Discovery of Novel Cyclophilin A Ligands Using an H/D Exchange– and Mass Spectrometry

Abstract

Cyclophilin A (CypA) is an overexpressed protein in lung cancer tumors and as a result is a potential therapeutic and diagnostic target. Described here is use of an H/D exchange– and a matrix assisted laser desorption/ionization (MALDI) mass spectrometry–based assay, termed single-point SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange), to screen 2 chemical libraries, including the 1280-compound LOPAC library and the 9600-compound DIVERSet library, for binding to CypA. This work represents the first application of single-point SUPREX using a pooled ligand approach, which is demonstrated here to yield screening rates as fast as 6 s/ligand. The false-positive and false-negative rates determined in the current work using a set of control samples were 0% and 9%, respectively. A false-positive rate of 20% was found in screening the actual libraries. Eight novel ligands to CypA were discovered, including 2-(α-naphthoyl)ethyltrimethyl-ammonium iodide, (E)-3-(4-t-Butylphenylsulfonyl)-2-propenenitrile, 3-(N-benzyl-N-isopropyl)amino-1-(naphthalen-2-yl)propan-1-one, cis-diammineplatinum (II) chloride, 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione, N-(3-chloro-1, 4-dioxo-1,4-dihydro-2-naphthalenyl)-N-cyclohexylacetamide, 1-[2-(3,4-dimethoxyphenyl)ethyl]-1H-pyrrole-2,5-dione, and 4-(2-methoxy-4-nitrophenyl)-1-methyl-10-oxa-4-azatricyclo[5.2.1.0~2,6~]dec-8-ene-3,5-dione. These compounds, which had moderate binding affinities to CypA (i.e., K_d values in the low micromolar range), provide new molecular scaffolds that might be useful in the development of CypA-targeted diagnostic imaging or therapeutic agents for lung cancer.

Keywords

cyclophilin A matrix-assisted laser desorption/ionization amide H/D exchange high-throughput screening

Introduction

The ready availability of natural and synthetic compound libraries of peptides, oligonucleotides, and small molecules (molecular weight [MW] <1000 amu) has created a new route to the discovery of novel ligands that bind to specific protein targets. This route, which typically involves the use of high-throughput screening (HTS) strategies to rapidly screen library members for protein-ligand binding, can be useful for the identification of new protein-targeted diagnostic and/or therapeutic agents. For example, the protein target in this work, CypA, is a peptidyl-prolyl cis-trans isomerase that has been shown to be overexpressed in lung cancer tumor cells^1,2 and as such is an attractive target for potential diagnostic imaging probes. It also appears to play an important role in lung tumor growth³ and thus could be an important therapeutic target as well.

A goal of this work was to identify novel CypA ligands that might be useful in the development of new CypA-targeted molecular imaging agents for the diagnosis of lung cancer. To this end, small-molecule compounds in 2 chemical libraries were screened for CypA binding. One library was the Library of Pharmacologically Active Compounds, or the LOPAC library, which consists of 1280 pharmacologically active compounds that span a range of cell signaling and neuroscience areas and that include a number of already marketed drugs. The other library was the 9600-member DIVERSet library, which consists of a highly diverse collection of drug-like small molecules selected through the use of a 3D pharmacophore analysis method to ensure broad coverage among the various types of interactions between target and ligand.

The HTS assay used in this work relies on an H/D exchange– and mass spectrometry–based methodology, termed single-point SUPREX, to detect the increase in a protein’s thermodynamic stability upon ligand binding. We initially developed the single-point SUPREX assay in the context of a model protein-peptide system,⁴ and the assay was used to screen the 880 compounds in the Prestwick Chemical Library for binding to CypA.⁵ Although no new CypA ligands were identified in the Prestwick Chemical Library screening, that work did establish a 3-min/ligand throughput of the single-point SUPREX protocol. As part of the current work, we describe a compound-pooling strategy that increases the screening rate of the single-point SUPREX protocol to less than 10 s/ligand. Our results demonstrate the feasibility of the new methodology for screening 1000- to 10,000-member libraries and identify 8 novel ligands to CypA.

Methods

Materials

The human CypA used in this work was obtained from T7 Express Escherichia coli transformed with pGEX-CypA, a plasmid encoding the glutathione S-transferase (GST) fusion protein of CypA. Briefly, the E. coli was grown in LB-Amp media (sodium chloride, tryptone, yeast extract, and ampicillin) at 37°C. Isopropyl β-D-1-thiogalactopyranoside (Mediatech, Inc., Mannassas, VA) was added to a final concentration of 0.125 mM to induce overexpression when the OD₆₀₀ of the culture reached 0.6 to 0.8 units. After 3.5 h, the E. coli was pelleted by centrifuging at 4000 g for 10 min. Cells were lysed with the addition of BugBuster reagent (EMD Chemicals, Inc., Gibbstown, NJ), and the insoluble material was pelleted at 17,000 g for 20 min. CypA was purified from the supernatant using GST·Bind Resin (EMD Chemicals, Inc.), and it was concentrated with a Vivaspin 20 centrifugal concentrator (Sartorius, Bohemia, NY). Thrombin (EMD Chemicals, Inc.) was added to remove the GST tag. The GST tag was subsequently removed via the GST·Bind Resin, and the thrombin was removed with a HiTrap Benzamidine FF column (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The CypA was further concentrated to about 100 µM. All CypA concentrations were determined using absorbance measurements at 280 nm and an extinction coefficient of 8730 M⁻¹ cm⁻¹ as calculated through ProtParam using the protein’s amino acid sequence (http://ca.expasy.org/tools/protparam.html).

