WO2001004277A9 - Compounds and methods for identifying compounds which inhibit a new class of aspartyl proteases - Google Patents
Compounds and methods for identifying compounds which inhibit a new class of aspartyl proteasesInfo
- Publication number
- WO2001004277A9 WO2001004277A9 PCT/US2000/018775 US0018775W WO0104277A9 WO 2001004277 A9 WO2001004277 A9 WO 2001004277A9 US 0018775 W US0018775 W US 0018775W WO 0104277 A9 WO0104277 A9 WO 0104277A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- tfpp
- type
- aspartyl protease
- prepilin
- peptidase
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K3/00—Use of inorganic substances as compounding ingredients
- C08K3/34—Silicon-containing compounds
- C08K3/346—Clay
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K7/00—Use of ingredients characterised by shape
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K9/00—Use of pretreated ingredients
- C08K9/04—Ingredients treated with organic substances
Definitions
- transme brane protease enzymes comprise two critical aspartic acid residues on the same side of the membrane at their active cleavage site.
- Members of this family of protease inhibitors can be identified by a consensus sequence at the C-terminal aspartic acid residue comprising G-X-G-D- (F or V or P or K or I or L or Y) .
- the mechanism of action of these aspartyl proteases is exemplified herein through the bacterial protease type 4 prepilin peptidase (TFPP) . Accordingly, these proteases are referred to herein as TFPP-like aspartyl proteases.
- the present invention relates to compositions and methods for identifying and designing compositions which inhibit TFPP-like aspartyl proteases by targeting the aspartic acid residues of the enzymatic active site or mimicking a region surrounding the cleavage site of a TFPP-like aspartyl protease substrate. These compositions are expected to be useful in inhibiting the activity of TFPP-like aspartyl proteases involved in diseases and infections.
- Anti-bacterial agents are developed by identifying unique targets not present in mammalian cells and then designing a drug to exploit that difference such that the bacterial cells are killed or neutralized while mammalian cells are left intact and unaffected.
- the goal of successful anti-bacterial drug therapy is to limit toxicity in the patient while maximizing the ability of the drug to invade the bacterial cells and neutralize those cells as selectively as possible.
- the major classes of anti-bacterial drugs available today target a variety of different cellular components and functions of bacteria such as the cell wall, protein synthesis, cell metabolism, DNA synthesis, and the bacterial cell membrane.
- Each of these target cellular components or functions is related in some way to the disease process of bacterial infections that involves first colonization of the bacteria, invasion of host cells, production of cellular toxins or inflammatory agents, and a host response to those toxins or agents .
- a fundamental process of all living cells, including bacteria, is the secretion of proteins across membranes. The majority of proteins that are secreted are synthesized as a precursor with an N-terminal signal sequence (or leader peptide) of about 16-24 amino acids in length. This leader sequence serves to promote recognition of the protein by the secretory apparatus of the cell and facilitates movement across the membrane. The leader sequence is subsequently processed by a leader peptidase to remove the sequence and allow release of the mature or active protein.
- Type 2 secretion systems of most bacteria involve a type 4 pilin for colonization pilus formation and type 4 pilin-like proteins for secretion of toxins and other factors associated with bacterial virulence and destruction of host tissue and enhancement of bacterial growth in the- host.
- Highly related type 4 pili serve as the major colonization factors for up to 50 different gram-negative bacterial species and type 4 pilin-like proteins have been found for a growing number of gram-positive bacteria as well.
- Type 4 pili are composed of a polymerized structure of type 4 pilin. The pilin is synthesized as a prepilin with a leader peptide that is very different from those of typical secreted proteins.
- a type 4 specific leader peptidase is required to process a type 4 prepilin leader sequence to allow secretion of the mature protein. Importantly, this secretion system including the type 4 leader peptidase itself is only found in bacteria and is not present in humans or other potential hosts of infection. Furthermore, mutating the type 4 prepilin peptidase (TFPP) renders the bacterium avirulent (March and Taylor 1998. Mol . Microbiol . 29:1481-1492).
- the type 4 signal peptide is highly conserved across all type 4 prepilin or prepilin-like proteins and is composed of 6 to 25 highly charged amino acids at the N-terminus followed by approximately 20 predominately hydrophobic amino acids.
