Inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase

Schoenen FJ, Whipple D, Baillargeon P, et al.

Publication Details

Malaria is one of the most prevalent human parasitic diseases and is a global health issue accounting for >600,000 deaths annually. For survival, the Plasmodium falciparum (Pf) malaria parasite requires the action of a number of metallo-aminopeptidases, including PfM1MAA (membrane alanine aminopeptidase), PfM17LAP (leucine aminopeptidase), and PfM18AAP (aspartyl aminopeptidase). Each enzyme displays restricted amino acid specificity, and they are thought to act in concert to degrade proteins (i.e., host erythrocyte hemoglobin) that the parasite uses to generate a pool of amino acids which are employed as building blocks for the synthesis of its own proteins. Since there were very few small-molecule inhibitors of PfM17LAP and no selective inhibitors relative to PfM1AAP (or PfM18AAP), we set out to identify new potent and selective small-molecule inhibitors of this enzyme. Biochemical assays employing enzymatically active recombinant PfM17LAP (rPfM17LAP), as well as recombinant Fasciola hepatica cathepsin L1 (rFhCTSL1), rPfM1MAA, rPfM18AAP, and human M17LAP (rhuM17), and cell-based parasite growth inhibition and cytotoxicity assays were used to identify CID 2466 (from the NIH MLSMR) as a viable starting point for SAR analysis. Three rounds of structure-activity relationship studies were performed to generate a panel of probe candidate compounds. Ultimately, the compound hit, CID 2466, also known as bufexamac, was nominated as the probe ML392. When the probe and analogues are used as recommended, they are “fit-for-purpose” and should be useful for advancing the search for new antimalarial drugs directed at PfM17LAP.

Assigned Assay Grant #: 1 R03 MH082342-01A1

Screening Center Name & PI: Scripps Research Institute Molecular Screening Center, Hugh Rosen; Southern Research Specialized Biocontainment Screening Center, E. Lucile White

Chemistry Center Name & PI: University of Kansas Specialized Chemistry Center, Jeffrey Aubé

Assay Submitter & Institution: John P. Dalton, Institute of Parasitology, McGill University, Quebec, Canada; Institute for Biotechnology of Infectious Diseases (IBID), University of Technology Sydney (UTS), Sydney, Australia; Donald L. Gardiner, Malaria Biology Laboratory, The Queensland Institute of Medical Research, Brisbane, Australia

PubChem Summary Bioassay Identifier (AID): 434965

Probe Structure & Characteristics

ML392.

ML392

1. Recommendations for Scientific Use of the Probe

Using this probe and analogues, researchers may evaluate the PfM17LAP enzyme as a target in culture for developing small-molecule anti-malaria drugs. Moreover, a more detailed understanding of the enzyme’s function in the parasite may be elucidated specifically and its putative role alongside other aminopeptidases (e.g. PfM1AAP and PfM18AAP) may be determined. Eventually, this could contribute to the development of multi-drug therapies targeting several malaria enzymes, therapies for which there might be a lower prospect of drug resistance developing.

More specifically, in live-cell morphological screening studies using light microscopy, parasitologists might use the compounds reported here to study when and where the PfM17LAP enzyme functions within malaria cells. Chemical biologists might use the compounds to design biotin, photo-affinity or fluorescent conjugates as an approach to illuminating the distinct biological roles for PfM17LAP, as has been demonstrated using bestatin-based conjugates. Crystallographers might use the highly soluble and selective compounds reported here for co-crystallization studies with the recombinant PfM17LAP and PfM1AAP enzymes, for which the X-ray crystal structures have been reported.

When the probe and analogues are used as recommended previously, they are “fit-for-purpose” [1] and should be useful for advancing the field.

2. Materials and Methods

See subsections for a detailed description of the materials and methods used for each assay.

Recombinant enzymes

Recombinant PfM17LAP, PfM18AAP, PfM1AAP, and human M17 were prepared at the University of Technology, Sydney, as described previously [2, 3]. Recombinant cathepsin L of the parasitic helminth Fasciola hepatica was prepared in the same laboratory as described previously [4].

