The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. In the malaria parasite Plasmodium falciparum, the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP. Since Plasmodium falciparum cells and infected host red blood cells rely on accelerated glucose flux they are dependent on G6PD activity of PfGluPho. The parasite enzyme is essential for Plasmodium proliferation and propagation and it differs structurally and mechanistically from the human orthologs. Thus PfGluPho fulfills the requirements for being a potential novel target for antimalarial drug design.

This project sought to identify selective inhibitors of the G6PD activity of PfGluPho (PfG6PDH). This Center Probe Report describes the screen of the ~340,000 compound Molecular Libraries Small Molecule Repository (MLSMR) and SAR elucidation that led to identification of the second described PfG6PDH inhibitor (190 nM IC₅₀) that was completely selective (>420-fold) vs. the human ortholog.

Resulting Publications

1.

Hershberger P. M., Hedrick M. P. et al. Imidazole-derived agonists for the neurotensin 1 receptor. Bioorg Med Chem Lett. 2014;24(1):262–267. [PMC free article: PMC3898338] [PubMed: 24332089]

Probe Structure & Characteristics

This Center Probe Report describes a second selective inhibitor, ML304, of the glucose-6-phosphate dehydrogenase activity of the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphoglucono-lactonase (PfGluPho) of the malarial parasite referred to herein as Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfG6PDH). ML304 is a ring-expanded chemical scaffold, related yet distinct from the first PfG6PDH inhibitor probe, ML276, that represents a 5-fold improvement in potency (robustly submicromolar) over ML304, with >420-fold selectivity against the human form (hG6PDH) and provides a different starting point for lead optimization efforts. Potency and selectivity characteristics are summarized for this probe in the summary table.

1. Recommendations for Scientific Use of the Probe

As with the first probe, ML276, this second probe, ML304, will also be of great value for further studying the structural, functional, and mechanistic characteristics of PfGluPho in direct comparison with its isofunctional host enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase. These analyses will be of importance for further validating PfGluPho as antimalarial drug target and for providing a basis for facilitated drug development. The improved potency selectivity, and different core scaffold of ML304 will provide a further improved tool for these studies and those listed later.

In particular, the probe will be used for detailed kinetic characterization of the mechanism of inhibition on both PfGluPho and human G6PD. To further substantiate this functional work, co-crystallization of this new inhibitor with PfGluPho and human G6PD followed by x-ray analysis is aimed at. The three-dimensional structure of such a complex would be the first enzyme-inhibitor complex of the malarial protein to be described, and would further stimulate drug design strategies and enhance our understanding of the enzymes’ comparative catalytic mechanism.

Furthermore, the probe will be used for detailed analyses of the compound on Plasmodium falciparum in cell culture. This would include the determination of IC₅₀ values on Plasmodium falciparum strains with various degrees of chloroquine resistance as well as synergy tests with clinically used antimalarials and novel redox-active antimalarial compounds currently under development. Also the effects of this probe, ML304, on parasite invasion and its stage specificity will be assessed.

It will be of great interest to further investigate the mechanism of antimalarial action of this probe ML304. For this purpose we aim at determining the thiol status and glutathione concentrations in parasites and host erythrocytes after treatment. Also, the effects of the compound on transcript and protein levels of PfGluPho as well as on other enzymes and pathways related to redox and energy metabolism in Plasmodium shall be investigated (pentose phosphate pathway, glutathione and thioredoxin dependent pathways, glycolysis). The probe will also be of interest for assessing changes in intraparasitic protein glutathionylation patterns that will provide information on the redox regulatory response of the cell to PfGluPho inhibition. The probe can furthermore be employed for studying the effects of a PfGluPho inhibitor on carbon flux through glycolysis and pentose-phosphate-pathway based on methods established in the Bode lab in collaboration with the Sanford-Burnham Institute for Medical Research. To further understand the mechanism of inhibition, the effects of ML304 on heme aggregation, which is affected by many antimalarial agents, will be determined as well as its effects on the intraparasitic redox potential using redox-active green fluorescent protein transfected into the parasite (a research tool recently established for P. falciparum in the Becker lab).

The probe will advance the field for the general research community interested in G6PD as a key enzyme in cell metabolism. The Plasmodium falciparum enzyme is of particular interest as a potential target for novel anti-malarial drugs. The enzyme G6PD in general has been associated with cancer and several other disorders, and a probe to inhibit this enzyme should spark interest as a research tool in various fields.

2. Materials and Methods

The details of the primary HTS and additional assays can be found in the “Assay Description” section in the PubChem BioAssay view under the AIDs as listed in Table 1. Additionally the details for the primary HTS are provided in the Appendix at the end of this probe report.

