2.1.1. rPfM18AAP QFRET-based Primary Biochemical High Throughput Assay (Primary Assay AID 1822)

The purpose of this assay is to identify compounds that inhibit the activity of M18 aminopeptidase of the malaria parasite Plasmodium falciparum (PfM18AAP). 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 measured at 340 nm excitation and 450 nm emission. 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. Test compounds were assayed in singlicate at a final nominal concentration of 7.35 micromolar. This assay was performed at the Scripps Research Institute Molecular Screening Center.

2.1.2. CTSL1 QFRET-based Counterscreen Biochemical High Throughput Assay (Primary Assay AID 1906)

The purpose of this assay is to identify compounds that inhibit the activity of recombinant Fasciola hepatica cathepsin L1 expressed in yeast. Like the HTS for PfM18AAP this assay also exploits fluorogenic enzyme substrates. This assay also serves as a counterscreen for a set of previous experiments entitled, “Fluorescence-based primary biochemical high throughput screening assay to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (rPfM18AAP)” (AID 1822). In this biochemical assay, a commercially available fluorogenic peptide substrate (Z-Leu-Arg-MCA) is incubated with purified recombinant cathepsin L1 protein in the presence of test compounds. Cleavage of the substrate by cathepsin L1 releases the fluorogenic MCA 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 singlicate at a final nominal concentration of 5.96 micromolar. This assay was performed at the Scripps Research Institute Molecular Screening Center.

2.1.3. rPfM18AAP QFRET-based Confirmatory Biochemical High Throughput Assay (Primary Assay AID 2170)

The purpose of this assay is to confirm activity of compounds identified as active in a set of previous experiments entitled, “QFRET-based primary biochemical high throughput screening assay to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (rPfM18AAP)” (AID 1822). 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 at a final nominal concentration of 7.35 micromolar. This assay was performed at the Scripps Research Institute Molecular Screening Center.

2.1.4. CTSL1 QFRET-based Confirmatory Counterscreen Biochemical High Throughput Assay (Primary Assay AID 2178)

The purpose of this counterscreen assay is to determine whether compounds identified as active in a set of previous experiments entitled, “QFRET-based primary biochemical high throughput screening assay to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (rPfM18AAP)” (AID 1822) also inhibit cathepsin L1. This assay also serves to confirm activity of compounds identified as active in a set of previous experiments entitled, “QFRET-based counterscreen for rPfM18AAP inhibitors: biochemical high throughput screening assay to identify inhibitors of the Cathepsin L proteinase (CTSL1)” (AID 1906). In this biochemical assay, a commercially available fluorogenic peptide substrate (Z-Leu-Arg-MCA) is incubated with purified recombinant cathepsin L1 protein in the presence of test compounds. Cleavage of the substrate by cathepsin L1 releases the fluorogenic MCA 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 at a final nominal concentration of 5.96 micromolar. This assay was performed at the Scripps Research Institute Molecular Screening Center.

2.1.5. rPfM18AAP QFRET-based Confirmatory Concentration-response Biochemical High Throughput Assay (Primary Assay AID 2195)

The purpose of this assay is to determine dose response curves for compounds identified as active in a set of previous experiments entitled, “QFRET-based biochemical high throughput confirmation assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (rPfM18AAP)” (AID 2170). 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 HGlu-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.

2.1.6. CTSL1 QFRET-based Confirmatory Countersceeen Concentration-response Biochemical High Throughput Assay (Primary Assay AID 2196)

The purpose of this counterscreen assay is to determine dose response curves for compounds identified as active in a set of previous experiments entitled, “QFRET-based biochemical high throughput confirmation assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (rPfM18AAP)” (AID 2178). In this biochemical assay, a commercially available fluorogenic peptide substrate (Z-Leu-Arg-MCA) is incubated with purified recombinant cathepsin L1 protein in the presence of test compounds. Cleavage of the substrate by cathepsin L1 releases the fluorogenic MCA 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.

2.1.7. Fluorescence-based rPfM18AAP Confirmatory Biochemical Assay (Hit Validation Assay AID 492974)

The purpose of this assay is to determine inhibitory activity of powder samples of compounds for recombinant P. falciparum M18AAP. In this assay, a fluorogenic peptide substrate (H-Glu-NHMec) that binds to the active site of rPfM18AAP was used to quantify the activity of rPfM18AAP in the presence of inhibitor compounds. The rate of hydrolysis of this substrate in the presence of 5 uM 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 rPfM18AAP 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.

2.1.8. Fluorescence-based Malarial Cell Lysate PfM18AAP Confirmatory Biochemical Assay (Hit Validation Assay AID 492975)

The purpose of this assay is to determine inhibitory activity of powder samples of compounds for PfM18AAP in a malarial cell lysate. In this assay, a fluorogenic peptide substrate (H-Glu-NHMec) that binds to the active site of PfM18AAP was used to quantify the activity of PfM18AAP in malaria cell lysate in the presence of inhibitor compounds. The rate of hydrolysis of this substrate in the presence of 5 uM 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 PfM18AAP 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.

2.1.9. Radiolabel-based Pf Parasite Growth Cell-based Assay (Hit Validation and SAR Assays AID 489015)

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 P. falciparum-infected red blood cells (RBC) 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. Test compounds were assayed in triplicate at 10 micromolar, and each compound was tested in two or three experiments. For the purposes of hit validation and SAR optimization, this assay was performed in the labs of the assay provider, Donald Gardiner.

2.1.10. Radiolabel-based Pf Parasite Growth Cell-based Assay (Hit Validation and SAR Assays AID 489011)

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 P. falciparum-infected red blood cells (RBC) 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 uM. For the purposes of hit validation and SAR optimization, this assay was performed in the labs of the assay provider, Donald Gardiner.