The LOPAC library (Sigma-Aldrich, St. Louis, MO) was obtained from the Small Molecule Synthesis and Screening Facility in the Center for Chemical Biology at Duke University. The library compounds were provided as 10-mM solutions in DMSO and were pipetted into 128 ten-member pools for a resulting concentration of 1 mM of each compound. The 9600-compound DIVERSet library was from ChemBridge Corp. (San Diego, CA) and was provided as 10-mM solutions in DMSO. The DIVERSet library compounds were combined into 960 ten-member pools for a resulting concentration of 1 mM of each compound. 3-[5-(4-Fluorobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]propanoic acid, N-(3-chloro-1,4-dioxo-1,4-dihydro-2-naphthalenyl)-N-cyclohexylacetamide, 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione, 4-(2-methoxy- 4-nitrophenyl)-1-methyl-10-oxa-4-azatricyclo[5.2.1.0~2,6~]dec-8-ene-3,5-dione, and 1-[2-(3,4-dimethoxyphenyl)ethyl]-1H- pyrrole-2,5-dione were from ChemBridge Corp. Cyclosporin A (CsA) was purchased from LKT Laboratories (St. Paul, MN). Deuterium oxide (D₂O; 99.9% atom D), sodium deuteroxide (40 wt% in D₂O, 99.9% atom D), sinapic acid (SA; 98% titration), and cis-diammineplatinum (II) chloride (cisplatin) were from Sigma-Aldrich. (E)-3-(4-t-butylphenylsulfonyl) -2-propenenitrile (Bay 11-7085) and 2-(α-naphthoyl)ethyltrimethylammonium iodide (α-NETA) were purchased from Alexis Biochemicals (Lausen, Switzerland). 3-(N-Benzyl-N-isopropyl)amino-1-(naphthalen-2-yl)propan-1-one hydrochloride (ZM 39923) was from Tocris Bioscience (Ellisville, MO). DMSO was from Acros Organics (Geel, Belgium), and phosphoric acid-d₃ was from Cambridge Isotope Laboratories, Inc. (Andover, MA). Guanidinium chloride (GdmCl) was purchased from EMD Chemicals, Inc., and trifluoroacetic acid (TFA) was from Halocarbon (River Edge, NJ). Acetonitrile (ACN) was purchased from Fisher (Fair Lawn, NJ). The chymotrypsin, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and trifluoroethanol were all from Sigma-Aldrich.

SUPREX buffer preparation

A deuterated solution of 20 mM sodium phosphate buffer, pD 7.4, containing 1.5 M GdmCl was used for the single-point SUPREX experiments. A series of solutions containing 20 mM phosphate buffer, pD 7.4, containing GdmCl concentrations ranging from 0 to 4 M was prepared for the full SUPREX analyses performed on CypA in the presence of the hit compounds. The GdmCl used for buffer preparation was previously deuterated by repeated dissolution and lyophilization (4 times total) of 19 g GdmCl in 25 mL D₂O. The pD of the buffers was adjusted by adding sodium deuteroxide and monitoring the pH using a Jenco microcomputer pH-Vision 6072 pH meter equipped with a Futura calomel pH electrode from Beckman (Fullerton, CA). The pD of the buffers was obtained by adding 0.4 to the pH reading to correct for isotope effects.⁶ The GdmCl concentration in the buffers was measured using a refractometer (Bausch & Lomb, Rochester, NY) using the method reported by Nozaki.⁷

Mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectra were acquired on an Ultraflex II TOF/TOF from Bruker Daltonics (Billerica, MA). Spectra were the sum of 100 laser shots collected in the linear mode using a 100-Hz Nd:YAG laser. Positive ion mass spectra were manually collected using the following parameters: an ion source 1 voltage of 25 kV, an ion source 2 voltage of 23.4 kV, a lens voltage of 6.5 kV, and a delay time of 100 ns. The MALDI matrix was SA, and it was prepared as a saturated solution containing 25% water and 75% ACN with 0.1% TFA.

Library screening (single-point SUPREX protocol)

For the screening assay, 1 µL of each 10-member pool containing 1 mM of each library compound in DMSO was combined with 9 µL of the deuterated exchange buffer containing 1.5 M GdmCl. A 1-µL aliquot of protonated CypA solution (i.e., CypA dissolved in H₂O-containing buffer), which was ~100 µM in CypA, was added to the resulting 10-µL volume of the D₂O-containing compound/exchange buffer, and the H/D exchange reaction proceeded for 35 min before it was quenched by transferring 1 µL of the exchange reaction to 9 µL of ice-cold matrix solution. A 1- to 2-µL aliquot of the quenched reaction mixture was spotted onto a MALDI sample target and dried with N₂. Five replicate MALDI mass spectra were collected to determine an average molecular mass of the deuterated CypA for each sample, and this was used to calculate an average ΔMass value. Average ΔMass values were obtained by subtracting the expected average molecular mass of the fully protonated CypA from the average molecular mass measured for each sample. Samples were prepared and analyzed in sets of 12 using a 12-channel pipette. Each set of 12 samples included 10 pools of 10 compounds, 1 positive control containing 1 mM CsA (K_d ~30-200 nM^8-12), and 1 negative control containing only DMSO. The H/D exchange reactions were staggered at 10-min intervals, so while 1 sample set was undergoing H/D exchange, MALDI mass spectra were being collected on the previous sample set.