Table 1

Summary of Assays and AIDs.

2.2. Probe Chemical Characterization

Chemical name of probe compound & structure including stereochemistry

The IUPAC name of the probe is (R)-N-((1-ethylpyrrolidin-2-yl)methyl)-4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide. The actual batch prepared, tested, and submitted to the MLSMR is archived as SID 134958857 corresponding to CID 56639562. The probe ML304 contains R-stereochemistry at the 2-position of the pyrrolidine ring. (See Figure 1)

Figure 1

Structure of ML304. Note R-stereochemistry as indicated around the 2-position of the pyrrolidine ring.

Availability from a vendor

This probe is not commercially available. A 25 mg sample of ML304 synthesized at SBCCG has been deposited in the MLSMR (Evotec) (see Probe Submission Table 3).

Table 3

Probe and Analog Submissions to MLSMR (Evotek) for P. falciparum G-6PDH antagonists.

Solubility and stability of probe in PBS at room temperature

The solubility of ML304 was investigated in aqueous buffers at room temperature. As noted in the Summary of in vitro ADME/T properties (see Table 4), ML304 has excellent solubility relative to its potency against PfG6PDH in aqueous buffer at all pH’s tested (>294 μM [>117 μg/mL], at pH 5, 6.2, and 7.4). To evaluate its potential hydrolytic instability an aliquot of ML304 was prepared in PBS or a acetonitrile:PBS (1:1) mixture and incubated at room temperature, and the amounts of the parent compound remaining at various times were analyzed by LC/MS (Figure 2 time course and Table 2). The results indicate that ML304 is entirely stable up to 48 hours at both conditions.

Table 4

Summary of in vitro ADME/T Properties of P. falciparum G6PDH inhibitor probe ML304.

Figure 2

Stability of ML304 in (△) PBS or (_●) 1:1 acetonitrile:PBS at room temperature.

Table 2

Hydrolytic stability of ML304 at ambient temperature.

Table 3 summarizes the deposition of the Probe and 5 analogs.

2.3. Probe Preparation

Synthesis and Structural Verification Information of probe SID 134958857 corresponding to CID 56639562 (See Scheme 1).

Scheme 1

Synthesis of ML304. Conditions: i) NaNO₂, HCl, Na₂S.9-H₂O, S₈, NaOH, H₂O; NaBH₄, THF; ii) Cs₂CO₃, 4-fluoro-3-nitrobenzoic acid, DMF; iii) Zn, HCl, dioxane, H₂O; iv) EDC.HCl, HOBt, Et₃N, (R)-(1-ethylpyrrolidin-2-yl)methanamine, DCM.

The synthesis of the key thiophenol was adapted from a previously reported procedure [25]. A mixture of sodium sulfide nonahydrate (2.6 g, 10.8 mmol) in 35 mL water was treated with powdered sulfur (0.34 g, 1.3 mmol) and was stirred 1 hour at 70ºC. To this was added 10 mL 1M aqueous sodium hydroxide solution followed by cooling in an ice/water bath. Meanwhile, a mixture of methyl 2-amino-3-methylbenzoate (1.0 g, 5.49 mmol) in 10 mL methanol was treated with 2 mL concentrated HCl and was cooled in an ice/water bath. To this a solution of sodium nitrite (0.72 g, 10.4 mmol) in 4 mL water was added dropwise. The diazonium salt was allowed to form at 0ºC for 1 hour prior to its dropwise addition to the chilled sulfide solution. The mixture was allowed to stand for 1 hour, was acidified with saturated sodium bisulfate, and the crude disulfide was extracted with ethyl acetate. The organic layer was dried over sodium sulfate then condensed in vacuo to provide a bright yellow-orange residue. The residue was dissolved in 8 mL 1:1tetrahydrofuran:methanol and was treated with sodium borohydride (0.33 g, 8.7 mmol) for 15 minutes at ambient temperature. The reaction was quenched with the addition of cold water followed by 1M NaOH. The alkaline solution was washed once with ethyl acetate, which was put aside. The alkaline phase was then acidified with HCl and extracted with ethyl acetate. Evaporation of solvent afforded methyl 2-mercapto-3-methylbenzoate (0.65 g, 59%). ¹H NMR (500 MHz, Chloroform-d) δ 7.95 – 7.89 (m, 1H), 7.35 – 7.27 (m, 1H), 7.08 (t, J = 7.7 Hz, 1H), 6.59 (s, 1H), 3.94 (s, 3H), 2.41 (s, 3H).

