacteria).23,24 The enzyme was found to perform the same catalytic functions of both CMS and MCS. The gene is referred to as the IspDF gene because IspD and IspF are commonly used to refer to CMS and MCS, respectively. IspDF is conserved in α- and ε-proteobacteria (Fig. 9),24 suggesting that inhibition of IspDF could be a possible drug treatment but it also means that inhibitors of IspD and IspF may not be effective treatments of infections caused by α- and ε-proteobacteria. It was also found that IspDF does not form 2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D- erythritol as IspF does in P. falciparum. It is also possible that CMK also forms a complex with

CMS and MCS but further studies are needed to verify this association and clarify the mechanism of association23.

19

Figure 9. Phylogenetic occurrence of the CMS and MCS genes in different bacterial species. Fused, linked and unlinked CMS and MCS genes are indicated by different arrows.24

Possible inhibitors of MCS were screen HTS.7 The inhibition of P. falciparum and M. tuberculosis MCS was the primary concern of the researchers but they used A. thaliana MCS because it was more stable and soluble, making it a more suitable surrogate for HTS. It was found that derivatives of thiazolopyrimidine were most effective in inhibiting MCS with low

7 IC50 values but showed weak inhibition with a red blood cell assay with active P. falciparum.

20 This is discrepancy is probably because the compounds have trouble entering the cell and inhibiting the enzyme.

(E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate synthase (HDS)

MEcPP, a MEP pathway intermediate, has demonstrated a stress-specific placid-to- nucleus retrograde signal. This phenomenon has been studied in Arabidopsis mutants (ceh1) with mutated HDS, which converts MEcPP to HMBPP, catalyzing the second to last step of the MEP pathway. HDS has a 4Fe-4S cluster that facilitates reductive dehydroxylation of MEcPP, but this cannot occur in the ceh1 mutants and the buildup of MEcPP causes an increase in nuclear stress response via activation of the HPL and SA genes, increasing the amount of SA. Plants with mutations that returned the function of the protein, termed (Rceh1, Rceh2, and Rceh3) were used to identify the DNA regions that dictate protein function. Rceh1 was the least recovered and

Rceh3 was the most recovered, behaving similarly to the wild type. Using a preexisting HDS model structure, determined that the Rceh1 and Rceh2 mutations occurred in the MEcPP binding domain, which is a TIM barrel. The Rceh3 mutation occured between the TIM barrel and the

4Fe-4S reductase domain. Next they engineered overpressed HDS in E. coli with mutations to emulate the revertants Rceh1, Rceh2, and Rceh3, so they could purify HDS and perform enzyme kinetics. They found that the WT showed the most activity (highest kcat/Km) and the revertants showed restored activity with Rceh3 recovering the most and Rceh2 the least recovered. The researchers suggest that Rceh3 could have a conformational change between the TIM barrel and the 4FE-4S cluster. They also performed an immunoblot to evaluate DXS expression in the genotypes and they found that ceh1 and Rceh1 contained more DXS. They conclude that this increase in DXS correlates with the accumulation of MEcPP. Knowing which residues to target to inhibit the MEP pathway could be useful in antimicrobial or malaria drug development.

21 Understanding the structural components that are vital for the functioning of the HDS enzyme could help develop an inhibitor to block the MEP pathway.25 These key structural features could potentially be targets for inhibition and drug treatment, but more research is needed. A 2013 study used the existing x-ray crystal structures and a tool called DoGSiteScorer to assign each enzyme in the MEP pathway a druggability score (Dscore). This Dscore is a measure of how efficiently an enzyme could be inhibited by ligands. The study identified several promising regions on DXS, CMS, MCS, and HDS enzymes that could be potentially be inhibited by allosteric inhibition.13 This study was of course limited by the availability of crystal structures and more studies are needed to evaluate the druggability of these enzymes.

(E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate reductoisomerase (HDR)

The last step of the MEP pathway converts (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate to both IPP and DMAPP in a 5:1 ratio and is catalyzed by HMBPP reductase

(HDR) via an iron-sulfur cluster. This occurs by reducing HMBPP to allylic anion and protonating either C2 to form IPP or C4 to form DMAPP.26,27

Researchers have found that an iron-sulfur cluster in HDR catalyzes this reaction. The iron-sulfur cluster was first characterized in E.coli by electron paramagnetic resonance (EPR).

The cloned HDR gene of E. coli was tagged with a histidine residue so that it could be purified with Ni2+ column chromatography. Gel filtration of the enzyme indicated that the protein is actually a dimer. The UV-vis spectrum of the enzyme indicated that iron and sulfur were members of a prosthetic group. Anerobic reconstructed of the iron-sulfur cluster showed an absorption indicative of a [4Fe-4S]2+ cluster.27

22

Figure 10. Proposed ligand binding of HMBPP to the A. aeolicus HDR enzyme. Proposed electrostatic interactions are shown with dashed red lines. Orange and yellow group indicates the iron-sulfur cluster. The red and orange region indicates the pyrophosphate group of the HMBPP substrate which interactions with the function residues of the enzyme, shown in teal and named with black text.26