The average molecular weight of CypA was determined from the MALDI–time of flight (TOF) mass spectra using either an Excel macro or MATLAB (The MathWorks, Inc., Natick, MA) script, both of which were developed in-house. Each takes the spectral data in text file form containing m/z and intensity values for each data point and performs a 19-point floating average smoothing of the data, a calibration using the internal standard, and a center of mass calculation for the CypA peak.

In the LOPAC library screening, the ΔMass values of the control data were averaged, and the cutoff value was determined by subtracting 2.5 standard deviations from the average negative control ΔMass value. In the DIVERSet library screening, the ΔMass values of the control data from the screen were smoothed using a 9-point central moving average. An average ΔMass value (i.e., a ΔMass_av value) for each negative control was calculated using the 9-point moving average and was subsequently used to calculate a cutoff value for each set of 10 pools. The average of all the 9-point standard deviations was calculated, multiplied by 2.5, and then subtracted from the ΔMass_av value to determine the cutoff values. Samples with ΔMass value less than or equal to the cutoff value were classified as preliminary hits (i.e., one of the compounds in the pool of 10 binds to CypA).

Z′ factors were calculated using equation (1).¹³

Z^{'} = 1 - \frac{(3 σ_{c +} + 3 σ_{c -})}{| µ_{c +} - µ_{c -} |} .

(1)

In equation (1), σ_c+ is the standard deviation of the positive control, σ_c− is the standard deviation of the negative control, µ_c+ is the mean of the positive control, and µ_c− is the mean of the negative control.

The preliminary pools classified as hits were subjected to a “second-tier” analysis, in which the initial hit pools were rescreened using the single-point SUPREX protocol described above. We have previously shown that such a 2-tier screening approach reduces the occurrence of false positives in single-point SUPREX.⁵ Each hit pool was rescreened under the same conditions as the initial screen. The individual compounds from the pools that were confirmed as hit pools in the second-tier screen were individually screened using the single-point SUPREX protocol, just as described above for the compound pools, except that the ligand stock solution only contained a single ligand at 1 mM.

Binding affinity measurements (full SUPREX analyses)

Full SUPREX analyses were performed on CypA in the absence and in the presence of the individual compounds selected as hits. In these full SUPREX analyses, 1 µL of a 1-mM DMSO solution of the hit ligand (or just DMSO in the case of the CypA analyses performed in the absence of ligand) was added to 9 µL of the SUPREX buffers, which in the full SUPREX analyses contained [GdmCl] concentrations ranging from 0 to 4 M (see above). A 1-µL aliquot of protonated CypA solution, which was ~100 µM in CypA, was added to each 10-µL volume of compound/exchange buffer, and the H/D exchange reaction proceeded for 35 min before it was quenched by transferring 1 µL of the exchange reaction to 9 µL of ice-cold matrix solution. A 1- to 2-µL aliquot of the quenched reaction mixture was spotted onto a MALDI sample target and dried with N₂. Ten replicate MALDI mass spectra were collected to determine an average molecular mass of the deuterated CypA for each sample, and this was used to calculate an average ΔMass value at each denaturant concentration for each ligand.

Plots of ΔMass versus [GdmCl] were constructed for the CypA in the presence and absence of the hit compounds. The data in the plots were fit to the following 4-parameter sigmoidal equation using a nonlinear regression in SigmaPlot (Systat Software, Inc., San Jose, CA).

Δ Mass = Δ M_{0} + \frac{a}{1 + e^{- (([GdmCl] - C_{SUPREX}^{1 / 2}) / b)}} .

(2)

In equation (2), ΔM₀ is the pretransition baseline (i.e., the change in mass of CypA before global exchange), a is the amplitude of the curve in Da, [GdmCl] is the molar concentration of GdmCl, is the [GdmCl] at the transition midpoint of the curve, and b is a parameter describing the steepness of the transition. These parameters were typically allowed to float when fitting the data to equation (2).

The values obtained from equation (2) were used in equation (3) to determine the magnitude of the increase in thermodynamic stability (i.e., decrease in folding free energy), assuming the m-value, which is a measure of a protein’s folding cooperability, is constant in the presence and absence of ligand.

Δ Δ G_{f} = - m \times Δ C_{SUPREX}^{1 / 2} .

(3)

In equation (3), ΔΔG_f is the change in the protein’s folding free energy (i.e., the protein’s free energy of binding), m is the m-value, and $Δ C_{S U P R E X}^{1 / 2}$ is the change in the $Δ C_{S U P R E X}^{1 / 2}$ value of the CypA upon binding to a ligand. Equation (3) was derived from the general form of the SUPREX equation found in Powell et al.¹⁴ The m-value was assigned a value of 3.7 kcal mol⁻¹ M⁻¹ based on an average of the values reported for CypA in Wang et al.¹²

The following equation was used to calculate dissociation constants, K_d, for the protein-ligand complexes.¹⁵

K_{d} = \frac{[L]}{(e^{- Δ Δ G_{f} / nRT} - 1)} .