- Cleavage occurs between the two domains immediately C-terminal of an invariant glycine and before the new N-terminal amino acid that is usually a methionine or a phenylalanine .
- processing by a type 4 peptidase occurs on the cytoplasmic side of the inner membrane (Strom and Lory, 1993. Ann . Rev. Microbiol . 47:565-596).
- proteases of this new family which are transmembrane protease enzymes comprising two critical aspartic acid residues on the same side of the membrane at their active cleavage site, wherein a consensus sequence at the C-terminal aspartic acid residue comprising G-X-G-D- (F or V or P or K or I or L or Y) is present, are referred to herein as "TFPP-like aspartyl proteases" . This term is meant to be inclusive of type 4 prepilin peptidases.
- an object of the present invention is to provide a method of designing inhibitors of TFPP-like aspartyl proteases which comprises synthesizing compounds which target the aspartic acid residues of the enzymatic active site of the TFPP-like aspartyl protease or mimic a region surrounding the cleavage site of the TFPP-like aspartyl protease substrate and inhibit cleavage activity of the protease. Also provided in the present invention are methods of selecting and screening compounds for inhibitory activity of these TFPP-like aspartyl protease enzymes . Compounds identified as inhibitors can be used to modulate activity of TFPP-like aspartyl proteases involved in disease and infection. In one embodiment, compounds identified as inhibitors are used as anti-bacterial agents in the inhibition of type 4 prepilin peptidases.
- Another object of the present invention is to provide new anti-bacterial agents which comprise compounds which target the aspartic acid residues of type 4 prepilin peptidase and inhibit cleavage activity of this peptidase.
- new anti-bacterial agents which comprise compounds which target the aspartic acid residues of type 4 prepilin peptidase and inhibit cleavage activity of this peptidase.
- such agents are also expected to be useful in inhibiting other TFPP-like aspartyl protease enzymes of this family which utilize the same cleavage mechanism as described herein for type 4 prepilin peptidase and/or contain the same active site.
- TFPP-like aspartyl protease enzymes of this family can be identified routinely by those of skill in the art based upon the presence of the consensus sequence comprising G-X-G-D- (F or V or P or K or I or L or Y) at the
- Another object of the present invention is to provide methods of inhibiting activity of TFPP-like aspartyl protease enzymes via administration of a compound which targets the aspartic acid residues of the enzymatic active site of the TFPP-like aspartyl protease or mimics a region surrounding the cleavage site of the TFPP-like aspartyl protease substrate and inhibits cleavage activity of the protease .
- bacterial infection in a host is inhibited by administering to a host infected with the bacteria a compound which targets the aspartic acid residues of type 4 prepilin peptidase and inhibits cleavage activity of this peptidase.
- Also related to this embodiment of the present invention are methods and compositions for decreasing development of drug resistant strains of bacteria through coadministration of a compound which targets the aspartic acid residues of type 4 prepilin peptidase and inhibits cleavage activity of this peptidase with a second known therapeutically effective anti- bacterial agent.
- Another object of the present invention is to provide TcpJ mutant constructs useful in defining the active cleavage site of type 4 prepilin peptidase and in X-ray crystal structure analysis of this enzyme.
- Yet another object of the present invention is to provide a homologous type 4 prepilin peptidase gene identified in Staphylococcus aureus.
- Figure 1 is a bargraph showing the percent of conserved homology of potential protease active site residues from 27 type 4 prepilin peptidase homologs .
- conserved residues which do not exist in TcpJ are in parenthesis and are designated with the relative position in TcpJ.
- Predicted membrane topology is indicated by C (cytoplasmic) , P (periplasmic) or M (transmembrane) below each residue. Shaded bars indicate the conserved residues which were altered in experiments described herein. Alignment was performed in DNASTAR MegAlign using the Clustal method.
- Figure 2 is a predicted TcpJ membrane topology model. The predicted topology of TcpJ was deduced by comparison with
- the type 4 prepilin peptidase is found only in bacteria and is responsible for the cleavage and N-methylation of a large number of secreted proteins known as type 4 prepilins, or prepilin-like proteins, that have a type 4 signal peptide at their N-terminus.
- the cleavage of the type 4 signal peptide is a necessary step to secretion of the type 4 prepilin.