2.1. Assays

Inhibitors of Plasmodium falciparum M17- Family Leucine Aminopeptidase (PfM17LAP) (Primary Assay AID 1619)

The purpose of this assay is to identify compounds that inhibit the activity of M17 aminopeptidase of the malaria parasite Plasmodium falciparum (PfM17LAP). In this simple biochemical assay a fluorogenic peptide substrate (H-Leu-NHMec) was used for quantifying the activity of M17LAP. The rate of hydrolysis of this substrate was measured by monitoring the release of the -NHMec fluorogenic leaving group at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. A kinetic assay was chosen to minimize interference by compounds that fluoresce at these wavelengths. As designed, compounds that inhibit PfM17LAP will block rPfM17LAP-mediated cleavage of H-Leu-NHMec and liberation of the NHMec leaving group from the substrate, resulting in decreased well fluorescence. For the primary screen, test compounds were assayed in singlicate at a final nominal concentration of 30 micromolar. For concentration-response confirmatory screening, the most potent inhibitors were tested using a 8-point dilution series starting at 100 micromolar. This assay was performed at the Southern Research Specialized Biocontainment Screening Center.

Inhibitors of Plasmodium falciparum M1-Family Alanyl Aminopeptidase (PfM1AAP) (Primary Assay AID 1445)

The purpose of this assay is to identify compounds that inhibit the activity of PfM1AAP aminopeptidase of the malaria parasite Plasmodium falciparum (PfM1AAP). This assay also serves as a counterscreen for the experiment titled, “Inhibitors of Plasmodium falciparum M17- Family Leucine Aminopeptidase (PfM17LAP, Primary Assay AID No. 1619)”. In this biochemical assay a fluorogenic peptide substrate (H-Leu-NHMec) was used for quantifying the activity of M1AAP. The rate of hydrolysis of this substrate was measured by monitoring the release of the -NHMec fluorogenic leaving group at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. A kinetic assay was chosen to minimize interference by compounds that fluoresce at these wavelengths. As designed, compounds that inhibit PfM1AAP will block rPfM1AAP-mediated cleavage of H-Leu-NHMec and liberation of the NHMec leaving group from the substrate, resulting in decreased well fluorescence. For the primary screen, test compounds were assayed in singlicate at a final nominal concentration of 30 micromolar. For concentration-response confirmatory screening, the most potent inhibitors were tested using a 8-point dilution series starting at 100 micromolar. This assay was performed at the Southern Research Specialized Biocontainment Screening Center.

Counterscreen for inhibitors of M1 and M17 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Cathepsin L proteinase (CTSL1) (Counterscreen Assay AID 2214)

The purpose of this assay is to determine cathepsin L1 dose response curves for compounds identified as active in one of the following set of experiments titled, “Inhibitors of Plasmodium falciparum M1-Family Alanyl Aminopeptidase (M1AAP)” (AID 1445), or “Inhibitors of Plasmodium falciparum M17-Family Leucine Aminopeptidase (M17LAP)” (AID 1619). This assay serves as a counterscreen to determine whether compounds are nonselective due to inhibition of cathepsin L1. In this biochemical assay, a commercially available N-terminally blocked fluorogenic peptide substrate (Z-Leu-Arg-NHMec) is incubated with purified recombinant cathepsin L1 protein in the presence of test compounds. Cleavage of the substrate by cathepsin L1 releases the fluorogenic NHMec leaving group, leading to an increase in well fluorescence. As designed, compounds that inhibit cathepsin L1 will prevent substrate cleavage and liberation of the fluorescent leaving group, resulting in decreased well fluorescence. Test compounds were assayed in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 59.6 micromolar. This assay was performed at the Scripps Research Institute Molecular Screening Center.

Counterscreen for inhibitors of M1 and M17 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) (Counterscreen Assay AID 2215)

The purpose of this assay is to determine rPfM18AAP dose response curves for compounds identified as active in the following set of experiments entitled, “Inhibitors of Plasmodium falciparum M1-Family Alanyl Aminopeptidase (M1AAP)” (AID 1445), or “Inhibitors of Plasmodium falciparum M17-Family Leucine Aminopeptidase (M17LAP)” (AID 1619). This assay serves as a counterscreen to determine whether compounds are nonselective due to inhibition of M18. In this biochemical assay, a commercially available fluorogenic peptide substrate (H-Glu-NHMec) is incubated with purified recombinant PfM18AAP enzyme (rPfM18AAP) in the presence of test compounds. Cleavage of the substrate by rPfM18AAP enzyme liberates the NHMec leaving group from the peptide, leading to increased well fluorescence. As designed, compounds that inhibit PfM18AAP will block rPfM18AAP-mediated cleavage of H-Glu-NHMec and liberation of the NHMec leaving group from the substrate, resulting in decreased well fluorescence as measured at 340 nm excitation and 450 nm emission. Test compounds were assayed in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 73.5 micromolar. This assay was performed at the Scripps Research Institute Molecular Screening Center.

Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PfM17LAP): fluorescence-based assay to identify inhibitors of rPfM17LAP (Hit Validation Assay AID 492977)

The purpose of this assay is to determine inhibitory activity of powder samples of compounds for recombinant P. falciparum M17LAP. In this assay, a fluorogenic peptide substrate (H-Leu-NHMec) that binds to the active site of rPfM17LAP was used to quantify the activity of rPfM17LAP in the presence of inhibitor compounds. The rate of hydrolysis of this substrate in the presence of 5 μM inhibitor compounds was measured by monitoring the release of the -NHMec fluorogenic leaving group at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. As designed, compounds that bind to rPfM17LAP will compete with binding of the fluorogenic peptide substrate, resulting in a decrease in the release of the fluorogenic leaving group and a decrease in well fluorescence. This assay was performed in the labs of the assay provider, John P. Dalton.

Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (M17LAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCs (Hit Validation Assay AID 492955)

The purpose of this assay is to determine the ability of powder samples of inhibitor compounds to inhibit the growth of Plasmodium falciparum in its asexual erythrocytic stage. In this assay, compounds are incubated with red blood cells (RBC) infected with P. falciparum parasites in hypoxanthine-free media. 3H-hypoxanthine is added, cells incubated 48 hours, and incorporation of 3H determined. As designed, compounds that inhibit the growth of P. falciparum in RBC will decrease the level of 3H incorporated. Three replicates at 10 μM were performed for each compound tested in each experiment, and each compound was tested in two or three experiments. This assay was performed in the labs of the assay provider, Donald L. Gardiner.

Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (M17LAP): radiolabel-based cell-based dose response assay to identify compounds that inhibit P. falciparum growth in RBCs (Hit Validation Assay AID 489016)

The purpose of this assay is to determine the potency of a powder sample of an inhibitor compound identified in a previous assay to inhibit the growth of Plasmodium falciparum in its asexual erythrocytic stage. In this assay, compounds are incubated with red blood cells (RBC) infected with P. falciparum parasites in hypoxanthine-free media. 3H-hypoxanthine is added, cells incubated 48 hours, and incorporation of 3H determined. As designed, compounds that inhibit the growth of P. falciparum in RBC will decrease the level of 3H incorporated. Compounds were tested in triplicate using a 5-point dilution series starting at a nominal concentration of 25 μM. This assay was performed in the labs of the assay provider, Donald L. Gardiner.

QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PfM17LAP) (SAR Assay AID 588688, AID 602216, AID 602309)

The purpose of this assay is to determine dose response curves for compounds identified as active. In this biochemical assay, a commercially available fluorogenic peptide substrate (H-Leu-NHMec) is incubated with purified recombinant PfM17LAP enzyme (rPfM17LAP) in the presence of test compounds. Cleavage of the substrate by rPfM17LAP enzyme liberates the NHMec leaving group from the peptide, leading to increased well fluorescence. As designed, compounds that inhibit PfM17LAP will block rPfM17LAP-mediated cleavage of H-Leu-NHMec and liberation of the NHMec leaving group from the substrate, resulting in decreased well fluorescence as measured at 340 nm excitation and 450 nm emission. Test compounds were assayed in triplicate in a 10-point 1:2 dilution series starting at a nominal test concentration of 100 micromolar. This assay was performed at the Southern Research Specialized Biocontainment Screening Center.

Counterscreen for inhibitors of PfM17LAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PfM1AAP) (Counterscreen Assay AID 588698, AID 602223, AID 602315)

The purpose of this assay is to determine dose response curves for compounds identified as active. In this biochemical assay, a commercially available fluorogenic peptide substrate (H-Leu-NHMec) is incubated with purified recombinant PfM1AAP enzyme (rPfM1AAP) in the presence of test compounds. Cleavage of the substrate by rPfM1AAP enzyme liberates the NHMec leaving group from the peptide, leading to increased well fluorescence. As designed, compounds that inhibit PfM1AAP will block rPfM1AAP-mediated cleavage of H-Leu-NHMec and liberation of the NHMec leaving group from the substrate, resulting in decreased well fluorescence as measured at 340 nm excitation and 450 nm emission. Test compounds were assayed in triplicate in a 10-point 1:2 dilution series starting at a nominal test concentration of 100 micromolar. This assay was performed at the Southern Research Specialized Biocontainment Screening Center.