Methyl 2-mercapto-3-methylbenzoate (0.65 g, 3.57 mmol) was dissolved in 15 mL dimethylformamide, at 60ºC and was treated with cesium carbonate (2.32 g, 7.13 mmol) followed by dropwise addition of a solution of 4-fluoro-3-nitrobenzoic acid (0.66g, 3.57 mmol) in 5 mL dimethylformamide. The mixture was maintained at 60ºC for 3 hours. Once allowed to cool to ambient temperature, the mixture was acidified with saturated sodium bisulfate and was extracted with ethyl acetate. The organic layer was washed twice with water then was dried with sodium sulfate prior to evaporation of solvent to provide the crude thioether. 4-(2-(methoxycarbonyl)-6-methylphenylthio)-3-nitrobenzoic acid. (0.98 g, 79%) ¹H NMR (500 MHz, Chloroform-d) δ 8.95 (d, J = 1.9 Hz, 1H), 7.96 (dd, J = 8.6, 1.9 Hz, 1H), 7.63 (dd, J = 6.4, 2.8 Hz, 1H), 7.53 – 7.48 (m, 2H), 6.79 (d, J = 8.6 Hz, 1H), 3.77 (s, 3H), 2.37 (s, 3H).

A solution of 4-(2-(methoxycarbonyl)-6-methylphenylthio)-3-nitrobenzoic acid (0.98 g, 2.8 mmol) in 10 mL dioxane and 2 mL conc. hydrochloric acid was stirred at 60ºC while powdered zinc (0.94 g, 14.3 mmol) was added slowly to control the vigorous reaction. Once the addition was complete, the mixture was transferred to a microwave reactor where the solution was heated to 120ºC for 10 minutes. The reaction volume was reduced to approximately 5 mL in vacuo and the mixture was diluted with 20 mL water. The resulting tan solids were filtered, washed with water, and were dried in vacuo. 4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylic acid. (0.74 g, 93%). ¹H NMR (500 MHz, DMSO-d6) δ 10.82 (s, 1H), 7.79 – 7.71 (m, 2H), 7.66 (dd, J = 8.0, 1.8 Hz, 1H), 7.46 (ddd, J = 16.9, 7.6, 1.5 Hz, 2H), 7.33 (t, J = 7.7 Hz, 1H), 2.54 (s, 3H).

A mixture of 4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxylic acid (200 mg, 0.70 mmol), triethylamine (200 uL, 1.43 mmol), 1-hydroxy benzotriazole (60 mg, 0.44 mmol), and EDC.HCl (140 mg, 0.73 mmol) in 8 mL dichloromethane was stirred at ambient temperature for 10 minutes prior to the addition of (R)-(1-ethylpyrrolidin-2-yl)methanamine (96 mg, 0.75 mmol). The mixture was stirred overnight then was diluted with 50 mL dichloromethane. The solution was washed with water, aqueous sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate and condensed in vacuo to provide a residue which was purified by RP-HPLC on a SunFire preparative C18 column (5 μm; 19 × 50 mm) using a stepwise gradient {10 % [(MeCN + 0.1% formic acid) in (water + 0.1% formic acid)] for 1 min.; 30 % for 1 min.; 50 % for 4 min.; 70 % for 1.5 min. and 98 % for 1.5 min.}. The fractions containing the expected molecular weight were collected, treated with an excess of solid potassium carbonate to provide the free base in solution, then were condensed in vacuo to provide a residue. The purified product was dissolved in dichloromethane then was decanted from the residual potassium salts (R)-N-((1-ethylpyrrolidin-2-yl)methyl)-4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide, 78 mg, 28%). ¹H NMR (500 MHz, Chloroform-d) δ 7.73 (d, J = 1.9 Hz, 1H), 7.69 – 7.60 (m, 2H), 7.51 (dd, J = 8.0, 1.9 Hz, 1H), 7.38 – 7.30 (m, 1H), 7.25 (t, J = 7.6 Hz, 1H), 3.73 (dd, J = 13.7, 2.9 Hz, 1H), 3.47 (s, 2H), 3.30 (dd, J = 13.8, 4.4 Hz, 1H), 3.15 (ddd, J = 9.3, 6.8, 2.7 Hz, 1H), 2.82 (dq, J = 12.0, 7.4 Hz, 1H), 2.67 (dddd, J = 8.8, 6.9, 4.2, 2.8 Hz, 1H), 2.59 (s, 3H), 2.29 – 2.12 (m, 2H), 1.95 – 1.84 (m, 1H), 1.77 – 1.54 (m, 3H), 1.10 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.67, 166.32, 140.82, 139.18, 138.40, 136.23, 135.83, 133.44, 133.30, 133.10, 129.21, 128.46, 123.51, 121.53, 62.26, 53.53, 48.08, 40.92, 28.11, 22.90, 21.12, 13.98. LRMS (ESI+ve): Calculated for C₂₂H₂₅N₃O₂S, [M+H] = 396.17, observed [M+H] = 395.97.