- Prepilin-like proteins are involved in DNA competence and/or uptake in B . subtilis, in phage morphogenesis in E. coli , and partially comprise the type 2 secretion system which is the main terminal branch of the general secretory pathway in gram negative bacteria (Pugsley, 1993.
- Type 4 pili serve as the major colonization factors for up to 50 different gram- negative bacterial species. Therefore, agents which interfere with, or inhibit, activity of type 4 prepilin peptidase are expected to be useful as anti-bacterial drugs and in the identification of anti-bacterial drugs. Further, given the role of prepilin-like proteins in DNA uptake, particularly in gram-positive bacteria (Dubnau, D. 1997. Gene 192:191-198; Wolfgang et al . 1999. Molecular Mi crobiology 31 (5) : 1345-1357) , it is believed that coadministration of these agents with a second anti-bacterial agent will inhibit uptake of antimicrobial agent resistance gene thereby reducing development of drug resistant strains of bacteria.
- type 4 prepilin peptidase specifically TcpJ, a type 4 prepilin peptidase of Vibrio cholerae, is a non-pepsin-like acid protease.
- a homologous gene has also now been identified in Staphylococcus aureus, a bacterium which is very difficult to treat due to its ability to become resistant to drugs.
- the nucleic acid sequence encoding this peptidase in Staphylococcus aureus is depicted as SEQ ID NO:l. The amino acid sequence of this
- Staphylococcus aureus peptidase is depicted as SEQ ID NO: 2. Contrary to prior art suggestions of cysteine residues being involved in the protease active site of this enzyme ( (Strom et al. 1993. Proc . Natl . Acad. Sci . USA 90:2404-2408), residues essential for cleavage activity of this peptidase have now been identified as two aspartic acid residues.
- TFPP-like aspartyl proteases of organisms other than bacteria, including humans, which comprise this novel active site and utilize a cleavage mechanism similar to that exemplified herein for type 4 prepilin peptidase can be identified. Accordingly, this peptidase is part of a novel subclass or family of non-pepsinlike acid proteases, referred to herein as TFPP-like aspartyl proteases.
- TFPP- like aspartyl protease it is meant a transmembrane protease enzyme comprising two critical aspartic acid residues on the same side of the membrane at its active cleavage site, wherein a consensus sequence at the C-terminal aspartic acid residue comprising G-X-G-D- (F or V or P or K or I or L or Y) is present. This term is meant to be inclusive of type 4 prepilin peptidases. Further, it has now been demonstrated that agents which target the aspartic acid residues of this active site inhibit the cleavage activity of proteases in this new family.
- inhibitors designed to target the aspartic acid residues, D125 and D189, which are essential to the cleavage activity of type 4 prepilin peptidase can be synthesized for use as anti-bacterial agents.
- the inhibitors are transition state analogues or other compounds that modify or competitively inhibit the aspartic acid residues thereby preventing cleavage activity.
- a mutational strategy was then used to determine the active site residues of type 4 prepilin peptidase of ViJbrio cholerae (Genbank Accession No. M74708) .
- the nucleic acid sequence of this gene is depicted herein as SEQ ID NO: 3; the amino acid sequence of this peptidase is depicted as SEQ ID NO: 4.
- Fifteen of the most highly conserved active site residues were mutated to either alanine or leucine in TcpJ, the type 4 prepilin peptidase of Vibrio cholerae .
- E. coli amber suppressor strains were utilized to determine the activity of TcpJ peptidases with a number of amino acid substitutions at the 125 and 189 positions.
- An amber (TAG) site was individually engineered into the 125 and 189 positions of the pCLlO plasmid and the resultant constructs were introduced into the Amber-Lys, Amber-Leu, Amber-Gin, Amber-Ser, Amber-Tyr amber suppressor strains.
- the strains were grown to mid-logarithmic phase and whole cell extracts were examined for TcpA and TcpJ by Western immunoblot analysis. No amino acid substitution at either 125 or 189 exhibited any cleavage activity.
- the protein levels of TcpJ were all comparable to wild-type TcpJ.
- TcpJ mutant activity was then assessed in an in vi tro assay modeled after the in vi tro peptidase assay devised for PilD of P. aeruginosa (Nunn, D.N. and S. Lory. 1991. Proc . Natl . Acad. Sci . USA 88:3281-3285).