(4)

In equation (4), [L] is the molar concentration of the free compound, n is the number of independent binding sites (equal to 1), R is the universal gas constant, and T is the temperature in K. Because the ligand concentration was present at 10-fold excess over the CypA, the total ligand concentration was used for the free ligand concentration.

Theoretical SUPREX curve construction

The theoretical SURPEX curves generated in this work ( Fig. 1 ) were generated exactly as described in Hopper et al.,⁵ with the exception that the pre- and posttransition baselines were set to 45 and 65 Da, respectively, to more accurately represent the pre- and posttransition baselines observed in the current work.

Fig. 1.

(A) Theoretical SUPREX curves generated for CypA in the presence and absence of a hypothetical CypA binding ligand (K_d value = 10 µM). The arrow indicates the [GdmCl] used in the single-point SUPREX experiments in this work. (B) Representative matrix-assisted laser desorption/ionization (MALDI) mass spectra of CypA from the screening assay. The top spectrum shows the CypA ion signal generated in the MALDI readout of a selected pool of LOPAC compounds that did not contain a hit, whereas the bottom spectrum shows the CypA ion signal generated in the MALDI readout of the pool containing compound 3, which was identified as a hit.

CypA inhibition studies

The ability of the hit compounds to inhibit the peptidyl-prolyl cis-trans isomerase activity of CypA was determined using a chymotrypsin-coupled enzyme assay that has been previously described.^16,17 In these assays, 875 µL of ice-cold buffer (50 mM HEPES, 100 mM NaCl, 1% DMSO, pH 8.0) containing 5 nM CypA and 10 µM of the hit compound were combined with 100 µL of an ice-cold chymotrypsin solution (30 mg/mL in 0.001 HCl) in a quartz cuvette. A 25-µL aliquot of a stock solution of the peptide substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, which was prepared in trifluoroethanol at a concentration of 3 mM, was added to the cuvette. The contents of the cuvette were rapidly mixed, and the absorbance at 390 nm was measured over 3 min to monitor the formation of p-nitroaniline upon chymotrypsin cleavage of the substrate peptide, which only occurs when the proline residue is in the trans-configuration. CypA catalyzes the cis-trans isomerization of proline residues, and thus the catalytic activity of CypA can be indirectly determined from the rate at which chymotrypsin cleaves the peptide substrate.

In our inhibition assay, it was possible to calculate the fraction of CypA that was free in solution (i.e., unbound to the test compounds). The calculation involved the use of equations (5) and (6):

[L] = L_{total} - \frac{P_{total} + L_{total} + K_{d} - \sqrt{{(P_{total} + L_{total} + K_{d})}^{2} - 4 P_{total} L_{total}}}{2} .

(5)

% Free = 100 - (\frac{L_{total} - [L]}{P_{total}} \times 100) .

(6)

In equations (5) and (6), [L] is the concentration of free ligand; P_total is the sum of the free and bound protein concentration; L_total is the sum of the free and bound ligand concentration; K_d is the dissociation constant of the CypA complex with the hit compound, which was determined in our binding affinity measurements (see above); and %Free is the fraction of CypA protein that is free in solution (i.e., not bound to ligand).

Results and Discussion

General strategy

Single-point SUPREX is an abbreviated version of the SUPREX technique, which exploits the H/D exchange properties of globally protected amide protons in a protein to measure the increase in a protein’s thermodynamic stability upon ligand binding in solution. In a full SUPREX analysis, the extent to which a protein, or protein complexed with ligand, undergoes amide H/D exchange during a specified amount of time is determined as a function of denaturant concentration (see Fig. 1 ). Ultimately, the denaturant concentration at the transition midpoint is used to ascertain the thermodynamic stability of the protein or protein-ligand complex. Proteins complexed with ligand have a SUPREX transition midpoint that is shifted to a higher denaturant concentration than that of the protein alone, and the magnitude of the shift is related to the binding affinity.

In single-point SUPREX, the extent to which a protein, or a protein in the presence of ligand, undergoes amide H/D exchange during a specified amount of time is determined at a single denaturant concentration. The denaturant concentration is chosen such that there is a maximum difference between the ΔMass values expected for the protein and for the protein complexed with ligands having the minimum binding affinity desired in the selection. Based on the SUPREX curves expected for CypA in the presence and absence of a hypothetical ligand that binds CypA with a K_d value of 10 µM (see Fig. 1 ), a 1.5 M denaturant concentration was chosen for the single-point SUPREX experiments in this work. This allowed for the selection of library compounds with K_d values ≤10 µM. We reasoned that ligands with K_d values up to ~10 µM would be useful lead compounds in our search for CypA-targeted diagnostic and imaging agents.