Counterscreen for inhibitors of PfM17LAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) (Counterscreen Assay AID 588697, AID 602218, AID 602316)

The purpose of this assay is to determine dose response curves for compounds identified as active in a PfM17LAP screen. In this biochemical assay, a commercially available fluorogenic peptide substrate (H-Glu-NHMec) is incubated with purified recombinant PfM18AAP enzyme (rPfM18AAP) in the presence of test compounds. Cleavage of the substrate by rPfM18AAP enzyme liberates the NHMec leaving group from the peptide, leading to increased well fluorescence. As designed, compounds that inhibit PfM18AAP will block rPfM18AAP-mediated cleavage of H-Glu-NHMec and liberation of the NHMec leaving group from the substrate, resulting in decreased well fluorescence as measured at 340 nm excitation and 450 nm emission. Test compounds were assayed in triplicate in a 10-point 1:2 dilution series starting at a nominal test concentration of 100 micromolar. This assay was performed at the Southern Research Specialized Biocontainment Screening Center.

Counterscreen for inhibitors of PfM17LAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Human M17LAP (huM17LAP) (Counterscreen Assay AID 602214, AID 602407)

The purpose of this assay is to determine dose response curves for compounds identified as active. In this biochemical assay, a commercially available fluorogenic peptide substrate (H-Leu-NHMec) is incubated with purified recombinant huM17LAP enzyme (rhuM17LAP) in the presence of test compounds. Cleavage of the substrate by rhuM17LAP enzyme liberates the NHMec leaving group from the peptide, leading to increased well fluorescence. As designed, compounds that inhibit huM17LAP will block rhuM17LAP-mediated cleavage of H-Leu-NHMec and liberation of the NHMec leaving group from the substrate, resulting in decreased well fluorescence as measured at 340 nm excitation and 450 nm emission. Test compounds were assayed in triplicate in a 10-point 1:2 dilution series starting at a nominal test concentration of 100 micromolar. This assay was performed at the Southern Research Specialized Biocontainment Screening Center.

Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (M17LAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCs (Hit Validation Assay AID 743274)

The purpose of this assay is to determine the ability of powder samples of inhibitor compounds to inhibit the growth of Plasmodium falciparum in its asexual erythrocytic stage. In this assay, compounds are incubated with red blood cells (RBC) infected with P. falciparum parasites in hypoxanthine-free media. 3H-hypoxanthine is added, cells incubated 48 hours, and incorporation of 3H determined. As designed, compounds that inhibit the growth of P. falciparum in RBC will decrease the level of 3H incorporated. Three replicates at 10 μM were performed for each compound tested in each experiment, and each compound was tested in two or three experiments. This assay was performed in the labs of the assay provider, Donald L. Gardiner.

Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (M17LAP): radiolabel-based cell-based dose response assay to identify compounds that inhibit P. falciparum growth in RBCs (Hit Validation Assay AID 743273)

The purpose of this assay is to determine the potency of a powder sample of an inhibitor compound identified in a previous assay to inhibit the growth of Plasmodium falciparum in its asexual erythrocytic stage. In this assay, compounds are incubated with red blood cells (RBC) infected with P. falciparum parasites in hypoxanthine-free media. 3H-hypoxanthine is added, cells incubated 48 hours, and incorporation of 3H determined. As designed, compounds that inhibit the growth of P. falciparum in RBC will decrease the level of 3H incorporated. Compounds were tested in triplicate using a 5-point dilution series starting at a nominal concentration of 25 μM. This assay was performed in the labs of the assay provider, Donald L. Gardiner.

Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M17 Leucine Aminopeptidase (PfM17LAP) (Counterscreen Assay AID 588707, AID 602227, AID 602317)

In this assay, we treated Vero E6 cells with compounds selected as hits in the PfM17LAP assay for 72 hours over a 10 point 2-fold dilution series, ranging from 0.19 μM to 100 μM. Following 72 hours of treatment, relative viable cell number was determined using Cell Titer Glo from Promega. Each plate contained 64 replicates of vehicle treated cells which served as negative controls. This assay was performed at the Southern Research Specialized Biocontainment Screening Center.