JPRED3, a software that predicts secondary structure, found that three cysteine residues were conserved over many organisms that anchor the iron-sulfur center. The conservation of these residues indicate that they are vital for the function of the enzyme. HMBPP was hypothesized to bind to this iron-sulfur cluster at the O4 position. The two conserved histidine residues were predicted to bind the diphosphate of HMBPP. An X-ray crystal structure for HDR from the bacterium A.aeolicus was obtained with a resolution of 1.65 A (Fig. 10). The structure shows α/β domains surrounding a 3Fe-4S cluster which have alternating alpha and beta secondary structures. These α/β domains form a cloverleaf-like structure, which is mostly planar

23 with a cavity on the front side where the substrate, HMBPP binds. The specifics of the enzyme- substrate binding have not yet been elucidated because HMBPP has yet to be crystallized with

HDR and the iron-sulfur center was thought to be 4Fe-4S, instead of 3Fe-4S. This structure indicates functional and conserved residues that are vital for the activity of the enzyme that may in the future be used to inhibit the MEP pathway.26

Before the X-ray crystal structure was determined an inhibitor was developed based off of the known components of the HDR. The researchers knew that HDR and HDS both contain

4Fe-4S clusters, so an inhibitor was designed to target this structure. Cationic diphosphates were found to inhibit IspH by directly attacking the iron-sulfur cluster. Since this inhibitor targets 4Fe-

4S clusters it would also be capable of inhibiting HDS, which contains the same moiety. These inhibitors were shown to be very effective, with the most inhibitory species having an IC50 value of 0.45 μM.28 This value alone would make these diphosphate inhibitors promising treatment avenues, but it targets the iron-sulfur center, which is ubiquitous in nature and the human body.

An in vivo study is necessary to determine the efficacy of these compounds as treatment options.

Conclusion

There is a growing body of research that indicates that the treatment of illnesses like malaria, tuberculosis, and other diseases is possible with the inhibition of the MEP pathway. The absence of the MEP pathway in humans and the reliance of illness causing organisms such as M. tuberculosis and Plasmodium falciparum on the pathway makes it a promising target for antimicrobial drugs. E. coli have been shown to depend on the MEP pathway so there is evidence that inhibiting the MEP pathway could treat bacterial infections.3 However, the MEP pathway is

24 lacking in fungi like Saccharomyces cerevisiae,29,30 so it couldn’t be used so inhibition of the pathway couldn’t be used as an antifungal treatment

A synthesized inhibitor, D-PheTrAP, shows clear inhibition of the MEP pathway and also deters E. coli proliferation. This shows that D-PheTrAP could be a useful drug, but it has yet to be tested on human cells to make sure that there are no toxic effects. While fosmidomycin was historically an effective anti-malaria drug, had1 strains of P. falciparum have grown resistant, due to the competitive inhibition mechanism of fosmidomycin. It is clear that a competitive inhibitor of DXR is useless against the newly resistant malaria strains and recent attempts to synthesize a new competitive inhibitor have proven unsuccessful. Future studies should investigate the application of an inhibitor that competitively inhibits the binding of the electron carrier NADPH instead of DXP or an uncompetitive inhibitor. A bisubstrate inhibitor similar to fosmidomycin was shown to be an effective inhibitor of DXR in had1 strains and was also effective in an in vivo mouse model of malaria. An inhibitor identified by Gao et al, domiphen bromine (DMB), was an effective inhibitor of CMS and is a true promising lead because DMB was shown to be an effective drug against Mycobacterium smegmatis, a model for

M. tuberculosis. However, the efficacy of DMB has not yet been tested on M. tuberculosis nor has it been tested with human cells to determine if there are any toxic effects.

Other inhibitors discussed in this paper may be effective in inhibiting the MEP pathway but there is a lack of testing to indicate if the inhibitors actually deter infection by these disease- causing pathogens. Studies have shown that the enzymes of the MEP pathway are vital for the survival of E. coli, P. falciparum, and M. tuberculosis,4,31,32 but there is a disconnect between this data and the inhibition studies examined in this paper. There are several drug delivery

25 options like dendrimers,33 silica nanoparticles,34 and gold nanoparticles,35 that could improve the bioavailability of these inhibitors and increase their efficacy.

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31 Abbreviations

MVA: mevalonate

MEP: 2-C-methylerythritol 4-phosphate

DXS (DXP): 1-Deoxy-D-xylulose 5-phosphate Synthase

DXR (IspC): 1-Deoxy-D-xylulose 5-phosphate reductoisomerase

CMS (YgbP, IspD): 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase

CMK (YchB, IspE): 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase

MCS (YgbB, IspF): 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase

HDS (GcpE, IspG): (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate synthase

HDR (LytB, IspH): (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate reductoisomerase

NADPH: Nicotinamide adenine dinucleotide phosphate

TPP: Thiamin pyrophosphate

DXP: 1-Deoxy-D-xylulose 5-phosphate

G3P: Glyceraldehyde 3-phosphate

IPP: Isopentenyl pyrophosphate

DMAPP: Dimethylallyl pyrophosphate

CTP: Cytidine triphosphate

CDP-ME: 4-diphosphocytidyl-2C-methyl-D-erythritol

MEcPP: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate

TB: Tuberculosis

PPi: Inorganic pyrophosphate

DMB: Domiphen bromide

HMBPP: (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate

32