The 1280 and 9600 compounds in the LOPAC and DIVERSet libraries, respectively, were screened for binding to CypA using the protocol outlined in Figure 2 . The protocol involved pooling 10 library compounds per well of a microplate prior to initiating the single-point SUPREX experiment. The library pools were prepared in 10 µL of deuterated buffer containing 1.5 M GdmCl and 10% DMSO, and the library compounds in each pool were present at a concentration of ~100 µM per compound. The single-point SUPREX protocol was initiated upon addition of CypA to each library pool. The CypA was allowed to undergo H/D exchange for 35 min in the presence of each library pool. Ultimately, the H/D exchange reactions were quenched upon the addition of a MALDI matrix solution, which also prepared the CypA protein in each sample for a MALDI-TOF analysis. The MALDI-TOF analysis was used to determine the number of amide protons in CypA that were exchanged with solvent deuterons during the 35-min H/D exchange reaction (i.e., the ΔMass value). The magnitude of the ΔMass value was used as described below to determine whether a given pool of library compounds contained binding ligand(s).

Fig. 2.

Schematic representation of the screening methodology used in this work. Library compounds were screened as 10-compound pools to improve throughput, and CypA was added and allowed to exchange. The mass increase was then used to determine whether a binding event had occurred. The bar graph plot represents a portion of actual data collected in this work.

Library screening

The pools of 10 library compounds, along with 2 sets of control samples, were screened for CypA binding using the single-point SUPREX protocol described above. The distributions of ΔMass values obtained in the analysis of the 1089 pools and 251 control samples are shown in Figure 3 . The control samples included 126 positive controls that contained CsA, which is a known tight binding ligand to CypA, and 125 negative controls that did not contain a ligand. The data obtained on these control samples, which were analyzed with every set of 10 pools, were used to generate the cutoff values for hit selection and to help establish the false-positive and false-negative rates of the strategy employed in this work. Figure 4 shows the ΔMass values obtained for the positive and negative controls obtained during the analysis of each library.

Fig. 3.

Distribution of ΔMass values recorded for the 1089 library pools (gray bars), 126 positive controls (black bars), and 125 negative controls (open bars) analyzed in the first tier screening of the 2 libraries used in this work. The solid lines represent the fit of each distribution to a normal Gaussian curve.

Fig. 4.

The ΔMass values from the positive (●) and negative (○) control samples analyzed with the (A) LOPAC library compounds and the (B) DIVERSet library compounds. The solid lines represent the average ΔMass values of the control data, and the dashed line represents the cutoff values used in this work. The negative control data point indicated with an arrow was determined to be an outlier and was not used to determine the cutoff value. Note that a 9-point moving average was used to determine the cutoff values in B. Shown in C is the distribution of Z′ factors observed in both library screenings.

In the LOPAC library screening, a cutoff value for the selection of hits in the first screening was set at 49.0 Da, which was 2.5 standard deviations below the average ΔMass value obtained for the negative controls. The ΔMass value of 1 negative control in the LOPAC library (see Fig. 4 ) was determined using the Q-test at the 95% level to be an outlier,¹⁸ and it was not used in the cutoff value calculation. The ΔMass value cutoffs used for the selection of hits in the DIVERSet library screening were calculated by subtracting 2.5 standard deviations from a 9-point moving average of the negative control data. Thus, a specific ΔMass value cutoff was determined for each set of 10 pools that were analyzed in the DIVERSet library screening. We previously showed that the use of such a moving average in the calculation of ΔMass cutoff values is useful in single-point SUPREX screening experiments that are performed over the course of multiple days, as was done in the DIVERSet library screening.⁵

Ultimately, a list of hit pools (i.e., those that yielded ΔMass values below the cutoff values) was generated. A total of 8 of the 131 LOPAC library pools and 13 of the 958 DIVERSet library pools were identified “hit pools” in this first tier of screening. The hit pools from each library were rescreened in a second tier of screening. We have previously shown that such a 2-tier screening strategy significantly reduces the false-positive rate using the single-point SUPREX assay.⁵ The 2-tier screening strategy is useful because the assay is subject to random error, which is reflected in the Gaussian distributions observed for the ΔMass values recorded here (see Fig. 3 ). The main sources of random error include the mass measurement uncertainty and the differential back-exchange (see below). After the second tier of screening, 5 hit pools were identified from the LOPAC library, and 5 hit pools were identified from the DIVERSet library.

The pooling strategy described here ultimately requires that separate analyses be performed on the individual compounds in the selected pools to determine which compound(s) yielded the “hit” (i.e., were responsible for the low ΔMass value). Thus, the 100 compounds from the 10 hit pools identified from the LOPAC and DIVERSet libraries were individually screened for binding to CypA using the single-point SUPREX protocol. Ultimately, 9 compounds were identified as CypA binding ligands in these individual screens (see Table 1 ). This included 1 compound from each of the 10 hit pools with the exception of 1 hit pool that did not yield an individual hit. The binding affinity of these 9 newly identified CypA ligands was then measured using the conventional SUPREX protocol.

Table 1.