2.1.11. rPfM18AAP QFRET-based Concentration-response Biochemical High Throughput Assay (SAR Assay AID 588678, AID 602222, AID 624177)

The purpose of this assay is to determine concentration response curves for compounds to support on-target SAR studies. 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.

2.1.12. rPfM1MAA QFRET-based Countersceeen Concentration-response Biochemical High Throughput Assay (Counterscreen Assay AID 588680 AID 602219, AID 624176)

The purpose of this assay is to determine concentration response curves for compounds to support off-target-selectivity SAR studies. In this biochemical assay, a commercially available fluorogenic peptide substrate (H-Leu-NHMec) is incubated with purified recombinant PfM1MAA enzyme (rPfM1MAA) in the presence of test compounds. Cleavage of the substrate by rPfM1MAA enzyme liberates the NHMec leaving group from the peptide, leading to increased well fluorescence. As designed, compounds that inhibit PfM1MAA will block rPfM1MAA-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.

2.1.13. rPfM17LAP QFRET-based Countersceeen Concentration-response Biochemical High Throughput Assay (Counterscreen Assay AID 588679, AID 602220, AID 624175)

The purpose of this assay is to determine dose response curves for compounds to support off-target-selectivity SAR studies. 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.

2.1.14. rhM18 QFRET-based Countersceeen Concentration-response Biochemical High Throughput Assay (Counterscreen Assay AID 588696, AID 602221, AID 624174)

The purpose of this assay is to determine dose response curves for compounds to support off-target-selectivity SAR studies. In this biochemical assay, a commercially available fluorogenic peptide substrate (H-Glu-NHMec) is incubated with purified recombinant human M18 enzyme (rhM18) in the presence of test compounds. Cleavage of the substrate by rhM18 enzyme liberates the NHMec leaving group from the peptide, leading to increased well fluorescence. As designed, compounds that inhibit hM18 will block rhM18-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.

2.1.15. Vero Cell-based Concentration-response Cytotoxicity Assay (Counterscreen Assay AID 588714, AID 602225, AID 624205)

In this assay, Vero E6 cells were treated with compounds selected as active in the PfM18AAP assay for 72 hours over a 10 point 2-fold dilution series, ranging from 0.19 uM to 100 uM. 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.

SAR Studies

Additional rounds of SAR could combine the structural features of the compounds listed in Table 3.4.9. For example, the first and third compound structures or the first and fourth compound structures could be combined to afford the compounds on the left and right, respectively, in the Figure 4.3.1.

Figure 4.3.1

Example Future SAR Studies.

Bioisosteric replacements for the catechol moiety, such as those popularized by Cohen, should also be revisited [20]. While many such catechol replacements were earmarked for synthesis, only a few were prepared due to the challenges associated with their preparation.

Mechanism of Action Studies

The mechanism of action for the probe or analogues could be explored using affinity-based protein profiling. This chemical biology strategy using bestatin-based probes has been used to illuminate the distinct roles for malaria PfM1MAA and PfM17LAP [27]. While the activity for the current probe is at the upper-end of what might be considered useful for such a probe, ideally, additional SAR studies should lead to significantly more active analogues, with activities more appropriate for such studies. The SAR studies to date suggest that the region of the inhibitor structure that is distal to the catechol moiety is a likely candidate for conjugation to biotin, photo-affinity, or fluorescent labels.

Co-Crystallization Studies using rPfM18AAP and the Probe or Analogues

Recently, the 3-D structure of the malaria recombinant PfM18AAP was resolved and showed that the enzyme exists as a large complicated multimeric structure [28]. Because of the lack of availability of a potent inhibitor, no structure for an inhibitor-enzyme complex was obtained. However, the activity and solubility of the current probe, and its analogues, should now fill this gap. One of the probe analogues, in particular, CID 53308676 (Table 3.4.5, entry 3, and Table J1, Appendix J), that shows potency in the low single-digit micromolar range (IC₅₀ against the rPfM18AAP) is quite soluble in aqueous solution across the pH range from 5.0 to 7.4 (>0.5 mM). Co-crystallization studies should allow a better understanding of the manner in which these compounds bind and interact with the malaria enzyme.

Fluorescent and Electron Microscopy Studies

Fluorescent and electron microscopy studies could be used to examine the capacity for the probe and analogues to disrupt the intracellular architecture of the malaria cell.

Studies on the Physical Properties of the Probe and Analogues and Their Selective Accumulation in the Parasitophorous Vacuole

The uptake and intracellular distribution properties of a compound are fundamentally important variables in achieving a potent and desirable biological response. The ability of compounds to cross cellular lipid bilayers is a necessary step in this process but rarely is it a sufficient one. This is because cells are highly compartmentalized entities with over 50% of their total volume comprised of membrane-bound organelles/compartments that can provide barriers between a probe and its intended target molecule.

Malaria parasite enzymes that interfere with hemoglobin digestion/metabolism are often presumed to be localized, at least partially, within the acidic digestive vacuole. Interestingly, PfM18AAP has been shown to be secreted into the parasitophorous space [16], which is also acidic due to the action of the V-H+-ATPase localized at the plasma membrane of the parasite [29]. Being relatively acidic, these compartments have the propensity to significantly concentrate weakly basic compounds according to an ion trapping-type mechanism. Accordingly, we hypothesize that compounds that have ideal structural and physicochemical properties that promote ion trapping (i.e., lysosomotropism) will have enhanced interactions with PfM18AAP relative to non-lysosomotropic counterparts. It is important to note that the presence or absence of lysosomotropic properties should not influence probe interactions with PfM18AAP in such assays. However, the lysosomotropic propensity of probes will be expected to favor interactions with PfM18AAP in the anti-malarial red blood cell cell-based assay since intracellular proton gradients are actively maintained. Interestingly, lysosomotropic compounds show enhanced potency in anti-malarial assays [23].

