Inhibitors of Leishmania GDP-Mannose Pyrophosphorylase Identified by

High-Throughput Screening of Small Molecule Chemical Library

Kurt Lackovic1, John P. Parisot

1, Nerida Sleebs

1, Jonathan B. Baell

2, Laurent Debien

2, Keith

G. Watson2, Joan M. Curtis

3, Emanuela Handman

3, Ian P. Street

1,4 and Lukasz Kedzierski

3,5*

1High-Throughput Chemical Screening Facility, Structural Biology Division;

2Medicinal

Chemistry Group, Structural Biology Division; 3Infection and Immunity Division;

4CRC for

Cancer Therapeutics; 5Cancer and Haematology Division, Walter+Eliza Hall Institute of

Medical Research, Melbourne, Australia

Running title: Leishmania GDP-mannose pyrophosphorylase inhibitors

*Corresponding author: Dr Lukasz Kedzierski

Walter+Eliza Hall Institute of Medical Research

1G Royal Pde.

Parkville 3052, Victoria

Australia

tel. +61 3 93452653

fax +61 3 93470852

email: kedzierski@wehi.edu.au

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.01634-09 AAC Accepts, published online ahead of print on 16 February 2010

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Abstract

Current treatment of leishmaniasis is based on chemotherapy, which relies on a handful of

drugs with serious limitations such as high cost, toxicity and lack of efficacy in endemic

regions. Therefore, development of new, effective and affordable antileishmanial drugs is a

global health priority. Leishmania synthesize a range of mannose-rich glycoconjugates that

are essential for parasite virulence and survival. A prerequisite for glycoconjugate

biosynthesis is the conversion of monosaccharides to activated mannose donor, GDP-

mannose, the product of a reaction catalysed by GDP-mannose pyrophosphorylase (GDP-

MP). The deletion of the gene encoding GDP-MP in Leishmania led to a total loss of

virulence, indicating that the enzyme is an ideal drug target. We developed a phosphate

sensor-based high-throughput screening assay to quantify GDP-MP activity, and screened a

library containing ~80,000 lead-like compounds for GDP-MP inhibitors. Based on their GDP-

MP inhibitory properties and chemical structure, 20 compounds, which were not toxic to

mammalian cells, were tested on ex vivo amastigotes and in macrophage amastigote assays.

The most potent compound identified in the primary screen (compound 3), a quinoline

derivative, demonstrated dose-dependent activity in both assays (IC50=21.9µM in macrophage

assay) and was shown to be non-toxic to human fibroblasts. In order to elucidate signs of

early SAR for this class of compounds, we obtained and tested analogues of compound 3, and

undertook limited medicinal chemistry optimization, which included a number of SAR probes

of the piperazinyl aryl substituent of compound 3. We have identified novel candidate

compounds for the design and synthesis of anti-leishmanial therapeutics.

3

Introduction

Leishmania are protozoan parasites shuttling between sandfly vectors and mammalian hosts,

causing a spectrum of diseases known as leishmaniases (13). Leishmaniasis is prevalent in

Africa, Latin America, Asia, the Mediterranean basin and the Middle East, with an estimated

12 million people currently infected and a further 350 million threatened by the disease in 88

countries. For many years, the public health impact of leishmaniasis has been underestimated,

as a substantial number of cases were never recorded. The expansion of leishmaniasis and the

sharp rise in prevalence is related to both environmental changes and migration of non-

immune people to endemic regions (1). Moreover, there is an increase in the overlap between

HIV infection and visceral leishmaniasis, especially in intravenous drug users of both South-

Western Europe and Brazil (6, 27). The situation may be much worse in Africa and Asia

where the prevalence and detection of HIV and Leishmania co-infections are still largely

underestimated.

Current treatment is based on chemotherapy, which relies on a handful of drugs with serious

limitations such as high cost and toxicity, difficult route of administration and lack of efficacy

in endemic areas (7). Extensive evidence from studies in animal models indicates that solid

protection can be achieved by immunisation, however, to date no vaccine is available in

clinical practice. Therefore, there is an urgent need to develop new, effective and affordable

antileishmanial therapeutics in order to control different forms of the disease.