Summary of the 9 CypA Ligands Selected in This Work

Compound	Library	K_d (µM)^a
2-(α-naphthoyl)ethyltrimethyl-ammonium iodide (compound 1)	LOPAC	4^b
(E)-3-(4-t-Butylphenylsulfonyl)-2-propenenitrile (compound 2)	LOPAC	16^b
3-(N-benzyl-N-isopropyl)amino-1-(naphthalen-2-yl)propan-1-one (compound 3)	LOPAC	4^b
cis-diammineplatinum (II) chloride (compound 4)	LOPAC	37^b
1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione (compound 5)	DIVERSet	≤10^b
3-[5-(4-Fluorobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]propanoic acid (compound 6)	DIVERSet	No binding^c
N-(3-chloro-1,4-dioxo-1,4-dihydro-2-naphthalenyl)-N-cyclohexylacetamide (compound 7)	DIVERSet	2^b
1-[2-(3,4-dimethoxyphenyl)ethyl]-1H-pyrrole-2,5-dione (compound 8)	DIVERSet	≤0.2^c
4-(2-methoxy-4-nitrophenyl)-1-methyl-10-oxa-4-azatricyclo[5.2.1.0~2,6~] dec-8-ene-3,5-dione (compound 9)	DIVERSet	4^b

Determined from data collected in the full SUPREX analyses performed in this work.

Inhibition of CypA’s isomerase activity was detected (see Fig. 6 ).

No inhibition of CypA’s isomerase activity was detected (see Fig. 6 ).

Full SUPREX analyses were performed on CypA in the presence of the selected compounds to determine their affinity for CypA ( Fig. 5 ). The individual data points in a full SUPREX curve are subject to the same ΔMass value uncertainties as those in the single-point SUPREX protocol. However, protein-ligand binding analyses using the full SUPREX protocol are much less sensitive to this error because multiple ΔMass values are used to define a full SUPREX curve (see Fig. 5 ). The full SUPREX analyses indicated that 8 of the 9 selected compounds yielded measurable binding interactions with CypA (i.e., a shift in the transition midpoint of the SUPREX curves). The SUPREX-derived K_d values of these 8 compounds ranged from 0.200 to 37 µM, with the majority being in the low micromolar range ( Table 1 ).

Fig. 5.

Full SUPREX curves obtained for CypA in the absence (●) and presence (○) of the 9 selected hit compounds. The solid and dashed arrows indicate the transition midpoints obtained in the absence and presence of ligand, respectively. Eight of the 9 had measureable binding due to a shift in the SUPREX curve. Compounds 5 and 8 did not produce a full SUPREX curve (see text), but the data points collected suggested a transition midpoint of at least 1.6 and 2.3 M, respectively. A measurable transition midpoint shift was not detected with compound 6.

Dissociation constants for 2 hit compounds, 5 and 8 from the DIVERSet library, could not be determined due to difficulties associated with obtaining MALDI ion signals for the CypA in the presence of these compounds at high [GdmCl] concentrations. At these elevated concentrations of denaturant, a MALDI ion signal for CypA was not observed, and multiple peaks at m/z 200 to 1000 Da higher than that expected for CypA were detected. However, the ΔMass values obtained at the lower denaturant concentrations used in our full SUPREX analyses of CypA in the presence of these individual compounds were consistent with those expected for a pretransition baseline, suggesting that binding was, in fact, detected and that the transition midpoint of the curve was at least 1.6 and 2.3 M for compounds 5 and 8, respectively. Binding was not detected in our full SUPREX analyses of CypA in the presence of one of the selected ligands, 6 (i.e., the SUPREX transition midpoints for CypA were similar in the presence and absence of this ligand), which appeared to be a false positive.

The 9 newly identified CypA ligands were also tested for their ability to inhibit CypA’s peptidyl-prolyl isomerase activity. Initial velocities of the chymotrypsin-catalyzed hydrolysis of the peptide substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were determined in the presence and absence of CypA and then in the presence of CypA and each newly identified CypA ligand. The substrate peptide is cleaved by chymo- trypsin when the Ala-Pro peptide bond is in the trans configuration. CypA increases the apparent rate that chymotrypsin hydrolyzes the scissile bond in this substrate by catalyzing the conversion of the cis isomer to the trans isomer. The degree to which CsA and the 9 hit compounds identified in this study affect the rate of this hydrolysis reaction in the presence of CypA was determined from initial rate measurements (see Fig. 6 ).

Fig. 6.

Results obtained in CypA inhibition studies. The closed bars and left y-axis represent the initial velocities determined for the chymotrypsin-catalyzed hydrolysis of the peptide substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in the presence and absence of CypA and in the presence of CypA and different ligands, including CsA and the 9 hit compounds identified in this study. The initial velocity values are the average of 4 to 6 determinations, and the error bars represent 1 standard deviation. The open bars and the right y-axis indicate the fraction of CypA protein expected to be free in solution in the assay (i.e., unbound to the target ligands) based on the CypA protein concentration in the assay and the measured K_d value (see Table 1 ).