For next-generation probe analogues we will rationally incorporate functional groups that will impart increasing degrees of lysosomotropic properties and evaluate them using in vitro enzyme inhibition and RBC-based anti-malarial assays as described, previously. There are two key physicochemical properties of drugs that influence the degree of ion trapping in acidified intracellular compartments that will be systematically modified. The first parameter is the pK_a value of the conjugate acid of the weak base [30]. The second is the ratio of the permeabilities of the base in the ionized versus unionized form [31]. We will experimentally estimate the ratio of permeabilities by evaluating octanol/water partition coefficients as a function of pH as previously described [31]. The pK_a values of probes will be estimated using spectrophotometric approaches. In addition, we will experimentally determine the lysosomotropic characteristics of the probes using a recently developed approach that has been validated using the fluorescent lysosomotropic probe Lysotracker Red (LTR). In this approach we comparatively evaluate the total cell uptake in cells before and after treatment with ionophores nigericin and monensin. The ionophores dissipate the lysosome-to-cytosol pH gradient that provides the driving force for ion trapping, and cells treated with ionophores have markedly reduced lysosomal sequestration of LTR. Using this data we can mathematically arrive at the percentage of total cell uptake that is driven by an ion trapping-based mechanism. For lysosomotropic molecules such as LTR, greater than 76% of its total cell uptake can be attributed to ion trapping. This measure of lysosomotropic potential will be evaluated for all probe analogues. Using these approaches, together with optimization of pK_a and permeability ratios of probe analogues, we expect that next-generation probes will have greatly enhanced intracellular distribution properties and therefore fully optimized anti-malarial activity if the lysosomotropic hypothesis is correct.

List of Reagents

• rPfM18AAP enzyme (supplied by Assay Provider)
• H-Glu-NHMec substrate (Bachem, part I-1180)
• 1536-well plates (Greiner, part 789176)
• Tris (Amresco, part 0497)
• CoCl₂ 6H₂0 (Univar, part D3247)
• ZnCl₂ (Sigma, part 208086)

Protocol Summary

Prior to the start of the assay, 2.5 microliters of assay buffer (50 mM Tris HCl pH7.5, 4 mM CoCl₂, 0.1% BSA) containing 5 micrograms/mL rPfM18AAP were dispensed into a 1536 microtiter plate. Next, 37 nL of test compound in DMSO, ZnCl₂ (2 mM final concentration), or DMSO alone (0.74% final concentration) were added to the appropriate wells. The plates were then incubated for 30 minutes at 25 degrees Celsius. The assay was started by dispensing 2.5 microliters of 100 micromolar H-Glu-NHMec substrate in buffer (50 mM Tris HCl, pH 8.8) into all wells. Well fluorescence was read immediately (T0) on the Viewlux (Perkin-Elmer) and again after 90 minutes (T90) of incubation at 25 degrees Celsius.

Prior to further calculations, T0 was subtracted from T90 for each individual well. The difference between RFU values read at T0 (RFU T0) and T90 (RFU T90), named delta RFU, was calculated as follows:

The percent inhibition for each well was then calculated as follows:

Where:

Test Compound is defined as wells containing test compound. Negative Control is defined as the median of the wells containing test compounds. Positive Control is defined as the median of the wells containing ZnCl₂.

A mathematical algorithm was used to determine nominally inhibiting compounds in the Primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score was normalized to 100% observed primary inhibition. Negative % inhibition values were reported as activity score zero.

The inactive compounds of this assay have activity score range of 0 to 28 and the active compounds have activity score range of 28 to 100.

List of Reagents

• Cathepsin L enzyme (supplied by Assay Provider)
• Z-Leu-Arg-MCA substrate (Peptides International, part MCA-3210-v)
• 1536-well plates (Greiner, part 789176)
• Tris (Amresco, part 0497)
• DTT (Invitrogen, part 15508-013)
• Z-Phe-Ala-diazomethylketone (Bachem, part N-1040)
• BSA (Calbiochem, part 126609)

Protocol Summary

Prior to the start of the assay, 2.5 microliters of assay buffer (25 mM Tris HCl pH7.5, 1 mM DTT, 0.1% BSA) containing 1.5 micrograms/mL cathepsin L were dispensed into a 1536 microtiter plate. Next, 30 nL of test compound in DMSO, Z-Phe-Ala-diazomethylketone (1micromolar final concentration), or DMSO alone (0.59% final concentration) were added to the appropriate wells. The plates were then incubated for 30 minutes at 25 degrees Celsius. The assay was started by dispensing 2.5 microliters of 100 micromolar Z-Leu-Arg-MCA substrate in buffer (25 mM Tris HCl, pH 7.5, 1mM DTT) into all wells. Well fluorescence was read immediately (T0) on the Viewlux (Perkin-Elmer) and again after 90 minutes (T90) of incubation at 25 degrees Celsius.

Prior to further calculations, T0 was subtracted from T90 for each individual well. The difference between RFU values read at T0 (RFU T0) and T90 (RFU T90), named delta RFU, was calculated as follows:

The percent inhibition for each well was then calculated as follows:

Where:

Test Compound is defined as wells containing test compound. Negative Control is defined as the median of the wells containing test compounds. Positive Control is defined as the median of the wells containing Z-Phe-Ala-diazomethylketone.

A mathematical algorithm was used to determine nominally inhibiting compounds in the Primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score was normalized to 100% observed primary inhibition. Negative % inhibition values were reported as activity score zero.