- Crude preparations of TcpA-containing membranes from JM109 p3Z-A and TcpJ-containing membranes from JM109 pCL9 were combined with cardiolipin and 5X assay buffer to commence cleavage of TcpA.
- TcpJ activity was defined as the amount of TcpJ-containing membrane required to cleave 50% of 0.5 ⁇ g of TcpA prepilin in 1 hour.
- pCLH ⁇ 35-81 was introduced into JM109 and a membrane preparation was made and tested in the in vi tro cleavage assay. This assay demonstrated that the TcpJ derivative expressed by the deletion retained activity. Western analysis using an anti-6His antibody confirmed that TcpJ ⁇ 35-81 was expressed and stable.
- TcpJ ⁇ 35-81 To determine the peptidase activity of TcpJ ⁇ 35-81 in vivo, pCLH ⁇ 35-81 and the wild-type control pCLll were introduced into J71K cells. The resultant strains were grown under TCP-inducing conditions. Whole cell extracts were examined by SDS-PAGE and Western immunoblot analysis with anti-TcpA antisera. The results showed that pCLH ⁇ 35-81 can complement J71K similar to pCLll. TcpJ ⁇ 35-81 was able to restore TCP biogenesis to J71K cells as seen by TEM with a negative stain of the cultures.
- domain 1 is not required for type 4 prepilin cleavage or assembly of the resulting mature pilin into the pilus structure.
- analysis of the protein sequence alignment of the type 4 prepilin peptidase family and the mutational strategy using the TcpJ mutant constructs coupled with the in vivo and in vi tro assays confirmed that aspartic acid residues 125 and 189 make up the active site contrary to prior art teachings relating to cysteine pairs.
- TcpJ in vi tro cleavage assay was performed with a number of chemical protease inhibitors to examine their ability to block TcpJ activity. Inhibitors were selected based upon this knowledge of the active site that were expected to effect or not effect this peptidase activity. The effect of chemical protease inhibitors on the peptidase activity of TcpJ was determined by incubating the inhibitor with an amount of membrane preparation known to contain 1 unit of activity of TcpJ for 30 minutes at room temperature. The TcpJ/inhibitor mixture was then tested for peptidase activity in the in vitro processing assay described in Example 3.
- % cleavage (amount of cleaved TcpA in inhibition assay/amount of cleaved TcpA in no inhibitor control assay) x 100.
- the inhibition of EDAC/Glycinamide is a two step protocol conducted in acidic conditions as described in Example 4. The inhibitors tested and the results are shown below in Table 1.
- TcpA cleavage site of TcpA.
- Peptides were tested that were wild-type, or that contained mutations that would prevent cleavage by TcpJ. Their ability to function in trans to compete for TcpJ processing activity of the coexistent wild-type preTcpA was then assessed in a standard western immunoblot which detects precursor and mature forms of TcpA.
- tcpA contained the following mutations relative to its +1 processed amino acid:
- these constructs were electroporated into JM109 cells.
- Antibiotic resistance on the pALTER-Ex2 backbone was to chloramphenicol, with the exception of CL1 which was to tetracycline .
- the wild-type TcpA for which processing was assessed was expressed from pGEM-3ZA/TCP- A which confers resistance to ampicillin. Expression of each peptide was driven via the T7 promoter and T7 RNA polymerase expressed upon infection of the cells with ⁇ DE3 phage.
- peptide is identical to the region surrounding the cleavage site of the target protein or substrate of the TFPP-like aspartyl protease or contains sufficient amino acid similarity such that the peptide mimics this region. As demonstrated herein, these peptide mimics inhibit the cleavage activity of the TFPP-like aspartyl proteases. In a preferred embodiment, these peptide mimics range in length from approximately 12 to 30 amino acids.
- TcpJ is a novel type of protease with two aspartic acid residues as the active site.
- This protease is completely resistant to pepstatin, a general aspartic acid protease inhibitor.
- the protein sequence analysis of the family of type 4 prepilin peptidases indicates that a common protease mechanism exists among these peptidases, regardless of the bacterial species tested. Further, it is believed that TFPP- like aspartyl proteases which utilize the same cleavage mechanism and/or contain the same active site as type 4 prepilin peptidase are present in other organisms, including humans .