The chymotrypsin-coupled enzyme assays revealed that 7 of the 9 compounds (including compounds 1-5, 7, and 9) inhibited CypA’s isomerase activity, and the degree to which each of these ligands inhibited CypA was generally consistent with the amount of free protein calculated based on each ligand’s measured K_d value, with the exception of compound 8, which appears to bind CypA with relatively high affinity but has a minimal effect on CypA’s isomerase activity. It is not surprising that such a compound was identified in this work, as the single-point SUPREX assay does not select compounds with specific activities but rather just binding interactions. The variety of compounds that can be selected in the single-point SUPREX assay (e.g., both those that alter a specific protein function and those that do not) can be an advantage of the methodology over other HTS approaches for certain applications, such as the one described here to identify molecular imaging agents. Interestingly, compound 6, which was not found to have a measurable CypA binding interaction in our full SUPREX analysis (K_d value >40 µM), did not significantly inhibit the isomerase activity of CypA isomerase. Compound 6 is most likely a false positive.

Throughput and efficiency

The first tier of the 1280-compound LOPAC library screening was accomplished by 2 researchers working over the course of 1 day for 3.5 hours, which was the time it took to perform the H/D exchange reactions and collect the mass spectral data. This equates to approximately 1.5 min per pool or less than 10 s/compound. The first tier of the 9600-compound Chembridge DIVERSet library screening was accomplished by 2 researchers working over the course of 4 days for a total of 22 hours, which again was the time it took to perform the H/D exchange reactions and collect the mass spectral data. This equates to a screening rate of approximately 1 min/pool or about 6 s/compound.

The overall throughput achieved in this work, 6 s/compound, is about 30 times faster than that previously reported for the single-point SUPREX protocol.⁵ In theory, the pooling strategy used here should increase the throughput of the original single-point SUPREX protocol by 10-fold. The additional 3-fold increase in throughput realized in this work is likely due to the use of a high-throughput MALDI-TOF instrument, which was equipped with a high repetition rate laser.

The false-positive and false-negative rates of a high-throughput screening assay can provide a measure of its efficiency. A total of 126 positive controls and 125 negative controls were analyzed during the single-point SUPREX screening experiments described here. Using the ΔMass cutoff values described above, false-positive and false-negative rates of 0% and 9% were determined from the control data, which are both identical to that previously observed for the control data in our Prestwick Chemical Library screening, which were 0 and 9%, respectively.⁵

During the screening of the pooled library samples, false negatives go undetected, and false positives are only detected after subsequent screens and/or analyses. A total of 10 hit pools were initially identified after our analysis of the 1089 ten-compound pools. Subsequent analyses of the individual ligands from these 10 hit pools identified hit compounds from all but 2 pools, suggesting a false-positive rate of 20%. Interestingly, 1 of the 2 false-positive hit pools consistently produced low ΔMass values upon reanalysis, even though it failed to produce a hit compound when the ligands from the pool were individually analyzed for CypA binding. This suggests that the higher occurrence of false positives in the pooled samples (i.e., ~20%) compared to the false positives in the controls (i.e., 0%), which were not pooled, may be a result of the pooling strategy. This is also supported by the observation that the 20% false-positive rate determined from the pooled library compounds in this work was larger than the 0% false-positive rate determined from the library compounds in our earlier screen of the Prestwick Chemical Library, which did not employ a pooling strategy.⁵ The 20% false-positive rate in this work is in line with typical pharmaceutical screens, which have been estimated to have false-positive rates of about 40%.¹⁹

A complicating issue in the pooling strategy is the potential for higher false-positive rates due to promiscuous aggregation and nonspecific binding of library compounds.^20,21 There have been reports of small molecules aggregating into particles with diameters ranging from 95 to 400 nm.²¹ Separate studies have shown that enzymes can be inhibited through reversible adsorption of enzyme onto the surface of such molecular aggregates.²⁰ This phenomenon is worse at higher concentrations of small molecules relative to protein concentration. Sequestration of the protein of interest in a screening assay by aggregate particles would thus increase the protein’s stability due to nonspecific binding. This could explain why some pools (with total small-molecule concentrations of 1 mM in the analysis buffers) were selected as positive hits, but then none of the individual compounds from the pools would be selected as binding ligands when they were analyzed individually (with the total small-molecule concentrations on the order of 100 µM). A measure of assay robustness is the Z′ factor. The Z′ factor is maximized by increasing the difference between the ΔMass values of hits and nonhits and by minimizing the standard deviations of the ΔMass values. The difference between hit and nonhit ΔMass values is largely a property of the specific protein under study, and it is directly related to the number of amide protons that are globally protected in the protein’s 3D structure. The standard deviations of the ΔMass values are limited by the mass spectrometer. The standard deviations obtained using the MALDI TOF mass spectrometer in this work averaged 3.7 Da during the screening of the LOPAC library and 3.4 Da in the DIVERSet. Generally, HTS assays with Z′ factors ≥0 are useful for screening large combinatorial libraries.¹³ The distribution of Z′ factors determined from the positive and negative controls in this work is shown in Figure 4C .