2.2. Probe Chemical Characterization

ML392 was purchased from Sigma-Aldrich or Fluka. Analytical characterization data (1H NMR, 13C NMR, HRMS, and elemental combustion analysis) were all consistent with the structure. The compound samples were 100% pure as measured on the basis of peak integration (area under the curve) from UV-Vis absorbance at 214 nm as determined using RP HPLC/UV/MS.

Probe Chemical Structure and Properties

Image ml392fu2

Aqueous Solubility

The aqueous solubility for the probe was measured in phosphate-buffered saline (PBS) at room temperature (23 °C). PBS by definition is 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic at a pH 7.4. The probe compound ML392 was found to have a solubility of 76 μg/mL, or 340 μM, under these conditions, which is 829X and 62X the IC50s in the biochemical and cell-based inhibition assays (rPfM17LAP and PfM17LAP, respectively).

Aqueous Stability & Thiol Stability

The aqueous stability for the probe compound was assessed in PBS (no antioxidants or other protectants, DMSO concentration 1%, room temperature) and the results are reported in Figure 2.2.1. The probe compound ML392 was found to be stable in PBS, whereby 100% of the compound remained after 88 hours, which is well-beyond the 72 hour timeframe for the assay with the longest duration, the Vero cell cytotoxicity assay (Figure 2.2.1, A). See the

Figure 2.2.1. Stability of ML392 in PBS & DTT Stability Assay.

Figure 2.2.1

Stability of ML392 in PBS & DTT Stability Assay. ML392 (CID 2466, SID 93577810) was tested over a time course in an aqueous stability assay and thiol stability assays. (A) Stability of the probe over 88 hours in PBS; (B) Stability of the probe (more...)

The thiol stability for the probe compound was assessed in PBS and the results are reported in Figure 2.2.1. The probe compound ML392 was found to be stable to DTT, whereby 100% of the compound remained after 8 hours (Figure 2.2.1, B).

Submission of the Probe and Analogues to the NIH MLSMR

Samples of the probe and five analogues were submitted to the NIH MLSMR compound collection on November 25, 2013.

2.3. Probe Preparation

ML392 was purchased from Sigma-Aldrich and was processed through a Model 00 Jet-O-Mizer jet mill (Fluid Energy Processing and Equipment Company, Telford, PA). The RP-UPLC purity was 100% (214 nm) and the 1H and 13C NMR spectra were consistent with the structure. Satisfactory elemental analysis results were also obtained.

3. Results

3.1. Dose Response Curves for Probe

Figure 3.1.1. Concentration-response Curves for the Probe ML392.

Figure 3.1.1Concentration-response Curves for the Probe ML392

ML392 (CID 2466, SID 93577810) was tested across a range of concentrations up to 100 μM in the primary and several secondary assays. Concentration response data was analyzed using a four parameter logistic fit to the data (GraphPad Prism). From these curves IC50 values were calculated. (A) rPfM17LAP biochemical assay (AID 1619, 602216), IC50 = 0.41 μM; (B) rPfM1AAP biochemical assay (AID 602223), IC50 = 14 μM; (C) rPfM18AAP biochemical assay (AID 602218), IC50 = 10; (D) rhuM17LAP biochemical assay (AID 602214) IC50 = 0.31 μM; (E) Red blood cell parasite growth assay (AID 489016) EC50 = 1.7 μM; (F) Vero cell cytotoxicity assay (AID 602317) EC50 > 50 μM

3.2. Cellular Activity

The malaria parasite growth inhibition assay and the Vero cell cytotoxicity assay are cell-based assays. Many of the compound analogues screened were active in the malaria parasite killing assay and were inactive in the Vero cell cytotoxicity assay. The cellular permeability (PAMPA) for ML392 was measured and was determined to be good. Specifically, the cellular permeability for the probe was determined to be approximately 187 × 10−6 cm/s, 182 × 10−6 cm/s, and 175 × 10−6 cm/s at pH 7.4, 6.2, and 5.0, respectively.

3.3. Profiling Assays

According to a search of PubChem as of February 13, 2014, the probe has been screened in 990 assays. For the 72 assays for which the probe was reported to be active, only 19 of the assays are unrelated to the current project or to activity against Plasmodium falciparum or malaria. As a result, the maximum “off-target” activity for the probe is in the neighborhood of 19/990 assays, or 1.9% of the assays.

