High-throughput assay for evaluating dipeptidyl peptidase I activity

This application claims benefit to U.S. Provisional Patent Application No. 61/107,046 filed Oct. 21, 2008, the contents of which are incorporated by reference herein in their entirety.

1. Field of the Invention

A method is provided for identifying agonists and antagonists of dipeptidyl peptidase I in a high-throughput format. The method uses a peptide substrate that is label-free and binds to multiple substrate binding sites of dipeptidyl peptidase I.

2. Description of the Related Art

Dipeptidyl peptidase I (DPPI) or cathepsin C is a member of the lysosomal papain-type cysteine protease family that also includes cathepsin B, K, H, L, O, and S (Bouma and Gruber, 1996, Biochem. Biophys. Acta 113: 350-358; Turk et al., 2000, Biochem. Biophys. Acta 1477: 98-111). This protease family activates serine proteinases in immune and inflammatory cells.

The physiological role of DPPI is to convert inactive proenzymes into active enzymes by removing two amino acids as a dipeptide unit from the N-terminal end of the proenzymes in immune and inflammatory cells (reviewed by Caughey, G. H., 2002, Molecular Immunology 38: 1353-1357; Wolters et al., 2001, J. Biol. Chem. 276: 18551-18556; McGuire et al., 1993, J. Biol. Chem. 268: 2458-2467; Pham and Ley, 1999, Proc. Natl. Acad. Sci. 96: 8627-8632; Pham et al., 2004, J. Immunol. 173: 7277-7281). DPPI activates many serine proteinases including chymase, tryptase, cathepsin G, elastase, and neutrophil-elastase, granzymes A and B from T-lymphocytes and natural killer cells; and rheumatoid arthritis proteases (Adkison et al., 2002, J. Clin. Invest. 109: 363-371). The enzymes or serine proteases activated by DPPI are needed for defense responses. In addition, the unregulated DPPI has been shown to cause over-activation of proteases associated with diseases including chronic obstructive pulmonary disease, Papillon-Lefevre Syndrome, Sepsis, arthritis and other inflammatory disorders. Therefore, DPPI is a potential target for therapeutic treatment.

It is proposed that DPPI consists of four identical monomers. Each monomer is produced from a precursor polypeptide, which is cleaved into a N-terminal exclusion domain, an activation peptide, and a papain-like domain. The papain-like domain is further cleaved into a heavy chain and a light chain. Subsequently, the N-terminal exclusion domain, the heavy and the light chains fold into a monomer, which tetramizes into the mature DPPI of about 200 kDa (Turk et al., 2001 EMBO J., 20: 6570-6582).

Recent crystallization of DPPI identified some structures, including an active site cleft and substrate binding sites, involved in the dipeptidyl proteolytic activity. The substrate binding sites reside on the external part of the papain-like structure and form hydrogen bonds with the substrates. The site that binds to the first amino acid at the N-terminal side of the cleaved bond of the substrate is referred to as the S 1 binding site, the site that binds to the second amino acid at the N-terminal side is referred to as the S 2 binding site. Further, the site that binds to the first amino acid at the C-terminal side of the cleaved bond of the substrate is referred to as the S 1 ′ binding site, and the site that binds to the second amino acid at the C-terminal side is referred to as the S 2 ′ binding site, etc (Turk et al., 2001, EMBO J., 20:6570-6582; Molgaard et al., 2007, Biochem. J., 401:645-650). The corresponding site in a DPPI substrate is referred to as P 1 , P 2 , P 1 ′, P 2 ′ etc, where P 1 of the substrate binds to the S 1 site of DPPI, P 2 of the substrate binds to S 2 of DPPI, and P 1 ′ of the substrate binds to the S 1 ′ site of DPPI, etc. Based on the crystallographic results of a binding complex of DPPI and a substrate of 6 amino acids, Molgaard et al. proposes a model where DPPI contains the S 2 -S 1 and S 1 ′-S 4 ′substrate binding sites. Since DPPI binds to larger substrates such as human prochymase of 228 amino acids in vivo; the substrate binding sites of DPPI may contain more than S 2 -S 1 -S 1 ′-S 4 ′, even to S 2 -S 1 -S 1 ′-S 18 ′.