Leishmania synthesize a range of mannose-rich glycoconjugates, which are considered

essential for both parasite virulence and survival. A prerequisite for glycoconjugate

biosynthesis in Leishmania, as in all eukaryotes, is the conversion of monosaccharides to

activated sugar nucleotides. This process is catalysed by several enzymes including

phosphomannose isomerase (PMI), phospomannomutase (PMM), GDP-mannose

pyrophosphorylase (GDP-MP) and dolicholphosphate-mannose synthase (DPMS) (9). The

4

consecutive action of PMM and GDP-MP transforms mannose 6-phosphate (M-6-P) to GDP-

mannose (GDP-Man), which acts as the mannose donor in the synthesis of molecules such as

lipophosphoglycan (LPG), proteophosphoglycan (PPG), glycoinositolphospholipids (GIPLs)

and N-glycans. Gene deletion studies in Leishmania have indicated that PMM and GDP-MP

constitute attractive targets for the development of novel therapeutics. Both PMM and GDP-

MP mutants are unable to establish infection in mice or survive in macrophages in vitro,

suggesting that these proteins are essential for the clinically relevant, intracellular form of the

parasite, the amastigote. However, these enzymes are not essential for survival of the

promastigotes, the insect vector stage of the parasite (10, 11). We have shown that

Leishmania GDP-MP forms a hexamer in solution, whose stability might be compromised at

both low ionic strength and high pH (8). We have also reported that leishmanial PMM shows

a high degree of similarity with its human isoforms, PMM1 and PMM2, suggesting that the

development of parasite-selective inhibitors would not be an easy task (21). Here, we report

the development of a highly sensitive, 384-well plate based enzyme activity assay utilizing

phosphate detection technology, and the use of high-throughput screening (HTS) of ~80,000

small, lead-like molecules to identify inhibitors of leishmanial GDP-MP. Although some of

these compounds demonstrated in vitro antileishmanial activity on both parasite life stages -

promastigotes and amastigotes - and may therefore be considered generally cytotoxic, one

class of quinoline derivatives showed promising and selective activity for the amastigote

stage. This class is exemplified by compound 3, which inhibited mouse macrophage infection

in a dose-dependent manner (IC50=21.9µM) and was non-toxic to human fibroblasts at

concentrations up to 100µM.

5

EXPERIMENTAL PROCEDURES

Chemical library.

Our “lead discovery chemical library” is a collection of ~80,000 compounds purchased from

commercial vendors, stored in neat dimethyl sulfoxide (DMSO) at a final compound

concentration of 10mM. The compounds represent a diverse set of molecules as judged by

Tanimoto dissimilarity analysis (T ≤ 0.85), and although simple filters based on the Lipinski

criteria were not used in the selection process, 89% of the compounds in the library are

Lipinski compliant (24) and 81% conform with Oprea’s criteria for “lead-likeness” (26).

GDP-MP high-throughput screening assay.

A high-throughput screen was conducted to identify lead-like compounds that inhibited L.

major GDP-MP from catalyzing the conversion of M-1-P to GDP-Mannose. The assay

ultimately detected inorganic phosphate produced from the enzymatic cascade shown in Fig.

1. Inorganic pyrophosphatase was added to the reaction in excess, to minimize the chance of

identifying inhibitors of this enzyme.

Recombinant L. major GDP-MP was expressed as a 6-His tagged protein in E. coli

BL21(DE3)pLysS. Purification of the fusion protein was performed by affinity

chromatography using TALON Metal Affinity Resin according to manufacturer’s instructions

(BD Biosciences). The purity and integrity of the protein was assessed on SDS-PAGE gels

stained with Coomassie Blue and enzymatic activity was assessed as previously described (8).