3. Results

3.1. Dose Response Curves for Probe

The multiple dose response titrations in Figure 3 (on the right) of the probe ML304 against PfG6PDH and hG6PDH highlight the high degree of selectivity the compound has for the Plasmodium falciparum enzyme.

Figure 3

Specificity of ML304 Inhibition for the Plasmodium vs. human G6PDH enzyme activity.

3.3. Profiling Assays

The probe was evaluated in a detailed in vitro pharmacology screen as shown in Table 4.

ML304 can achieve concentrations >294 μM in aqueous buffer between a pH range of 5.2–7.4, which is more than 1,550-fold its IC₅₀ of 190 nM for PfG6PDH.

The PAMPA (Parallel Artificial Membrane Permeability Assay) assay is used as an in vitro model of passive, transcellular permeability. An artificial membrane immobilized on a filter is placed between a donor and acceptor compartment. At the start of the test, drug is introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments is measured using UV spectroscopy. Consistent with its solubility data, ML304 exhibits high permeability with increased pH of the donor compartment. When incubated with an artificial membrane that models the blood-brain-barrier (BBB), ML304 was found to be highly permeable.

Plasma protein binding is a measure of a drug’s efficiency to bind to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Highly plasma protein bound drugs are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues. ML304 was highly plasma protein bound which may contribute to its high plasma stability.

Plasma stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which strongly can influence the in vivo efficacy of a test compound. Drug candidates are exposed in plasma to enzymatic processes (proteinases, esterases), and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. ML304 appears quite stable in plasma thus increasing its exposure to blood-borne parasites such as P. falciparum.

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. This assay addresses the pharmacologic question of how long the parent compound will remain circulating in plasma within the body. ML304 is almost completely metabolized in human and mouse liver homogenates within 1 hour.

ML304 shows no toxicity (>50 μM) toward human hepatocytes.

Profiling against other GPCRs

The probe, ML304 (CID 56639562), was submitted to the Psychoactive Drug Screening Program (PDSP) at the University of North Carolina (Bryan Roth, PI) and the data against a GPCR binding assay panel is shown in Figure 4. Preliminary results indicate ML304 shows a moderate level of promiscuity across a range of GPCRs. It is not known whether these activities in binding assays are translated into functional modification of the activities of these receptors or how relevant they are with regard to the use of the probe for mechanistic studies, antimalarial assessment in RBC models and development of future antimalarials.

Figure 4

Profile of ML304 against the Psychoactive Drug Screening Program (PDSP) % inhibition at 10 μM.

4. Discussion

Parasite and Plasmodium-infected host cells demonstrate elevated rates of glucose consumption compared to uninfected red blood cells. As this is a novel target and pathway, we expect a different mode of action and lack of susceptibility to resistance mechanisms encountered by the current antimalarials (vide infra). Tropical malaria caused by the protozoan parasite Plasmodium falciparum is responsible for up to one million deaths annually [1]. Due to increasing regional distribution and resistances against the clinically used antimalarials, novel antimalarial drugs – which have new mechanisms of action and are suitable for combination therapies – are urgently required [2-5]. Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfGluPho) is a potential novel target for antimalarial drug design [6]. The glucose-6-phosphate dehydrogenase (G6PD) reaction is the first and rate-limiting step in the pentose phosphate pathway (PPP). In Plasmodium falciparum a unique bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP [7], a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. Plasmodium falciparum cells and infected host RBCs rely on accelerated glucose flux and are dependent on glucose-6-phosphate dehydrogenase activity of PfGluPho [13-24]. Our analyses of the reactions indicate that the single G6PDH part of PfGluPho is functionally very similar to the G6PDH activity in the complete bifunctional enzyme. However, PfGluPho differs mechanistically and structurally from the human G6PDH - these differences are pronounced enough to allow specific targeting of the parasite enzyme [8]. The parasite enzyme is essential for Plasmodium proliferation and it is distinct from the human enzymes, thus making it an excellent target for novel antimalarial drug design. Prior to this study PfGluPho protein was unavailable and therefore, selective and specific inhibitors for it are non-existent. Given this gap, development of an HTS screen to find chemical probes that inhibit Plasmodium G6PD activity may lead to novel anti-malarial therapies.

5. References

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Appendix. Supplementary Information ¹H NMR and LC-MS spectra of ML304

¹H NMR Spectrum of ML304 (500 MHz, CDCl₃)

¹³C NMR Spectrum of ML304 (125 MHz, CDCl₃)

LC-MS for ML304