The activity score range for active compounds was 100-14, for inactive 14-0.

List of Reagents

• rPfM18AAP enzyme (supplied by Assay Provider)
• H-Glu-NHMec substrate (Bachem, part I-1180)
• Tris (Amresco, part 0497)
• CoCl₂ 6H₂0 (Univar, part D3247)
• ZnCl₂ (Sigma, part 208086)
• BSA (Calbiochem, part 126609)
• 1536-well plates (Greiner, part 789176)

Protocol Summary

Prior to the start of the assay, 2.5 microliters of assay buffer (50 mM Tris HCl pH7.5, 4 mM CoCl₂, 0.1% BSA) containing 5 micrograms/mL rPfM18AAP were dispensed into a 1536 microtiter plate. Next, 37 nL of test compound in DMSO, ZnCl₂ (2 mM final concentration), or DMSO alone (0.74% final concentration) were added to the appropriate wells. The plates were then incubated for 30 minutes at 25 degrees Celsius. The assay was started by dispensing 2.5 microliters of 100 micromolar H-Glu-NHMec substrate in buffer (50 mM Tris HCl, pH 8.8) into all wells. Well fluorescence was read immediately (T0) on the Viewlux (Perkin-Elmer) and again after 90 minutes (T90) of incubation at 25 degrees Celsius.

Prior to further calculations, T0 was subtracted from T90 for each individual well. The difference between RFU values read at T0 (RFU T0) and T90 (RFU T90), named delta RFU, was calculated as follows:

The percent inhibition for each well was then calculated as follows:

Where:

Test Compound is defined as wells containing test compound. Negative Control is defined as the median of the wells containing test compounds. Positive Control is defined as the median of the wells containing ZnCl₂.

The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the primary screen was declared active.

The reported PubChem Activity Score was normalized to 100% observed primary inhibition. Negative % inhibition values were reported as activity score zero.

The activity score range for active compounds was 100-28, for inactive 28-0.

List of Reagents

• Cathepsin L enzyme (supplied by Assay Provider)
• Z-Leu-Arg-MCA substrate (Peptides International, part MCA-3210-v)
• Tris (Amresco, part 0497)
• DTT (Invitrogen, part 15508-013)
• Z-Phe-Ala-diazomethylketone (Bachem, part N-1040)
• BSA (Calbiochem, part 126609)
• 1536-well plates (Greiner, part 789176)

Protocol Summary

Prior to the start of the assay, 2.5 microliters of assay buffer (25 mM Tris HCl pH7.5, 1 mM DTT, 0.1% BSA) containing 1.5 micrograms/mL cathepsin L were dispensed into a 1536 microtiter plate. Next, 30 nL of test compound in DMSO, Z-Phe-Ala-diazomethylketone (1 micromolar final concentration), or DMSO alone (0.59% final concentration) were added to the appropriate wells. The plates were then incubated for 30 minutes at 25 degrees Celsius. The assay was started by dispensing 2.5 microliters of 100 micromolar Z-Leu-Arg-MCA substrate in buffer (25 mM Tris HCl, pH 7.5, 1mM DTT) into all wells. Well fluorescence was read immediately (T0) on the Viewlux (Perkin-Elmer) and again after 90 minutes (T90) of incubation at 25 degrees Celsius.

Prior to further calculations, T0 was subtracted from T90 for each individual well. The difference between RFU values read at T0 (RFU T0) and T90 (RFU T90), named delta RFU, was calculated as follows:

The percent inhibition for each well was then calculated as follows:

Where:

Test Compound is defined as wells containing test compound. Negative Control is defined as the median of the wells containing test compounds. Positive Control is defined as the median of the wells containing Z-Phe-Ala-diazomethylketone.

The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the primary screen (AID 1906) was declared active.

The reported PubChem Activity Score was normalized to 100% observed primary inhibition. Negative % inhibition values were reported as activity score zero.

The activity score range for active compounds was 100-18, for inactive 13-0.

List of Reagents

• rPfM18AAP enzyme (supplied by Assay Provider)
• H-Glu-NHMec substrate (Bachem, part I-1180)
• 1536-well plates (Greiner, part 789176)
• Tris (Amresco, part 0497)
• CoCl₂ 6H₂0 (Univar, part D3247)
• ZnCl₂ (Sigma, part 208086)
• BSA (Calbiochem, part 126609)

Protocol Summary

Prior to the start of the assay, 2.5 microliters of assay buffer (50 mM Tris HCl pH7.5, 4 mM CoCl₂, 0.1% BSA) containing 5 micrograms/mL rPfM18AAP were dispensed into a 1536 microtiter plate. Next, 37 nL of test compound in DMSO, ZnCl₂ (2 mM final concentration), or DMSO alone (0.74% final concentration) were added to the appropriate wells. The plates were then incubated for 30 minutes at 25 degrees Celsius. The assay was started by dispensing 2.5 microliters of 100 micromolar H-Glu-NHMec substrate in buffer (50 mM Tris HCl, pH 8.8) into all wells. Well fluorescence was read immediately (T0) on the Viewlux (Perkin-Elmer) and again after 90 minutes (T90) of incubation at 25 degrees Celsius.

Prior to further calculations, T0 was subtracted from T90 for each individual well. The difference between RFU values read at T0 (RFU T0) and T90 (RFU T90), named delta RFU, was calculated as follows:

The percent inhibition for each well was then calculated as follows:

Where:

Test Compound is defined as wells containing test compound. Negative Control is defined as the median of the wells containing rPfM18AAP. Positive Control is defined as the median of the wells containing ZnCl₂.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC₅₀ values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 73.5 micromolar) did not result in greater than 50% inhibition, the IC₅₀ was determined manually as greater than 73.5 micromolar. Compounds with an IC₅₀ greater than 10 micromolar were considered inactive. Compounds with an IC₅₀ equal to or less than 10 micromolar were considered active.

Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

The activity score range for active compounds was 100-1. There were no inactive compounds.

List of Reagents

• Cathepsin L enzyme (supplied by Assay Provider)
• Z-Leu-Arg-MCA substrate (Peptides International, part MCA-3210-v)
• 1536-well plates (Greiner, part 789176)
• Tris (Amresco, part 0497)
• DTT (Invitrogen, part 15508-013)
• Z-Phe-Ala-diazomethylketone (Bachem, part N-1040)
• BSA (Calbiochem, part 126609)

Protocol Summary

Prior to the start of the assay, 2.5 microliters of assay buffer (25 mM Tris HCl pH7.5, 1 mM DTT, 0.1% BSA) containing 1.5micrograms/mL cathepsin L were dispensed into a 1536 microtiter plate. Next, 30 nL of test compound in DMSO, Z-Phe-Ala-diazomethylketone (1 micromolar final concentration), or DMSO alone (0.59% final concentration) were added to the appropriate wells. The plates were then incubated for 30 minutes at 25 degrees Celsius. The assay was started by dispensing 2.5 microliters of 100 micromolar Z-Leu-Arg-MCA substrate in buffer (25 mM Tris HCl, pH 7.5, 1mM DTT) into all wells. Well fluorescence was read immediately (T0) on the Viewlux (Perkin-Elmer) and again after 90 minutes (T90) of incubation at 25 degrees Celsius.

Prior to further calculations, T0 was subtracted from T90 for each individual well. The difference between RFU values read at T0 (RFU T0) and T90 (RFU T90), named delta RFU, was calculated as follows:

The percent inhibition for each well was then calculated as follows:

Where:

Test Compound is defined as wells containing test compound. Negative Control is defined as the median of the wells containing Cathepsin L. Positive Control is defined as the median of the wells containing Z-Phe-Ala-diazomethylketone.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC₅₀ values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 59.6 micromolar) did not result in greater than 50% inhibition, the IC₅₀ was determined manually as greater than 59.6 micromolar. Compounds with an IC₅₀ greater than 10 micromolar were considered inactive. Compounds with an IC₅₀ equal to or less than 10 micromolar were considered active.

Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

There are no active compounds in this assay.

List of Reagents

• rPfM18AAP (Assay Provider)
• H-Glu-NHMec (Bachem Chemical Co., catalog I-1180)
• Tris buffer (Supplier name, BioShop Canada, catalog TRS001-1)
• 96-well plates (Costar Incoporated USA, catalog 3904)

Protocol Summary

Prior to the start of the assay, 5 uL of rPfM18AAP were dispensed into 96-well black non-binding surface plates. This was followed by 90 uL of assay buffer (25 mM Tris HCl, pH 7.5) and then 5 uL of compound. After 10 minutes, 100 uL of 100 uM fluorogenic peptide substrate (H-Glu-NHMec) in assay buffer was added to each well. The final concentrations in the reaction were 50 uM H-Glu-NHMec, 1 ug/ml rPfM18AAP, 25 mM Tris-HCl (pH 7.5), and 5 uM compound. The test plate was immediately transferred to a Synergy HT fluorimeter with microplate reader and fluorescence was measured at an excitation wavelength of 370 nm and an emission wavelength of 460 nm at 60-second intervals for 30 minutes (30 readings). Each plate had 4 control wells in the eight outside columns with 2 wells containing the complete reaction mixture with carrier control (positive control) and 2 wells in which the rPfM18AAP had been left out (background).

The % inhibition for each well was then calculated as follows:

Where:

Test = RFU in wells with rPfM18AAP treated with test compound. Background Control = mean count of wells containing substrate in assay buffer only. Positive Control = mean count of wells containing rPfM18AAP with substrate but no compound added.

PubChem Activity Outcome and Score:

Compounds resulting in greater than 50% inhibition of rPfM18AAP were considered active.

The reported PubChem Activity Score was normalized to 100% observed inhibition. Negative % inhibition values were reported as activity score zero.

The PubChem Activity Score range for active compounds was 100-55, and for inactive compounds 54-0.

List of Reagents

• Soluble malaria cell extract (Assay Provider)
• H-Glu-NHMec (Bachem Chemical Co., catalog I-1180)
• Tris buffer (Supplier name, BioShop Canada, catalog TRS001-1)
• 96-well plates (Costar Incoporated USA, catalog 3904)

Protocol Summary

Prior to the start of the assay, 5 uL of malaria cell lysate were dispensed into 96-well black non-binding surface plates. This was followed by 90 uL of assay buffer (25 mM Tris HCl, pH 7.5) and then 5 uL of compound. After 10 minutes, 100 uL of 100 uM fluorogenic peptide substrate (H-Glu-NHMec) in assay buffer was added to each well. The final concentrations in the reaction were 50 uM H-Glu-NHMec, 10 ug/ml malaria cell lysate, 25 mM Tris-HCl (pH 7.5), and 5 uM compound. The test plate was immediately transferred to a Synergy HT fluorimeter with microplate reader and fluorescence was measured at an excitation wavelength of 370 nm and an emission wavelength of 460 nm at 60-second intervals for 30 minutes (30 readings). Each plate had 4 control wells in the eight outside columns with 2 wells containing the complete reaction mixture with carrier control (positive control) and 2 wells in which the malaria lysate had been left out (background).

The % inhibition for each well was then calculated as follows:

Where:

Test = RFU in wells with malaria lysate treated with test compound. Background Control = mean count of wells containing substrate in assay buffer only. Positive Control = mean count of wells containing malaria lysate with substrate but no compound added.