Regarding off-target activity for the probe, with respect to the recently reported class II HDAC inhibitory activity for the probe, in the article published by Drewes [5], the probe (i.e., bufexamac) is reported to inhibit the HDACs 1, 2, 3, 6, 8, and 10, with Kdapp of NI (no inhibition), NI, 341 μM, 10.7 μM, 235 μM, and 12.3 μM, respectively, using a chemoproteomics binding assay. While it is true that the probe is “active” against HDACs 6 and 10, the reported potencies of 10.7 μM and 12.3 μM, respectively, are 764X and 878X less potent than the 14 nM activity against the rPfM17LAP (vide infra, Section 4.1, mechanism of action). Within the MLPCN, the probe was screened against the Histone Deacetylase 3 (PubChem AID 2718), and negative results were reported, which is consistent with the observations of Drewes.

With respect to the reported activity of the probe against cyclooxygenases (see PubChem AID 7184 and AID 7185), the probe is reported to inhibit 5-lipoxygenase with an IC50 of 27 μM in an in vitro assay. While it is true that the probe is “active” against 5-lipoxygenase, the reported potency of 27 μM is 1,928X less than the 14 nM activity against the rPfM17LAP.

With respect to potential off-target metalloprotease activity for the probe, as of February 13, 2014, the probe ML392 is reported in PubChem to have been screened in a total of 990 assays. Within this set of 990 assays, negative results were reported for the following five metalloproteases: (1) matrix metalloproteinase 13 (MMP13) (PubChem AID 570), (2) ADAM17 (PubChem AID 720648), (3) matrix metalloproteinase 1 (PubChem AID 618), (4) metallo-beta-lactamase IMP-1 (PubChem AID 1556), and (5) VIM-2 metallo-beta-lactamase (PubChem AID 1527), suggesting that the probe compound is selective versus metalloproteases.

Finally, with respect to general off-target protease activity for the probe, also within the set of 990 PubChem assays, negative results were reported for the following eleven proteases (excluding the metalloproteases above): (1) 3C-like protease (PubChem AID 1706), (2) cysteine protease ATG4B isoform (PubChem AID 504462), (3) Ubiquitin-specific Protease USP2a (PubChem AIDs 927), (4) Sentrin-specific protease 8 (SENP8) (PubChem AID 2540), (5) membrane-associated serine protease Rv3671c in M. tuberculosis (PubChem AID 2606), (6) Botulinum neurotoxin light chain F protease (PubChem AID 588459), (7) Sentrin-specific protease 7 (PubChem AID 434973), (8) Lethal Factor Protease (PubChem AID 588461), (9) Sentrin-specific protease 6 (PubChem AID 2599), (10) Escherichia coli DNA-binding ATP-dependent protease La (eLon) (PubChem AID 602123), and (11) SUMO protease, SENP1 (PubChem AID 624204), suggesting that the probe compound is selective versus a range of general proteases..

The probe compound was submitted to the Sanford-Burnham Medical Research Institute for various levels of exploratory pharmacology profiling (i.e., aqueous solubility in Pion’s buffer (pH 5.0, 6.2, and 7.4), aqueous solubility in 1x PBS, cell permeability, (PAMPA), plasma stability (human and mouse), plasma protein binding (human and mouse), hepatic microsome stability (human and mouse), and toxicity towards Fa2N-4 immortalized human hepatocytes). Overall, the probe showed good aqueous solubility (76 μg/mL, 340 μM; at pH 7.4), good permeability that is consistent across the pH range for which measurements were collected (187 × 10−6 cm/s, 182 × 10−6 cm/s, 175 × 10−6 cm/s at pH 7.4, 6.2, 5.0, respectively), good stability to human/mouse plasma (67%, 71% remaining after 3 h; human, mouse), moderate binding to human/mouse plasma proteins (82%, 74% of bound compound at 10 μM; human, mouse), very good/moderate stability to human/mouse hepatic microsomes (80%, 30% remaining after 1 h; human, mouse), and no toxicity toward immortalized human hepatocytes (LC50 > 50 μM against Fa2N-4 immortalized human hepatocytes).

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

The prior art was investigated by searching the Chemical Abstracts database using the SciFinder software. Abstracts were obtained for all references returned from the search and were analyzed for relevance to the current project. For all references that were deemed relevant, the articles were analyzed and the results are summarized. The search results are current as of December 2, 2013. The structures for reported inhibitors of PfM17LAP along with their reported activities are shown in the Figure 4.1.1 and Table 4.1.1.