4 mg/ml GDP-MP was stored in aliquots at -80°C. Aliquots were thawed on ice immediately

prior to use. Assay buffer components Tween®

20, albumin from chicken egg white (OVA),

inorganic pyrophosphatase from bakers yeast, α-D(+)mannose-1-phosphate and guanosine 5’-

triphosphate were purchased from Sigma Aldrich. Additional buffer components AnalR

water, NaCl and MgCl2.6H2O were purchased from Merck. Tris-HCl and phosphate sensor

6

were purchased from Invitrogen. Phosphate sensor is a purified form of recombinant E. coli

phosphate binding protein labeled with an N-[2-(1-maleimidyl)ethyl]-7-

(diethylamino)coumarin-3-carboxamide (MDCC) fluorophore that is sensitive to changes in

its environment (15). Upon binding phosphate, the fluorescence intensity of the bright blue

MDCC fluorophore increases dramatically. Phosphate binding is tight (Kd ~100nM), enabling

sub-µM detection of phosphate. In addition, as the binding is rapid, this technology enables

detection of phosphate production in real time.

The assay buffer comprised of 25mM Tris-HCl (pH 8.0), 4mM MgCl2, 150mM NaCl, 0.02%

v/v Tween®

20, with the pH adjusted to 7.5. On each experimental day, OVA was added to a

final concentration of 0.4 mg/ml and the buffer then filtered through a 0.45 µm filter

(Millipore).

Individual enzymatic assays were performed in a total reaction volume of 5µl, in low volume,

black, 384-well plates (Greiner). Optimization experiments determined the Km for M-1-P to

be 4.6µM, and for GTP to be 1.5µM (Fig. 2).

High-throughput screening reactions were performed close to the Km for both substrates, with

a final M-1-P concentration of 5µM and GTP concentration of 1.3µM. Initially, 2.5µl of

10µM M-1-P with 5µM phosphate sensor in assay buffer was dispensed into columns 1-23 of

a 384-well plate, and a solution lacking M-1-P (negative control) into column 24.

Subsequently, 22nl (14% interplate coefficient of variance determined by fluorimetric quality

assurance protocols) of individual 10mM library compounds were added to columns 1-22 of

the 384 plate, with 22nl of neat DMSO vehicle added to the remaining columns 23-24

(positive and negative controls, respectively). Finally, 2.5µl of 0.0652U/ml inorganic

pyrophosphatase, 4µg/ml GDP-MP, and 2.6µM GTP in assay buffer were added to all wells

of the plate, starting the enzymatic reaction in the assay plates, except for the negative control

wells. Final library compound and DMSO concentrations were 44µM and 0.44% (v/v)

7

respectively. We determined that up to 1% (v/v) DMSO had no effect on the rate of reaction.

Fluorescence was determined each minute for 10 minutes in an Envision 2103™ Multilabel

Plate Reader (Perkin Elmer), with Ex430/8 nm and Em480/30 nm filters. Rate of phosphate

production was quantified using ActivityBase™ v5.4 with XLfit (IDBS). “Hit” compounds

were determined on a plate-by-plate basis and defined as compounds reducing the rate of

phosphate production by >50% relative to both the positive and negative controls wells. Z’, a

measure of high-throughput assay quality taking into consideration both the signal window

and the variation within positive and negative controls, was determined for each plate. Plates

with Z’<0.4 were repeated. Fig. 3 shows both the average positive and negative control values

and calculated Z’ values for individual library compound plates.

Initial confirmation of active compounds.

Initial confirmation experiments were performed in the same manner as for the primary

screen. Primary screen “hit” compounds, determined as described previously, were hit-picked

from a separate 10mM DMSO solution of the library compound for confirmation assays.

Dose-response determination and counter screen.

Confirmed inhibitors of GDP-MP were repurchased from the original commercial vendor.

These compounds were prepared in pure DMSO to a final compound concentration of 10mM.