As DPPI is a potential therapeutic target, several assays for analyzing DPPI activity have been commonly used (Gelman et al., J. Clin. Invest. 1980, 65: 1398-1406; (Tran et al., Arch. Biochem. Biophys. 2002, 403: 160-170). Gelman et al. uses a labeled dipeptide of Gly-Phe-β-naphthylamide as substrate and determined DPPI activity by measuring the concentration of free β-naphthylamide with a spectrophotofluorometer. Similarly, Tran et al. uses several dipeptides labeled with 7-amino-4-methylcoumarin as DPPI substrates and determined DPPI activity by measuring the concentration of free methylcoumarin with a spectrophotofluorometer. Tran et al. shows that the dipeptide substrate of Ala-Hph-7-amino-4-methylcoumarin is the best substrate for DPPI, even though the substrate contains a non-physiological residue Hph (homophenylalanine).

These commonly used dipeptide substrates with fluorescence labels have several problems. First, the dipeptide substrate only binds to the S 1 and S 2 sites. This is partial or incomplete in comparison to biological substrates in vivo which binds to a binding site in addition to the S 1 and S 2 sites. The incomplete or partial binding may be non-specific and hence lead to the identification of false positive or negative compounds. Also, the additional step of fluorescence detection is needed to detect the DPPI activity. This additional step makes the assay difficult to be adapted for use in a high-throughput format.

Therefore, there is still a need to develop an assay that uses a substrate which is label-free and biologically related for analyzing DPPI activity. Also, there is a need to develop a DPPI assay suitable for use in a high-throughput format to enable efficient screening of a large number of compounds or agents for drug development.

The label-free technology has been combined with a mass spectrometry system to analyze enzymatic reactions. Quercia et al. (J. Biomol. Screen. 12: 473-480, 2007) identifies the kinase AKT1/PKBα inhibitors using mass spectrometry in multiple reaction monitoring. Similarly, Forbes et al. (J. Biomol. Screen. 11: 628-634, 2007) evaluates phosphortidylserine decarboxylase in 96-well plates using mass spectrometry in multiple reaction monitoring. A mass spectrometry detection system in a high-throughput format has not been applied for analyzing DPPI activity.

The present application provides a method for assaying DPPI activity. The method utilizes a label-free peptide substrate that comprises biologically related amino acid sequences and binding specificity. The assay may be detected using a mass spectrometry system and may be operated in a high-throughput format, which reduces processing time and increases throughput which are desired for screening compounds. The method is also useful for differential tracking of fragment-based compounds for drug development and evaluating other dipeptidyl proteolytic reactions for functional studies.

An object of the present application is to provide a method for screening compounds which modulate DPPI activities. The method comprises the steps of adding a substrate of DPPI to a reaction mixture which comprises DPPI and the compounds, wherein the substrate has 3 to 100 amino acids and binds to a binding site of DPPI in addition to the S 1 -S 2 site, and measuring a change in the molecular weight of the substrate and/or a change in the mass ratio of the substrate and products, wherein the change in molecular weight of the substrate or mass ratio is indicative of the presence of DPPI activity.

According to one embodiment of the present invention, the compound may be 2-(piperidin-1-ylcarbonyl)phenol or 2-(piperidin-1-ylcarbonyl)aniline.

Another object is to provide label-free DPPI substrates useful for screening compounds which modulate DPPI activity. Exemplary DPPI substrates may be selected from the group consisting of SEQ ID NOs: 1-7. The DPPI substrate may preferably comprise a peptide having 20 amino acids. The DPPI substrate is preferably label-free.

Other objects and features of the present invention will become apparent from the following detailed description considered in conjunction with the accompanying drawings. It is to be understood, however, that the drawings are designed solely for purposes of illustration and not as a definition of the limits of the invention, for which reference should be made to the appended claims. It should be further understood that the drawings are not necessarily drawn to scale and that, unless otherwise indicated, they are merely intended to conceptually illustrate the structures and procedures described herein.