- Such enzymes can be identified by the presence of the consensus sequence G-X-G-D- (F or V or P or K or I or L or Y) at the C-terminal aspartic acid residue.
- Wild-type VcpD is known to be capable of at least partially cleaving this prepilin.
- wild-type, D147A and D212A forms of pJM294 were introduced into an E. coli JM109 strain carrying the TcpA-expressing plasmid pRTH3G7. Strains were grown to idlog phase; whole cell protein extracts were made from the cultures; and the extracts were examined by SDS-PAGE and western immunoblot analysis with anti-TcpA antiserum. Wild-type VcpD cleaved the TcpA prepilin completely under these conditions. In contrast, no cleavage occurred in the D147A and D212A mutants.
- one of skill can identify compounds capable of inhibiting activity of type 4 prepilin peptidases and other TFPP-like aspartyl proteases which utilize this same cleavage mechanism and/or contain the same active site. These compounds are expected to be useful as inhibitors of TFPP-like aspartyl proteases. In one embodiment, these compounds can be used as anti-bacterial agents in the inhibition of the type 4 prepilin proteases.
- One example of a method for identifying such inhibitors comprises determining the inhibitory activity of a test compound in an in vi tro cleavage assay.
- a bacterial cell membrane preparation which expresses type 4 prepilin peptidase is incubated with a test compound.
- the amount of cleavage activity in the preparation in the presence of the test compound is then compared to cleavage activity in a preparation which does not contain the test compound.
- Test compounds which decrease or inhibit cleavage activity are inhibitors of type 4 prepilin peptidase as well as other TFPP- like aspartyl proteases. For example, using this method, EDAC in combination with glycine amide was identified as an inhibitor of type 4 prepilin peptidase.
- peptides corresponding to the region surrounding the cleavage site of the prepilin substrate were also demonstrated to be effective inhibitors of type 4 prepilin peptidase.
- High throughput screening assays can also be developed in accordance with well known methodologies to identify these inhibitors. Identified inhibitors are believed to be useful as anti-bacterial agents. These agents are also expected to be useful in inhibiting the cleavage activity of other TFPP-like aspartyl proteases in this subclass which utilize the same cleavage mechanism and/or contain the same active site. Further, knowledge of the chemical nature of test compounds identified as inhibitors and the target site at which these inhibitors must act on the enzyme to inhibit cleavage activity is useful in the identification and/or design and development of additional inhibitory agents .
- test compounds with structures known or suspected to target the aspartic acid residues of type 4 prepilin peptidase or to mimic peptides corresponding to the region surrounding the cleavage site of the prepilin substrate can be identified.
- new test compounds with structures designed to target aspartic acid residues of type 4 prepilin peptidase or to mimic peptides corresponding to the region surrounding the cleavage site of the prepilin substrate can be synthesized.
- the ability of these test compounds to inhibit cleavage activity of this peptidase is then determined via well known methods.
- inhibitory activity is determined via an in vi tro cleavage assay such as that described herein. Similar approaches to those used in the design and selection or identification of protease inhibitors for the treatment of HIV can also be utilized in the design and selection or identification of -these new anti-bacterial agents.
- Test compounds identified as inhibitors of peptidase cleavage activity are believed to be particularly useful as anti-bacterial agents.
- Pharmaceutical compositions comprising an active test compound and a pharmaceutically acceptable vehicle can be formulated in accordance with well known techniques. The compounds can then be administered to inhibit virulence factor production by bacteria and to inhibit bacterial infections in a host.
- host it is meant to include humans.
- active test compounds will also be useful in inhibiting development of drug resistant strains of bacteria when administered in combination with a second known therapeutically effective anti-bacterial agent. In this embodiment, it is preferred that the active test compound be administered just prior to or at the same time as the second anti-bacterial agent.
- the present invention also relates to compositions comprising a compound which inhibits type 4 prepilin peptidase activity and a second known therapeutically effective anti-bacterial agent .
- Example 1 Membrane Preparation Membrane preparation protocol was adapted from the cell fractionation protocol described by Pfau (1998. J. Bact . 180 (17) :4724-33) . Overnight cultures were placed on ice for 20 minutes, then pelleted at 5000 x g for 10 minutes. The cell pellet was resuspended in 1 ml 200 mM Tris pH 8.0 to which 1 ml 50 mM Tris pH 8.0 , 1 M sucrose, 2 ml water, 20 ⁇ l 0.5 M EDTA, and 20 ⁇ l lysozyme (10 mg/ml) were added. The cell suspension was allowed to incubate on ice for 30 minutes.