Compounds were titrated 1:2 in DMSO, and subsequently added to the enzymatic assay in the

same manner as for the primary screen, enabling quantification of IC50 values for each

compound. To determine whether the hit compounds were interfering with the phosphate

sensor detection technology, a counter screen was employed where titrated compounds were

assayed in the presence of 2.5µM phosphate sensor spiked with 1µM of Na2HPO4 in assay

buffer.

8

In vitro testing on Leishmania parasites.

L. major V121 (MHOM/IL/67/JERICHO II) (WHO Reference Centre for Leishmaniases,

Jerusalem, Israel) was maintained as promastigotes at 26oC in M199 medium (Invitrogen)

supplemented with 10% heat inactivated fetal bovine serum (HI-FBS) (Trace Biosciences).

Amastigotes were harvested from lesions of L. major infected hypothymic CBA nu/nu mice

as previously described (12). Amastigotes were resuspended in M199 medium and

immediately used for in vitro experiments.

The CellTiter Blue Cell Viability Assay (Promega, Madison, WI, USA) was used to screen

for antileishmanial activity as previously described (20). Compounds were dissolved in

DMSO at 10mM working concentration and titrated across a range of concentrations (2-fold

dilutions from 100µM to 49nM) in culture medium. The assay was performed in duplicate in

96-well plates containing 106 parasites per well. Cell viability was assessed

spectrophotometrically at 550nm with a reference wavelength of 630nm. Amphotericin B

(Sigma) was used as a reference antileishmanial agent.

Macrophage invasion assay was performed as previously described (20). Briefly, mouse, bone

marrow-derived macrophages were plated on coverslips placed in 24-well plates and allowed

to adhere for 48h. Adherent cells were exposed to promastigotes (20:1, parasite/cell ratio) for

5h and non-attached cells and free promastigotes were washed, followed by the addition of

medium containing the compound. The intracellular proliferation drug sensitivity assay was

performed using amastigotes for macrophage invasion as described in Vermeersch et al. (30).

Infected cells (duplicate coverslips) were exposed to the compounds for 48h, and then washed

and fixed with methanol. Following Giemsa staining of the cells, coverslips were examined

microscopically and the percentage of infected cells counted.

9

Toxicity of candidate compounds.

Primary human foreskin fibroblasts (a gift from Dr. Christopher Tonkin, WEHI) were

maintained in DME medium containing 10% HI-FBS at 37oC, in 5% CO2. The CellTiter Blue

Cell Viability Assay (Promega, Madison, WI, USA) was used to examine fibroblast toxicity

in the presence of compounds.

10

RESULTS

High-throughput screen to identify inhibitors of GDP-MP. The high-throughput screen of

~80,000 compounds from the WEHI Bio21 lead discovery library identified 442 compounds

that inhibited the activity of GDP-MP by more than 50% at a compound concentration of

44µM, yielding a hit rate of ~0.6%. These compounds were subsequently picked from a fresh

10mM copy of the compound library, and re-tested in triplicate at 44µM. 223 of the 442

compounds re-tested demonstrated confirmed activity, yielding a hit confirmation rate of

50%.

Compound dose-response curves for the 223 primary hits were conducted with the GDP-MP

assay and with a counter screen assay containing buffer, phosphate sensor, and 1µM

Na2HPO4 only. Compounds that exhibited similar IC50 values in both the GDP-MP and

counter screen assays were designated as false positives and eliminated from further

consideration. The most potent compounds were found to have IC50 values less than 1µM in

the GDP-MP assay and no activity in the counter screen assay.

In vitro antileishmanial activity. Following counter-screening and validation of the original

hits, we chose 20 compounds for further testing on parasites, based primarily on their

inhibitory potency against GDP-MP. We obtained 39 analogues from commercial sources to

develop structure-activity relationships. The structures of the 25 compounds discussed in

more detail below are given in Fig. 4.