PubChem Activity Outcome and Score:

Compounds with greater than 50% inhibition of PfM18AAP in malaria cell lysate were considered active.

The reported PubChem Activity Score was normalized to 100% observed inhibition. Negative % inhibition values were reported as activity score zero.

The PubChem Activity Score range for active compounds was 100-53, and for inactive compounds 49-0.

List of Reagents

• Plasmodium falciparum clone 3D7
• Hypoxanthine Mono-Hydrochloride, [3H(G)]-(Perkin Elmer code Net177)
• Plate Microtiter 96 well flat bottom Sterile (Costar, catalog 3595)
• Special Gas Mix (5% CO₂ 5% O₂ 90% N2) (BOC Gas)
• Culture media (85% RPMI, 4% Sodium Bicarbonate, 10% human sera) (GIBCO, catalog 31800-089)

Protocol Summary

Prior to the start of the assay, 200 uL of PBS was added to the outside wells of a 96 well flat-bottomed microtiter plate. 100 uL hypoxanthine-free RPMI 1640 media plus 10% serum was added to all other wells except those that contained diluted compound or vehicle control. An appropriate volume of compound (10 uM) or vehicle control (DMSO <= 1%) in hypoxanthine-free RPMI 1640 media plus 10% serum was added to remaining wells. A parasite suspension (1% parasitemia, 2% hematocrit) was prepared in hypoxanthine-free RPMI media containing 10% serum and added to all test wells (except RBC controls) before the addition of 0.5 uCi/well of 3H-hypoxanthine (10 uL). Uninfected RBC controls (in triplicate) were included on each assay plate. Plates were incubated at 37 °C for 48 hours in an atmosphere of 90% N2, 5% CO₂, and 5% O₂. A cell harvester and Beta counter were used to determine the amount of hypoxanthine incorporated for each well. Three replicates at 10 uM were performed for each compound tested in each experiment, and each compound was tested in two or three experiments.

The % inhibition for each well was then calculated as follows:

Where:

Test = count (3H-hypoxanthine incorporation) by parasites treated with test compound. Background Control = mean count of uninfected RBC control tests. Positive Control = mean count (3H-hypoxanthine incorporation) by untreated parasites exposed to vehicle only.

PubChem Activity Outcome and Score:

Compounds achieving >= 50% growth inhibition for any replicate were considered active; compounds achieving < 50% growth inhibition in all replicates were considered inactive.

List of Reagents

• Plasmodium falciparum clone 3D7
• Hypoxanthine Mono-Hydrochloride, [3H(G)]-(Perkin Elmer code Net177)
• Plate Microtiter 96 well flat bottom Sterile (Costar, catalog 3595)
• Special Gas Mix (5% CO₂; 5% O₂; 90% N2) (BOC Gas)
• Culture media (85% RPMI, 4% Sodium Bicarbonate, 10% human sera) (GIBCO, catalog 31800-089)

Protocol Summary

Prior to the start of the assay, 200 uL of PBS was added to the outside wells of a 96 well flat-bottomed microtiter plate. 100 uL hypoxanthine-free RPMI 1640 media plus 10% serum was added to all other wells except those that contained diluted compound or vehicle control. An appropriate volume of compound (10 uM) or vehicle control (DMSO <= 1%) in hypoxanthine-free RPMI 1640 media plus 10% serum was added to remaining wells. A parasite suspension (1% parasitemia, 2% hematocrit) was prepared in hypoxanthine-free RPMI media containing 10% serum and added to all test wells (except RBC controls) before the addition of 0.5 uCi/well of 3H-hypoxanthine (10 uL). Uninfected RBC controls (in triplicate) were included on each assay plate. Plates were incubated at 37 C for 48 hours in an atmosphere of 90% N2, 5% CO₂, and 5% O₂. A cell harvester and Beta counter were used to determine the amount of hypoxanthine incorporated for each well.

The % inhibition for each well was calculated as follows:

Where:

Test = count (3H-hypoxanthine incorporation) by parasites treated with test compound. Background Control = mean count of uninfected RBC control tests. Positive Control = mean count (3H-hypoxanthine incorporation) by untreated parasites exposed to vehicle only

For each test compound, percent inhibition was plotted against compound concentration. The reported IC₅₀ value was generated from a fitted curve by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value.

PubChem Activity Outcome and Score:

Compounds with an IC₅₀ greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.

Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds was 100-43, and for inactive compounds 1-0.

List of Reagents

• rPfM18AAP enzyme (supplied by Assay Provider)
• H-Glu-NHMec substrate (Bachem, part I-1180)
• Tris (Amresco, part 0497)
• CoCl₂ 6H₂0 (Univar, part D3247)
• BSA (Calbiochem, part 126609)

Protocol Summary

2.5 uL of rPfM18AAP reagent mix, which included the fluorogenic peptide substrate (H-Glu-NHMec) in assay buffer, was added to each well of the previously compound dosed 1536-well plates. The reaction was initiated with the addition of 2.5 uL of the rPfM18AAP diluted in assay buffer. The final concentrations in the reaction were 0.1 mM H-Glu-NHMec and 5 ug/ml rPfM18AAP diluted in assay buffer (50 mM Tris-HCl (pH 7.5), 2 mM CoCl₂, 0.1% BSA, 0.01% Triton X-100, and 2% DMSO). The test plate was incubated at room temperature for 90 minutes, then transferred to a Perkin Elmer Envision microplate reader and fluorescence (RFU) was measured at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. Each plate had 256 control wells in the eight outside columns with 128 wells containing the complete reaction mixture with carrier control (Full Rxn) and 128 wells in which the rPfM18AAP had been left out (Bkg).