- Membrane preparations were made from overnight cultures at 42°C of the E. coli K38 that does not express TcpA and E. coli K38 p3Z-A, pGPl-2 which overexpresses TcpA prepilin.
- P3Z-A provides tcpA under the control of a T7 promoter
- GPl-2 provides the heat-inducible T7 RNA polymerase that drives transcription of the tcpA on p3Z-A.
- the volume of the 3 ⁇ g trypsin band, the TcpA prepilin band, the corresponding location of the TcpA prepilin band in the TcpA negative control lane (K38) , and ' an approximately 28 kDa band from both K38 and K39 p3Z-A, pGPl-2 were measured while adjusting to a single background value.
- the mass of the TcpA prepilin present in the K30 p3Z-A, pGPl-2 lane was determined by first subtracting the volume of the TcpA prepilin band from the volume of the corresponding area in the K38 TcpA prepilin-lacking lane.
- TcpA prepilin band value to determine the ⁇ g TcpA/ ⁇ l membrane preparation.
- the TcpA membrane preparation was diluted with 50 mM Tris buffer to a concentration of 0.2 ⁇ g/ ⁇ l.
- Example 3 In Vi tro Cleavage Assay A membrane preparation from the E. coli strain K38 p3Z- A, pGPl-2, which was determined to contain 0.2 ⁇ g TcpA prepilin/ ⁇ l, was the source of prepilin in the in vi tro TcpJ proteases assay instead of purified prepilin.
- a membrane preparation of the TcpJ-expressing strain JM109, pCL9, both wild-type and mutant alleles was the source of the wild-type and mutant TcpJ protein for the in vi tro cleavage reactions.
- the 100 ⁇ l processing reaction is performed by combining a 50 ⁇ l substrate fraction with a 50 ⁇ l enzyme fraction and incubating at 37°C for 1 hour.
- the substrate fraction was prepared by combining 5 ⁇ l of the TcpA-containing membrane preparation (1 ⁇ g TcpA), 10 ⁇ l 0.5% w/v cardiolipin, 20 ⁇ l of 2X assay buffer (125 mM triethanolamine HC1 pH 7.5, 2.5% v/v Triton-X-100) , and brought up to the total volume with water.
- the enzyme fraction was prepared by combining varying volumes of TcpJ-containing membrane preparation and water to bring the volume to 50 ⁇ l .
- the cleavage reaction was stopped by the addition of 100 ⁇ l of 5X protein sample buffer.
- the EDAC/glycinamide inhibition protocol is a two step chemical reaction that modifies carboxylic acid functional groups, such as those that partly make up the R-group of aspartic acid and glutamic acid. This protocol is based on well known procedures set forth for the selective modification of carboxyl groups in proteins .
- TcpJ containing membranes containing the equivalent of 6 units of TcpJ activity were incubated in 80 ⁇ l with 25 mM potassium biphthalate, pH 4.0, and 100 mM EDAC (l-ethyl-3- (3-dimethlaminopropyl) carbodiimide hydrochloride) for 30 minutes at room temperature.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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EP00947169A EP1200567A4 (en) | 1999-07-12 | 2000-07-11 | Compounds and methods for identifying compounds which inhibit a new class of aspartyl proteases |
CA002379269A CA2379269A1 (en) | 1999-07-12 | 2000-07-11 | Compounds and methods for identifying compounds which inhibit a new class of aspartyl proteases |
US10/030,808 US6887677B1 (en) | 1999-07-12 | 2000-07-11 | Compounds and methods for identifying compounds which inhibit a new class of aspartyl proteases |
US11/071,972 US8609087B2 (en) | 1999-07-12 | 2005-03-04 | Compounds and methods for identifying compounds which inhibit a new class of aspartyl proteases |
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US14335599P | 1999-07-12 | 1999-07-12 | |
US60/143,355 | 1999-07-12 |
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US11/071,972 Continuation-In-Part US8609087B2 (en) | 1999-07-12 | 2005-03-04 | Compounds and methods for identifying compounds which inhibit a new class of aspartyl proteases |
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