We initially investigated whether these compounds affected in vitro survival and proliferation

of wild type L. major promastigotes. GDP-MP knockout parasites are viable in culture,

therefore we were not expecting to see growth inhibition of wild type parasites if the

compounds were specifically targeting parasite GDP-MP. Parasites were incubated for 48h in

the presence of test compounds and their growth rate was compared to that of non-treated

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controls. Compound 21 showed relatively potent anti-promastigote activity, while three others

(compound 7, 17, 23) displayed leishmaniostatic activity (confirmed by microscopy), whereas

all other compounds had no anti-promastigote activity (data not shown).

We next examined the 20 most potent GDP-MP inhibitors for activity against L. major lesion-

derived ex vivo amastigotes, the clinically relevant form of the parasite. The amastigotes were

purified from lesions of infected hypothymic CBA nu/nu mice (12) and were incubated for

24h in the presence of different concentrations of compounds and their growth rate was

compared to that of non-treated controls. The majority of compounds did not display

significant antileishmanial activity at the concentrations tested, however, 9 compounds

inhibited parasitic growth (Table 1). The most active compounds were compound 20 and

compound 17 with in vitro IC50 of 23.7µM and 25.1µM, respectively. Compounds 24, 3 and

25 were characterized by in vitro IC50 of 52.6µM, 62.3µM and 73.4µM, respectively, whereas

four other compounds demonstrated inhibitory activity only at 100µM. Amphotericin B was

used as a reference antileishmanial agent (IC50 1.5µg/ml) and DMSO used at a range of

concentrations (0.0005-1%) showed no adverse effect on amastigote viability (Table 1).

Human primary fibroblasts were used for the determination of compound toxicity. The 10

compounds tested showed no significant effects on the fibroblasts across the range of

concentrations investigated (Table 1).

Nine compounds with activity against Leishmania lesion-derived amastigotes were also tested

for activity in intracellular assays. Initially, promastigote-infected macrophages (macrophage

invasion assay) were incubated for 48h in the presence of 30µM test compound, a

concentration chosen based on the IC50 of the two most potent compounds in the amastigote

assay. A significant reduction in the number of promastigote-infected macrophages was

observed for compounds 3 (P<0.001), 25 (P=0.003) and 17 (P=0.01), whereas other

compounds showed only moderate activity in this assay (Fig. 5A). An amastigote invasion

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assay (intracellular drug sensitivity assay) yielded identical results, however, statistically

significant inhibition (P<0.001) was also observed for compounds 6 and 24 (Fig. 5D). Despite

having no adverse effect on human fibroblasts, compounds 20 and 13 displayed an adverse

effect on mouse macrophages at 30µM (data not shown).

Compound 3 showed moderate activity against amastigotes in vitro, but excellent activity in

intracellular drug sensitivity assays. Therefore, we were particularly interested in

comprehensive testing of its efficacy. This compound was further tested in the macrophage

assay at 10, 20, 30 and 40µM, together with purchased structural analogues (compounds 2

and 4) tested at 10, 20 and 30µM. With the exception of compound 2 at 10µM, treatment with

all three compounds at all concentrations tested showed a significant reduction in the number

of infected macrophages (P<0.001), and comparable activity against the ex vivo amastigotes.

This reduction occurred in a dose dependant manner for all 3 compounds, regardless of the

parasite stage used for macrophage invasion, either promastigotes (Fig. 5B) or amastigotes

(Fig. 5E). A third analogue, compound 5, was subsequently synthesized and examined more

thoroughly for activity against amastigotes. As shown in Table 1, this compound displayed

antileishmanial activity against ex vivo amastigotes (IC50=32.6µM) and displayed excellent

selectivity, showing no signs of toxicity to fibroblasts even at concentrations of 100µM.

Compound 5 also showed excellent activity in the macrophage invasion assay (IC50=17.6µM)

(Fig. 5C) or intracellular proliferation assay (IC50=11.7µM) (Fig. 5F). Results of all tests are

summarized in Table 1.