Figure 4.1.1. Reported inhibitors of rPfM17LAP.

Figure 4.1.1

Reported inhibitors of rPfM17LAP.

Table 4.1.1. Compounds reported in the literature to inhibit PfM17LAP or hM17LAP.

Table 4.1.1

Compounds reported in the literature to inhibit PfM17LAP or hM17LAP.

Using the recombinant PfM1AAP and PfM17LAP enzymes, bestatin, a natural Phe-Leu dipeptide analog isolated from the fungus Streptomyces olivoretticuli and a known aminopeptidase inhibitor, was shown to be a potent inhibitor of both P. falciparum rPfM17LAP (0.025 μM) and rPfM1AAP (0.478 μM) [2, 6].

During studies aimed at using a bestatin-based chemical biology strategy to investigate the distinct roles for malaria M1- and M17-family aminopeptidases, one probe, PNAP (Phe-Napthyl), showed some selectivity for PfM17LAP over PfM1AAP (i.e., Ki’s of 29 nM versus 330 nM, respectively) [7]. These in vitro potency results were consistent with the phenotypes observed using live-cell morphological screening.

The diasteromeric and racemic phosphinate dipeptide compound mixtures, LeuP[CH2]Leu, hPheP[CH2]Gly, hPheP[CH2]Phe, and hPheP[CH2]Tyr, known inhibitors of mammalian aminopeptidase N, were shown to be double- and triple-digit nanomolar inhibitors of rPfM17LAP [9], although these compounds were reported to also inhibit rPfM1AAP [8]. The compound mixtures hPheP[CH2]Phe and hPheP[CH2]Tyr were also reported to reduce parasite growth in culture (Dd2 and 3D7 P. falciparum parasites) with IC50s ranging between 13 and 75 μM. The compound mixture hPheP[CH2]Phe was assessed for anti-malarial activity in vivo using the rodent P. c. chabaudi model, and, at a dose of 100 mg/kg twice a day, the compound significantly reduced parasite burdens in mice, although the researchers state that there is a curious discrepancy between the activity observed for the compound in cell culture and in vivo.

Approximately 35 α-aminoalkylphosphonate compounds showed a range of activities against cultured P. falciparum parasites with the most potent (1-amino-4-cyclohexylbutyl)phosphonic acid) showing 98% inhibition at 10 μM and approximately 30% inhibition at 0.1 μM against PfM17LAP and PfM1AAP [11].

During research around phosphonic arginine mimetics as inhibitors of the rPfM17LAP and rPfM1AAP aminopeptidases, studies directed at understanding the interactions necessary for a dual inhibitor, the phosphonic acid arginine mimetics 6 and 8 showed good potency and selectivity for rPfM17LAP over rPfM1AAP (i.e., Ki’s of 11 nM versus 104 μM, and 160 nM versus >1000 μM, respectively) [10]. Cell-based activity for these compounds is not disclosed.

Starting with tosedostat, also known as CHR-2797, a potent inhibitor of a number of intracellular mammalian aminopeptidases [12], Skinner-Adams identified CHR-2863 and CHR-6768 as potent and selective inhibitors of rPfM17LAP over rPfM1AAP (i.e., Ki’s of 76 nM versus 1413 nM, and 26 nM versus 2409 nM, respectively) which kill P. falciparum parasites in culture and showed that the former is efficacious against rodent malaria in vivo [13].

At the start of the project, the only prior art that had been reported was for bestatin, the phosphinate dipeptide compound mixtures, and the α-aminoalkylphoshonate class. All of these compounds were non-selective for PfM17LAP versus PfM1AAP. The PfM17LAP-selective PNAP, phosphonic acid arginine mimetics, and CHR-2863/CHR-6768 were disclosed after initiating the present work on the compound hit and analogues. With respect to the prior art, in all cases, the compounds are not selective relative to human M17. For most of the prior art compounds, activity against the human M17 was the impetus for testing the compound classes for activity against the Plasmodium falciparum parasite.

Based on the previous analysis, the compounds bestatin and tosedostat/CHR-2797 were chosen as benchmark compounds against which to compare the activity and selectivity for ML392. Table 4.1.2 captures the results, and supports the best-in-class status for ML392.

Table 4.1.2. Activity and selectivity for ML392 compared to select benchmark compounds.