128 background control wells containing the peptide substrate only and 128 full reaction control wells containing peptide substrate and 5 ug/ml rPfM18AAP were included on each assay plate and used to calculate a Z' value for each plate and to normalize the data on a per plate basis. Data were analyzed using the IDBS Activity Base software. Results for each concentration were expressed as percent inhibition (% Inhibition) and was calculated as: 100*((Med Full Rxn RFU- Med Bkg RFU) - (Cmpd RFU - Med Bkg RFU))/ ((Med Full Rxn RFU - Med Bkg RFU)). The dose response data was analyzed using a four parameter logistic fit to the data (Excel Fit equation 205) with the maximum and minimum locked at 100 and 0. From these curves IC₅₀ values were calculated.

Compounds showing 30% or greater inhibition at any concentration were considered “Active”. IC₅₀ values were calculated for these compounds and used to determine the relative score. The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score: Compounds in the primary screen are scored on a scale of 0-40 based on % activity; a score of 40 corresponds to 100% activity. In the confirmatory dose response screen of primary screen hits, active compounds are scored on a scale of 41-80 based on IC50 result while compounds where activity was not confirmed are given the score 0. Confirmatory dose response and secondary screens of purified and/or resynthesized compounds, indicating the highest degree of confidence) are scored on a scale of 81-100 based on IC50 result. Inactive compounds are given the score 0.

List of Reagents

• rPfM1MAA enzyme (supplied by Assay Provider)
• H-Glu-NHMec substrate (Bachem, part I-1180)
• Tris (Amresco, part 0497)
• CoCl₂ 6H₂0 (Univar, part D3247)
• BSA (Calbiochem, part 126609)

Protocol Summary

2.5 uL of rPfM1MAA reagent mix, which included the fluorogenic peptide substrate (H-Leu-NHMec) in assay buffer, was added to each well of the previously compound dosed 1536-well plates. The reaction was initiated with the addition of 2.5 uL of the rPfM1MAA diluted in assay buffer. The final concentrations in the reaction were 0.1 mM H-Leu-NHMec and 5 ug/ml rPfM1MAA diluted in assay buffer (50 mM Tris-HCl (pH 7.5), 2 mM CoCl₂, 0.1% BSA, 0.01% Triton X-100, and 2% DMSO). The test plate was incubated at room temperature for 90 minutes, then transferred to a Perkin Elmer Envision microplate reader and fluorescence (RFU) was measured at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. Each plate had 256 control wells in the eight outside columns with 128 wells containing the complete reaction mixture with carrier control (Full Rxn) and 128 wells in which the rPfM1MAA had been left out (Bkg).

128 background control wells containing the peptide substrate only and 128 full reaction control wells containing peptide substrate and 5 ug/ml rPfM1MAA were included on each assay plate and used to calculate a Z' value for each plate and to normalize the data on a per plate basis. Data were analyzed using the IDBS Activity Base software. Results for each concentration were expressed as percent inhibition (% Inhibition) and was calculated as: 100*((Med Full Rxn RFU- Med Bkg RFU) - (Cmpd RFU - Med Bkg RFU))/ ((Med Full Rxn RFU - Med Bkg RFU)). The dose response data was analyzed using a four parameter logistic fit to the data (Excel Fit equation 205) with the maximum and minimum locked at 100 and 0. From these curves IC₅₀ values were calculated.

Compounds showing 30% or greater inhibition at any concentration were considered “Active”. IC₅₀ values were calculated for these compounds and used to determine the relative score. The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score: Compounds in the primary screen are scored on a scale of 0-40 based on % activity; a score of 40 corresponds to 100% activity. In the confirmatory dose response screen of primary screen hits, active compounds are scored on a scale of 41-80 based on IC₅₀ result while compounds where activity was not confirmed are given the score 0. Confirmatory dose response and secondary screens of purified and/or resynthesized compounds, indicating the highest degree of confidence) are scored on a scale of 81-100 based on IC₅₀ result. Inactive compounds are given the score 0.

List of Reagents

• rPfM17LAP enzyme (supplied by Assay Provider)
• H-Glu-NHMec substrate (Bachem, part I-1180)
• Tris (Amresco, part 0497)
• CoCl₂ 6H₂0 (Univar, part D3247)
• BSA (Calbiochem, part 126609)

Protocol Summary

2.5 uL of rPfM17LAP reagent mix, which included the fluorogenic peptide substrate (H-Leu-NHMec) in assay buffer, was added to each well of the previously compound dosed 1536-well plates. The reaction was initiated with the addition of 2.5 uL of the rPfM17LAP diluted in assay buffer. The final concentrations in the reaction were 0.1 mM H-Leu-NHMec and 5 ug/ml rPfM17LAP diluted in assay buffer (50 mM Tris-HCl (pH 7.5), 2 mM CoCl₂, 0.1% BSA, 0.01% Triton X-100, and 2% DMSO). The test plate was incubated at room temperature for 90 minutes, then transferred to a Perkin Elmer Envision microplate reader and fluorescence (RFU) was measured at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. Each plate had 256 control wells in the eight outside columns with 128 wells containing the complete reaction mixture with carrier control (Full Rxn) and 128 wells in which the rPfM17LAP had been left out (Bkg).