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DISCUSSION

The pentavalent antimonials such as sodium stibogluconate and meglumine antimoniate have

been recommended for the treatment of leishmaniasis for over 70 years. It is thus not

surprising, that resistance to this class of drugs is increasing, and in some endemic areas their

use is limited due to a lack of efficacy. Second line drugs used in the treatment of

leishmaniasis include aromatic diamidines (Pentamidine) and amphotericin B, but similarly to

the pentavalent antimonials, these drugs are toxic, with severe (sometimes life-threatening)

side effects (22). Since the introduction of miltefosine, at the beginning of this century, which

is the only orally active drug against leishmaniasis, no new antileishmanial compounds have

been approved for human treatment. Therefore, the development of safe, effective and

affordable antileishmanial chemotherapies is a critical global public-health priority.

Leishmania synthesize unique mannose-rich glycoconjugates that are essential for their

survival in the mammalian host. In search of novel antileishmanial compounds, we have

employed a novel high-throughput screen involving a small molecule chemical library of

~80,000 drug-like molecules to identify inhibitors of leishmanial GDP-MP, an important

enzyme in mannose-containing glycoconjugate biosynthesis (8). Several compounds were

identified that inhibited GDP-MP activity with sub-µM or low-µM IC50 and were therefore

worthy of further investigation. We have successfully screened against other parasitic targets

with the guiding philosophy behind the selection criteria for the design of the chemical library

being that lead-like simplicity rather than drug-like complexity is better for furnishing more

optimizable screening hits, which are better starting points for drug development and better

cover diversity space. We have commented in more detail on this elsewhere (17, 18). “Lead-

like” chemical structures have been defined as having simple molecular structures that are

chemically unreactive, synthetically accessible and have “drug-like” properties, that might

14

bind only relatively weakly in the low-µM range to target proteins but that are also highly

optimizable.

Figure 4 depicts the structures of the twenty most potent GDP-MP inhibitors that were tested

for activity against ex vivo amastigotes, along with some purchased or synthesized analogues

that were also investigated further. Eight of the compounds are singletons and belong to their

own structural class. However, the remaining compounds populate three distinct classes:

pyrazinylquinolines (A), thiadiazole-like derivatives (B), and pyrazoline-3,5-diones (C).

Thiadiazole-containing compounds, in particular 1,3,4-thiadiazoles, comprise by far the

biggest group of compounds. Scrutiny of the structures does not yield clear SAR and neither

does potent GDP-MP activity translate to good anti-amastigote activity in vitro. In particular,

we note that almost identical compounds to those discussed here, which belong to the same

sub-class of 1,3,4-thiadiazoles bearing a ring-fused 1,3,4-triazole, have recently been reported

as screening (wet and in silico) hits for p38 MAP kinase (5), factor-inhibiting HIF-1 (23),

CYP17 (2) and shikimate kinase (28). We therefore view these as likely pan assay

interference compounds (3) and in light of this, did not progress these compounds any further.

However, we note that the 1,3,4-thiadiazole heterocycle per se is present in compounds with

good oral bioavailability (14) as well as marketed drugs (4) and it may be that specific sub-

classes of 1,3,4-aminothiadiazoles may be optimizable screening hits while others, such as

those fused with a 1,3,4-triazole, may be pan assay interference compounds.

Of the 20 compounds (initial hits) tested on lesion-derived amastigotes, the nine most active

were tested in a murine macrophage model of L. major infection. The most potent, compound

3, belongs to the quinoline class of GDP-MP inhibitors that we have identified here. As

shown in Fig. 5A and 5D, this compound reduced macrophage infection in a dose-dependent

manner, and by around 70-80% at a concentration of 30µM. This is more efficacious than its

anti-ex vivo amastigote activity might suggest (IC50 62.3µM). While basic compounds such as

15

piperazines can accumulate intracellularly in a parasitological setting (see (17) and references

therein), it is also possible that this apparent discrepancy is simply due to different assay

formats. However, it is also possible that ex vivo amastigotes maintained in axenic media

underwent conversion to the promastigote form, which is not affected by the compounds

tested in this study (data not shown). Previous studies suggested that around 30% of L. major

lesion-derived amastigotes become flagellated while maintained for 24h at 37oC and pH 7.3,

conditions very similar to those in the current study (16). Nevertheless, our data suggest

(unpublished observations) that ex vivo amastigotes maintained in the axenic medium have

the ability to infect dendritic cells, which is lost following 48h incubation at 37oC. This is

consistent with previous reports indicating that amastigotes, but not promastigotes, have the

ability to invade dendritic cells (31). Nonetheless, the results obtained from the intracellular

proliferation assay are of greater importance since they reflect the compound’s activity

against the true clinically relevant form of the parasite, an amastigote proliferating in the

parasitophorous vacuole.

In order to elucidate signs of early SAR for this class of compounds, we obtained two

analogues of compound 3, namely compounds 2 and 4 (structures given in Fig. 4). These

compounds showed similar activity to compound 3 in the intracellular assays and were also

inhibitory in a dose-dependent manner (Fig. 5B and 5E). This suggests that an aryl substituent

on the piperazine ring may be important for activity. In order to test this hypothesis, we made

the truncated analogue (compound 1) shown in Fig 4. In support of the hypothesis that an aryl

substituent is important, compound 1 was completely inactive, providing useful negative

SAR. Such negative SAR is important because it supports the notion that most of the features

of the original hit are essential for activity and that this activity arises from specific

engagement of the test compound with GDP-MP. On this basis, we undertook some limited

medicinal chemistry optimization, which included a number of SAR probes of the piperazinyl

16

aryl substituent. The best of these was compound 5, which bore a para-phenyl ring from the

benzyl group. As shown in Table 1 and Fig. 5C and F, this compound showed good activity

against ex vivo amastigotes and in an intracellular drug sensitivity assay, with IC50 of 32.6µM

and 11.7µM, respectively, and yet was completely non-toxic to fibroblasts at concentrations

as high as 100µM. This compound has been selected for further testing and optimization. The

key to optimizing this compound will be to increase the ligand-binding efficiency (19) by

improving activity without significantly increasing the molecular weight.

Two other classes of compounds showed statistically significant activity in the murine

macrophage model, namely compounds 25 and 17. These belong to phenolic mannich base

and pyrazoline-3,5-dione classes, respectively. Recent analysis of our historical screening

data has shown these compound classes to be problematic in high-throughput screening, in

that they may appear to be genuine hits, but in fact may be selectively protein reactive: in the

former class, via formation of quinone methides and in the latter, by acting as a Michael

acceptor for protein nucleophiles. We have coined the acronym PAINS for such compounds

(Pan Assay Inteference Compounds) and caution readers that these compound classes are

likely to represent poor choices for optimization. In addition, compounds 6 and 24 showed

significant activity in reducing amastigote infection in macrophages. Compound 6 has been

assigned to the problematic thiadiazole category (group B), but in fact it is distinct in structure

from the other compounds in this group that are fused with 1,2,4-triazole. This compound

might still be of potential interest for further optimization, and interestingly resembles the

antihelmintic drug levamisole (32), that also shows putative anticancer efficacy through its

immunomodulatory action (25). Compound 24 belongs to the class of cyanopyridones that we

have found to be PAINS, possibly by being protein reactive and/or redox reactive. Therefore,

we would treat it cautiously as a candidate for further optimization.

17

In general, the compounds identified in the primary screen, which showed excellent inhibitory

activity in the enzyme-based assay were less active in anti-parasite assays. Possible

explanations might be inefficient uptake of the compounds by the parasites, rapid intracellular

degradation or compound exclusion due to permeability constraints. The lack of correlation

between enzyme inhibition and antiparasitic activity was not unexpected, and is not

uncommon when altering assay formats in drug discovery and as such has been reported

previously (29). In addition, without biomarker information for GDP-MP inhibition, it cannot

be ruled out that antiparasitic activity may arise through off-target activity. In any case, it is

unlikely that the non-optimized screening hits would exhibit potent antileishmanial activity

without further iterative rounds of medicinal chemistry optimization of their enzyme

inhibition activity, permeability and solubility. Interestingly, our macrophage invasion assay

using promastigotes for invasion over 5 hours, provided equivalent outcomes to the

intracellular proliferation assay recently labeled as a “gold standard” for in vitro testing of

antileishmanial compounds (30).

Studies using parasites from which the gene for GDP-MP had been deleted, validated GDP-

MP as a drug target that is involved in a biosynthetic pathway essential for parasite survival in

the mammalian host. Given the increasing incidence of leishmaniasis and the need for potent

yet minimally toxic treatment alternatives, identification of several GDP-MP inhibitors

amenable to medicinal chemistry optimization is encouraging and warrants further

investigation. These inhibitors all belong to a class of 4-pyrazin-4-yl quinolines and are drug-

like and selectively toxic towards Leishmania amastigotes.

ACKNOWLEDGMENTS

This work was supported by an NHMRC Project Grant 406631 and by the Program Grant

406601.

18

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FIGURE LEGENDS

FIG 1. High-throughput screening enzymatic cascade. The assay ultimately detected

production of inorganic phosphate. Inorganic pyrophosphatase was added to the reaction in

excess, to minimize the chance of identifying inhibitors of this enzyme.

FIG 2. Determination of the Km values. Km for M-1-P (A) was determined to be 4.6µM and

Km for GTP (B) was determined to be 1.5µM, as indicated by the dashed lines.

FIG 3. Average control values for compound plates in chronological order of plates screened.

(A) Average positive (squares) and negative (triangles) controls and (B) calculated Z’ values,

for individual library compound plates screened. Z’>0.4 was considered acceptable.

FIG 4. Structures of compound focus sets discussed herein. Three distinct classes can be

discerned: 4-pyrizinylquinolines (A), thiadiazole-like (B), and pyrazolin-3,5-diones (C)

FIG 5. Treatment of L. major-infected, mouse bone marrow-derived macrophages with GDP-

MP inhibitors. (A and D) Infected macrophages were treated for 48h with 30µM of test

compounds. Data are plotted for 7 compounds (two additional compounds tested had adverse

effects on macrophages (see Results section) and were not included); (B and E) Infected

macrophages were treated with various concentrations of compound 3 and its structural

analogues; (C and F) Infected macrophages were treated with different concentrations of

compound 5, Amphotericin B was used as a reference compound. Panels A-C, macrophages

were infected with promastigotes; Panels D-F, macrophages were infected with amastigotes.

The number of macrophages infected with at least one amastigote was determined by

24

microscopy from fixed specimens in duplicates. Assays were performed at least twice in

duplicates. Error bars indicate SEM, * indicated statistically significant difference.

25

TABLE 1. Summary of antileishmanial activity of GDP-MP inhibitors.

Compound # HTS

IC50 (µM) Amastigotes

IC50 (µM) Fibroblasts

CC50 (µM)

Intracellular

Proliferation Assay

IC50 (µM)

1 NT >100 NT NT

2 NT 70.6 NT >30

3 0.58 62.3 >100 21.9

4 NT 62.2 NT >30

5 NT 32.6 >100 11.7

6 0.49 78.04 >100 >30

7 4.45 >100 NT NT

8 2.93 >100 NT NT

9 1.09 >100 NT NT

10 3.53 >100 NT NT

11 1.26 >100 NT NT

12 2.98 >100 NT NT

13 1.37 88.9 >100 NA

14 3.95 >100 NT NT

15 1.05 >100 NT NT

16 10.67 90.7 >100 >30

17 5.75 25.1 >100 >30

18 5.53 96.8 >100 >30

19 7.28 >100 NT NT

20 1.56 23.7 >100 NA

21 8.25 >100 NT NT

22 7.54 >100 NT NT

23 5.31 NT NT NT

24 2.44 52.6 >100 >30

25 4.12 73.4 >100 >30

Amphotericin B NT 1.5 µg/ml NT >0.5 µg/ml

DMSO (%) NT >1 >1 >1

NT, not tested

NA, not available