Table 4.1.2

Activity and selectivity for ML392 compared to select benchmark compounds.

Moreover, based on the Ki data collected in the Dalton labs (summarized for comparison purposes in the Tables 4.1.1 and 4.1.2), the key advantages of ML392 compared to the prior art compounds are that ML392 is: (1) equally as potent against rPfM17LAP (Ki = 14 nM) as any prior art compound, (2) more selective versus rPfM1AAP (Ki = 48 μM) than all but two of the most recent prior art compounds (the phosphonic acid arginine mimetic compounds for which no “cell-based” data was reported), (3) more selective versus rPfM18AAP (Ki could not be obtained since the probe compound does not significantly inhibit the enzyme and Vmax could not be measured), and (4) more selective versus the rhuM17LAP (Ki = 420 nM).

Mechanism of Action Studies

The assays used in this project included a combination of on- and off-target biochemical and cell-based assays. Recombinant PfM17LAP was used for the biochemical target assay, while recombinant FhCTSL1, PfM1AAP, PfM18AAP, and human M17LAP were used for the biochemical anti-target assays. Malaria parasite growth inhibition in red blood cells and cytotoxicity against Vero cells were used as the cell-based target and anti-target assays, respectively. The probe ML392 and compound analogues (data not shown) show activity across this suite of assays that is consistent with an on-target mechanism of action. In general, the correlation between activity against rPfM17LAP in the biochemical assay and activity in the RBC-based parasite growth assay is quite good. For the few compounds for which activity against rPfM17LAP in the biochemical assay and activity in the RBC-based parasite growth assay does not correlate (data not shown), cytotoxicity is observed in the Vero cell assay, suggesting a plausible explanation for the discrepancy.

ML392 and 19 select analogues were further characterized in the Dalton labs for molecular mechanism of action, by the method of Dixon, and ML392 was found to be a competitive inhibitor. Furthermore, ML392 was found to be a quite potent and selective inhibitor for rPfM17LAP compared to rPfM1AAP and rhuM17LAP, exhibiting Ki’s of 14 nM, 48 μM, and 420 nM, respectively, equating to selectivity of 3,357X and 30X, respectively (see Figure 4.1.2 for the Dixon plots).

Figure 4.1.2. Dixon Plots for the Probe ML392.

Figure 4.1.2

Dixon Plots for the Probe ML392. Inhibition of rPfM17LAP, rPfM1AAP, and rhM17LAP by ML392 (CID2466, SID93577810) was performed at pH 7.5 using the substrate L-Leu-NHMec. Dixon plots were generated using the SigmaPlot software, where the reciprocal of (more...)

There is still considerable debate regarding the exact role of PfM17LAP in the parasite. One reason for nominating the compound hit (CID 2466, ML392, bufexamac) as the probe is that there are two reports in PubChem, and one in the primary scientific literature, for phenotypic high-throughput malaria parasite killing assays where bufexamac was identified as an active compound. In conjunction with the biochemical assay information presented here, this additional information could help to illuminate the exact role for PfM17LAP. The nature of these three assays will be described briefly.

In PubChem AID 504834, “Primary qHTS for delayed death inhibitors of the malarial parasite plastid, 96 hour incubation”, a cell-based assay that measures parasite growth based on the expression of an integrated copy of a firefly luciferase reporter, which detects small molecules that cause the “delayed death” phenotype, where erythrocytes infected with the luciferase-expressing parasites were incubated with compounds for either one or two generations, corresponding to 48 and 96 hours, respectively, compounds that inhibit parasite growth in the second generation, but not the first, should be enriched in antimalarials that target the apicoplast, and bufexamac is reported to be an approximately 0.2 μM inhibitor.

In PubChem AID 504749, “qHTS profiling for inhibitors of Plasmodium falciparum proliferation”, to profile malarial isolates for differential chemical responses and to identify inhibitors of malarial growth, an assay that measured P. falciparum growth and replication within erythrocytes was employed. The assay used a DNA-binding fluorescent dye (SYBR Green) to detect parasite DNA following cell lysis. Because mature erythrocytes lack DNA, only Plasmodium-derived DNA is detected. Bufexamac was reported as active in this assay [14].

From a phenotypic screen using P. falciparum parasites that stably express cytoplasmic firefly luciferase, Lucimi [15] identified bufexamac as a 2.77 μM inhibitor of parasite growth.

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