In the drawings:

FIG. 1 . Initial velocity analysis for DPP1 kinetic constant in reaction with a 20-mer substrate (A) and a dipeptide substrate (B). (A) About 2 nM of DPPI was used in the reaction with the 20-mer substrate of GEIIGGTECKPHSRPYMAYL (SEQ ID NO:1) and the reaction was monitored using a mass spectrometry system. (B) About 0.4 nM of DPP1 was used in the reaction with the dipeptide substrate of GR-α-methylcoumarin and the reaction was monitored using a rate-based format monitoring fluorescence at E M =460 nm and E X =380 nm. Initial rates are taken from kinetic data where less than 7% substrate turnover was achieved.

FIG. 2 . Evaluating the DPPI-modulating activity with two test compounds using a dipeptide substrate (A) and using a 20-mer peptide substrate (B). (A) About 0.4 nM of DPP1 and about 10 μM of dipeptide substrate GR-α-methylcoumarin were used in a rate-based format and monitored with fluorescence at E M =460 nm and E X =380 nm. (B) About 2 nM of DPPI and 20 μM of the 20-mer peptide substrate GEIIGGTECKPHSRPYMAYL (SEQ ID NO:1) were used in a reaction and monitored with a mass spectrometry system. 2-(piperidin-1-ylcarbonyl)phenol is represented by solid squares and 2-(piperidin-1-ylcarbonyl)aniline is represented by solid circles. R 2 -values is >0.95.

To detect the activity of dipeptidyl peptidase I (DPPI) in the presence of an agonist or an antagonist of DPPI, a peptide which is used as a substrate of DPPI, is added into a reaction mixture such that the reaction mixture contains a reaction buffer, an agonist or an antagonist of DPPI, DPPI and a substrate. A buffer system of PIPES, HEPES, sodium phosphate or any buffering system where the pKa is about 7.0 can be used for the DPPI assay. A HEPES buffer system is preferred. For example, a 10× reaction buffer is 500 mM HEPES at pH 7.0, 1 M NaCl, and 0.05% Tween-20. Other buffer systems commonly known to a person skilled in the art may also be used. Prior to the reaction, DTT or glutathione is added to the 1× reaction buffer to a final concentration of 2 mM.

DPPI may be obtained from a commercial source, produced by recombinant DNA technology, or isolated or purified from cells by methods commonly known to a person skilled in the art.

Any peptides of at least 3 amino acids, preferably 5 to 100 amino acids and most preferably 10 to 50 amino acids that binds to more than the S 1 -S 2 substrate binding site of DPPI may be used as a substrate for detecting the proteolytic activity of DPPI. The number of the substrate binding sites of the peptide substrate may be 3 to 100, preferably 5 to 50 and most preferably 6 to 20. Therefore, the peptide substrate binds to DPPI at the S 1 -S 2 substrate binding site and at least one additional substrate binding site of S 1 ′-S 98 ′, preferably S 1 ′-S 48 ′ and most preferably S 1 ′-S 4 ′.

The peptide substrate of the present application is not labeled or modified with any flurogenic or chromogenic agents. The peptide substrate may be synthesized chemically, expressed using recombinant DNA technology, or isolated or purified from cells by methods commonly known to a person skilled in the art.

As used herein, the term polypeptide refers to three or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 naturally occurring amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.

A reaction volume of 5-50 μls, preferably 10 or 20 μls, is aliquoted into a tube or a plate commonly used in the laboratory and assayed for DDPI activity by measuring the substrate turnover. As used herein, the substrate turnover refers to the reduction in the quantity of substrate and the subsequent increase in the quantity of product. The substrate turnover may be measured by monitoring the molecular mass of the substrate and the product. A mass spectrometry system is preferred. By way of example, the concentrations of substrates and products can be determined by single ion monitoring (SIM) technology using the Agilent MSD with Agilent Chemstation software or the like known to a person of ordinary skill in the art. Data was analyzed using Agilent Chemstation and BioTrove Rapidfire Integrator. The assay was designed to tolerate less than 10% substrate turnover for compound testing.

An equilibrium of a general enzymatic reaction is illustrated below:

wherein E is an enzyme, S is a substrate for the enzyme and P is the product of the reaction.