The Z′ factor values varied in this work mainly because the amplitude of the assay signal (i.e., ΔMass value difference between the positive and negative controls) varied over the course of the screening (see Fig. 4B ). This was a result of differential back-exchange (a random effect of not being able to determine the ΔMass values in the MALDI-TOF experiment at the same time after they are quenched) and the fact that the standard deviations for the mass measurements of some controls were large and clearly outliers. Although the moving average procedure accounted for this differential back-exchange to set the cutoff values in the screen, it is clear that the back-exchange reaction negatively affects the Z′ value. Although there was a large range of Z′ values (i.e., −1.3 to 0.7), the large majority of Z′ values were positive with most clustered between 0.1 and 0.5 (see Fig. 4C ) using the protocol described here. The distribution of Z′ factors observed here using our pooling strategy is very similar to that observed in our previous application of the single-point SUPREX assay that did not use a pooling strategy.⁵ In theory, the Z′ factors in the single-point assay should be normally distributed. The higher than expected number of low Z′ factors used here ( Fig. 4C ) and in our previous work⁵ is likely due to the back-exchange reaction, which compromises Z′ values by reducing the separation between hits and nonhits. These problems associated with the back-exchange reaction can be minimized by recording ΔMass values of the H/D exchanged protein samples in the MALDI-TOF experiment as soon after they are quenched as is possible.

Identification of novel CypA ligands

Full SUPREX analyses of the 9 hit compounds selected in our screen ( Fig. 4 ) revealed that 8 of the 9 selected compounds bound CypA with K_d values in the low micromolar to high nanomolar range ( Table 1 ). These 8 hit compounds are all novel CypA ligands. It is interesting to find that CypA binds to compound 4 (cisplatin) because this drug is a commonly used antitumor agent. CypA has been found to be overexpressed in many cancer types, including non–small cell lung cancer,^1,2 pancreatic cancer,^22,23 endometrial carcinoma,²⁴ and oral cancer.²⁵ In addition, CypA has been shown to be associated with the progression of breast cancer.²⁶ The mode of action for cisplatin-induced apoptosis is not completely understood but is at least partly understood through the generation of reactive oxygen species.²⁷ It has previously been observed that cells overexpressing CypA have been more resistant to cisplatin-induced death by minimizing stress-induced apoptosis while offering no additional resistance to other anticancer drugs.²⁸ We hypothesize that this may be the result of CypA binding to cisplatin as shown in our results, thus decreasing the amount of cisplatin available to the cell to generate reactive oxygen species.

It has also been shown that compound 2 (Bay 11-7085) inhibits the tumor necrosis factor–α (TNFα)–induced phosphorylation of IκBα, the inhibitor form of nuclear factor kappa B (NFκB), which serves to decrease expression of the inflammatory molecules ICAM-1, VCAM-1, and E-selectin.²⁹ The specific protein target of Bay 11-7085 was not determined in the study. However, it was speculated that the phosphorylation inhibition might be due to inhibition of a signaling protein upstream of the IκBα. It has been demonstrated that CypA has the ability to induce phosphorylation of IκBα in inflammation sites caused by rheumatoid arthritis,³⁰ and thus it is feasible to hypothesize that the mode of action of Bay 11-7085 involves CypA being inhibited by Bay 11-7085, which in turn decreases the phosphorylation of IκBα and in turn the activation of NFκB-activated expression of these inflammatory molecules. The other selected compounds from the LOPAC library have known roles. Compound 3 (ZM 39923 HCl) is a selective Janus Kinase 3 (JAK3) inhibitor, binding to JAK3’s adenosine triphosphate binding site.³¹ It has also been found to inhibit transglutamase during an HTS assay.³² Compound 1 (α-NETA) is a selective, fluorescent inhibitor of choline acetyltransferase and is used for the investigation of acetylcholine synthesis.³³ To date, the hits from the Chembridge DIVERSet library have no previously known function to the best of our knowledge.

This study demonstrated the use of single-point SUPREX for small (<10,000 compounds) libraries. Using a pooling strategy, the throughput was increased more than 10-fold compared to what has been previously reported. However, one drawback of the pooling strategy was a higher false-positive rate of 20%. Although the pooling strategy did not specifically affect Z′ factors of the single-point SUPREX assay, it is important to recognize that the Z′ values determined in this work were generally between 0 and 0.5, with approximately one-third of the values being less than 0. As a result of these relatively low Z′ values, the assay requires at least a 2-tier screening strategy. This was feasible for the relatively small (1000-10,000) compound libraries studied here. However, the relatively low Z′ values of the assay may limit applications to larger compound libraries. The results of this study confirmed the assay’s capacity to identify novel ligands to cyclophilin. In this case, 8 novel CypA ligands were discovered, with 1 compound estimated to have a K_d of less than 200 nM. In addition, the use of a traditional biochemical assay showed that 7 of the 8 novel ligands also inhibited the isomerase activity of CypA. The assay is unique among HTS assays in its generality (i.e., ligand selection is based solely on the introduction of structural stability and not necessarily on the modulation a protein’s specific biological activity).

Footnotes

Acknowledgements

The authors are very grateful to Vladimir Poltoratsky for his help in pooling the DIVERSet library.

This work was supported by an NIH grant to MCF (R21-CA-127064).

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Discovery of Novel Cyclophilin A Ligands Using an H/D Exchange– and Mass Spectrometry–Based Strategy

Abstract

Keywords

Introduction

Methods

Materials

SUPREX buffer preparation

Mass spectrometry

Library screening (single-point SUPREX protocol)

Binding affinity measurements (full SUPREX analyses)

Theoretical SUPREX curve construction

CypA inhibition studies

Results and Discussion

General strategy

Library screening

Throughput and efficiency

Identification of novel CypA ligands

Footnotes

Acknowledgements

References