128 background control wells containing the peptide substrate only and 128 full reaction control wells containing peptide substrate and 5 ug/ml rPfM17LAP were included on each assay plate and used to calculate a Z' value for each plate and to normalize the data on a per plate basis. Data were analyzed using the IDBS Activity Base software. Results for each concentration were expressed as percent inhibition (% Inhibition) and was calculated as: 100*((Med Full Rxn RFU- Med Bkg RFU) - (Cmpd RFU - Med Bkg RFU))/ ((Med Full Rxn RFU - Med Bkg RFU)). The dose response data was analyzed using a four parameter logistic fit to the data (Excel Fit equation 205) with the maximum and minimum locked at 100 and 0. From these curves IC₅₀ values were calculated.

Compounds showing 30% or greater inhibition at any concentration were considered “Active”. IC₅₀ values were calculated for these compounds and used to determine the relative score. The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score: Compounds in the primary screen are scored on a scale of 0-40 based on % activity; a score of 40 corresponds to 100% activity. In the confirmatory dose response screen of primary screen hits, active compounds are scored on a scale of 41-80 based on IC₅₀ result while compounds where activity was not confirmed are given the score 0. Confirmatory dose response and secondary screens of purified and/or resynthesized compounds, indicating the highest degree of confidence) are scored on a scale of 81-100 based on IC₅₀ result. Inactive compounds are given the score 0.

List of Reagents

• rhM18 enzyme (supplied by Assay Provider)
• H-Glu-NHMec substrate (Bachem, part I-1180)
• Tris (Amresco, part 0497)
• CoCl₂ 6H₂0 (Univar, part D3247)
• BSA (Calbiochem, part 126609)

Protocol Summary

2.5 uL of rhM18 reagent mix, which included the fluorogenic peptide substrate (H-Glu-NHMec) in assay buffer, was added to each well of the previously compound dosed 1536-well plates. The reaction was initiated with the addition of 2.5 uL of the rhM18 diluted in assay buffer. The final concentrations in the reaction were 0.1 mM H-Glu-NHMec and 5 ug/ml rhM18 diluted in assay buffer (50 mM Tris-HCl (pH 7.5), 2 mM CoCl₂, 0.1% BSA, 0.01% Triton X-100, and 2% DMSO). The test plate was incubated at room temperature for 90 minutes, then transferred to a Perkin Elmer Envision microplate reader and fluorescence (RFU) was measured at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. Each plate had 256 control wells in the eight outside columns with 128 wells containing the complete reaction mixture with carrier control (Full Rxn) and 128 wells in which the rhM18 had been left out (Bkg).

128 background control wells containing the peptide substrate only and 128 full reaction control wells containing peptide substrate and 5 ug/ml rhM18AAP were included on each assay plate and used to calculate a Z' value for each plate and to normalize the data on a per plate basis. Data were analyzed using the IDBS Activity Base software. Results for each concentration were expressed as percent inhibition (% Inhibition) and was calculated as: 100*((Med Full Rxn RFU- Med Bkg RFU) - (Cmpd RFU - Med Bkg RFU))/ ((Med Full Rxn RFU - Med Bkg RFU)). The dose response data was analyzed using a four parameter logistic fit to the data (Excel Fit equation 205) with the maximum and minimum locked at 100 and 0. From these curves IC₅₀ values were calculated.

Compounds showing 30% or greater inhibition at any concentration were considered “Active”. IC₅₀ values were calculated for these compounds and used to determine the relative score. The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score: Compounds in the primary screen are scored on a scale of 0-40 based on % activity; a score of 40 corresponds to 100% activity. In the confirmatory dose response screen of primary screen hits, active compounds are scored on a scale of 41-80 based on IC₅₀ result while compounds where activity was not confirmed are given the score 0. Confirmatory dose response and secondary screens of purified and/or resynthesized compounds, indicating the highest degree of confidence) are scored on a scale of 81-100 based on IC₅₀ result. Inactive compounds are given the score 0.

Protocol Summary

Cell Culture: Vero E6 cells were subcultured every 7 days in E-MEM with 10% fetal bovine serum and 2 mM glutamine (complete growth medium), incubated at 37 degrees C in 5% carbon dioxide, and maintained for no more than 20 passages.

Compound Dosing/Plating: Carrier control / compounds were diluted in complete growth medium to prepare a 6X concentrated dosing solution which was dispensed into 384-well black clear-bottom tissue culture treated plates (5 uL volume).

Cell Plating: Twenty uL of complete growth medium containing 3000 cells were dispensed per well. Plates were incubated at 37 C, 5% CO₂ for 72h prior to endpoint detection.

Endpoint/Detection: At the end of the treatment period, assay plates were removed from the incubator and equilibrated to room temperature for 10 min. Twenty-five uL of Cell Titer Glo reagent was added and plates were incubated for an additional 10 min in the dark. At the end of the incubation, assay plates were analyzed using a PerkinElmer Envision microplate reader in luminescence mode with an integration time of 0.1 s.

Data Analysis: Sixty-four control wells containing cells treated with DMSO vehicle and were included on each assay plate. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100*(Cmpd Lum-Med background)/(Med Cell Ctrl - Med background). The normalized % viability was plotted against the tested concentrations. The CC₅₀ values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0, respectively and allowing extrapolation to identify weakly active compounds.

Compounds that showed <80% cell viability for at least one concentration were defined as “Active” (toxic). If the % viability at all doses was <80%, the compound was defined as “Inactive” (non-toxic). Instances where replicate data sets conflicted using these criteria are listed as “Inconclusive”. The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score: Compounds in the primary screen are scored on a scale of 0-40 based on % activity; a score of 40 corresponds to 100% activity. In the confirmatory dose response screen of primary screen hits, active compounds are scored on a scale of 41-80 based on CC50 result while compounds where activity was not confirmed are given the score 0. Confirmatory dose response and secondary screens of purified and/or resynthesized compounds, indicating the highest degree of confidence) are scored on a scale of 81-100 based on CC50 result. Inactive compounds are given the score 0