The equilibrium is modified to illustrate the dipeptidyl proteolysis reaction in the DPPI assay with a peptide substrate of the present application:

When initiated, DPPI cleaves and removes two amino acids G-E or Gly-Glu from the N-terminus of the peptide substrate having 20 amino acids, G-E-I-I-G-G-T-E-C-K-P-H-S-R-P-Y-M-A-Y-L (SEQ ID NO:1). This results in a change of molecular weight or mass of the peptide substrate from 2,224 Da to 2,037 with a difference of 187 Da. The molecular weight or mass of the peptide substrate in the reaction may be detected by a mass spectrometry using single ion monitoring. The change or difference of the peptide substrate detected by SIM is from 1,112 amu (atomic mass unit) to 1,018.5 amu. This may be used to determine the value for the substrate turnover.

Once the rate of the substrate turnover under the initial velocity condition, the kinetic constants for DPPI and the peptide substrate are calculated using the following formula (I):

Y=V MAX (( X+Et+K M )−(((( Et+X+K M ) 2 )−(4 XEt )) 0.5 ))/(2 Et )

wherein

Y is concentration of substrate turnover X is substrate concentration Et is DPPI concentration K M is substrate concentration at ½ V MAX V MAX is substrate turnover at substrate saturation

For screening test compounds which modulate DPPI activity, the inhibition of DPPI activity may be calculated using the following formula:

Quantity of Substrate Turnover in a Positive Control:

[ S ] × ( ( SIM PC - SIM NC ) ( SIM SPC - SIM SNC ) + ( SIM PC - SIM NC ) ) )

Quantity of Substrate Turnover in a Sample Containing a Test Compound:

[ S ] × ( ( SIM P - SIM NC ) ( SIM S + ( SIM P - SIM NC ) ) ) Percent ⁢ ⁢ inhibition =

⁢ ( 1 - ( ( ( SIM P - SIM NC ) ( SIM S + ( SIM P - SIM NC ) ) ) × ( ( SIM PC - SIM NC ) ( SIM SPC + ( SIM PC - SIM NC ) ) ) - 1 ) ) × 100 OR ⁢

( 1 - ( ( μM ⁢ ⁢ substrate ⁢ ⁢ turnover ⁢ ⁢ of ⁢ ⁢ sample ) × ( μM ⁢ ⁢ substrate ⁢ ⁢ turnover ⁢ ⁢ of ⁢ ⁢ pos . ⁢ control ) - 1 ) × 100

wherein

[S] is concentration of the substrate (μM) SIM S is SIM of the substrate in sample (area under curve from amu) SIM SPC is SIM of the substrate in positive control samples (area under curve from amu) SIM P is SIM of the product in sample (area under curve from amu) SIM PC is SIM of product for positive control (area under curve from amu) SIM SPC is SIM of the substrate in positive control samples (area under curve from amu) SIM NC is SIM of product for negative control (area under curve from amu) SIM SNC is SIM of substrate for negative control (area under curve from amu)

wherein a positive control is a reaction without any test compound, a negative control is a reaction without DPPI or substrate, a sample is a reaction containing a test compound.

When a candidate compound is identified, the IC 50 of the DPPI inhibition by the candidate compound is calculated using the formula (II):

S i = S o + ( S min - S o ) ⁡ [ I ] h IC 50 h + [ I ] h

wherein

S i is net substrate turnover signal of sample S o is net substrate turnover signal of positive control S min is net am signal of negative control I is concentration of a candidate compound

wherein a positive control is a reaction without any test compound and a negative control is a reaction without DPPI or substrate.

The above described assay may be used in a high-throughput format for screening test compounds, and identifying candidate compounds which modulate the activities of DPPI. In such case, the reaction mixture is first aliquoted to micro-titer plates containing a plurality of wells such as 96- or 386-well plates, then the plates are loaded to a detection system such as a mass spectrometry or the like, which reads the reaction in each well of each plate to generate numeric data.

Thus, while there have been shown and described and pointed out fundamental novel features of the invention as applied to a preferred embodiment thereof, it will be understood that various omissions and substitutions and changes in the form and details of the compounds, methods and devices illustrated, and in their operation, may be made by those skilled in the art without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps which perform substantially the same function in substantially the same way to achieve the same results are within the scope of the invention. Moreover, it should be recognized that structures and/or elements and/or method steps shown and/or described in connection with any disclosed form or embodiment of the invention may be incorporated in any other disclosed or described or suggested form or embodiment as a general matter of design choice. It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto.