This invention concerns a method for measuring the activity of dual substrate enzymes, and for identifying compounds that regulate such enzymes, and as such are useful for treating bacterial infections, disorders of lipid metabolism and cellular proliferation.
Millions of individuals are afflicted with diseases that are caused by the inability to regulate important cellular processes such as fatty acid biosynthesis and cell proliferation. For example, the inability to regulate fatty acid biosynthesis results in lipid metabolism disorders that play a major role in diseases that include obesity, chronic hepatic encephalopathy, lipid diabetes, insulin resistant diabetes, and coronary heart disease. Failure to regulate cell cycle events can lead to unregulated cellular proliferation resulting in the growth of cancerous tissue and the malignant state. Some well known cancers that plague our society include breast cancer, prostate cancer and colon cancer. Thus, much research is carried out to understand fatty acid biosynthesis and cell proliferation to fulfill the need to treat these diseases and to maintain normal regulation of these processes. Much of the regulation of fatty acid biosynthesis and cell proliferation is carried out by enzymes. Often, too much or too little activity of specific enzymes can disrupt regulation of the processes they control. Since abnormal levels of activity of even a single enzyme can lead to the diseases described above, a great deal of research is being carried out to identify compounds that modulate the activity of key enzymes. For example, the enzyme acteyl CoA carboxylase (ACC) produces malonyl-CoA, which regulates fatty acid biosynthesis in cells. Mice lacking ACC were recently shown by Abu-Elheiga et al. (Science 291:2613-2616, 2001) to suffer major decreases in body fat (50%) despite increased food intake. Thus, inhibition of ACC might allow individuals suffering from obesity to lose weight while maintaining normal caloric intake.
Bacterial enzymes are also the focus of research to identify compounds that modulate enzyme activity, and thus can be used to treat bacterial infections. Resistance to commonly used antibiotics is widely prevalent and a growing concern in clinics and hospitals, thus causing a great demand today to identify new antibacterial compounds that are not plagued with resistance problems. Recently, much attention has been focused on targeting bacterial enzymes involved in fatty acid biosynthesis. In particular, enzymes involved in the bacterial fatty acid biosynthetic pathway are a popular target for the development of antibacterials for treating infectious diseases.
While some enzymes require a single substrate to function, others require two substrates. These are called dual substrate enzymes, or two-substrate enzymes. It is estimated that more than half of known biochemical reactions in mammals are catalyzed by dual substrate enzymes. Therapeutic areas whose drug discovery efforts focus on two-substrate enzymes include cancer, lipid metabolic disorders and anti-infectives. For example, some enzymes implicated in various cancers are kinases such as MEK, which are dual substrate enzymes. Enzymes involved in the fatty acid biosynthesis (FAB) pathway in the bacterial pathogen E. coli include malonyl coenzyme A-acyl carrier protein transacylase (FabD), B-ketoacyl-ACP reductase (FabG), B-ketoacyl-ACP synthase III (FabH) and enoyl-ACP reductase (FabI).
The current assays for identifying compounds that inhibit dual substrate enzymes have significant limitations. A new assay for the B-ketoacyl-ACP synthase III (FabH) was recently reported by He X. et al., 2000, Anal. Biochem. 282: 107-114. However, that assay is limited to using substrates that are radiolabeled with tritium, and substrates that are radiolabeled with other isotopes such as carbon-14 cannot be used in that assay. Assays for the identification of inhibitors of kinases often rely upon precipitation of a radiolabeled peptide or protein with trichloroacetic acid. Performing these assays requires the processing of large amounts of corrosive liquid, which is damaging to the instrumentation as well as the environment, and presents a high level of risk to the researcher. In addition, the use of viscous liquid scintillation cocktails in assays requiring radiometric detection presents a difficult challenge for high throughput screening processes. Individuals who perform these assays experience frequent equipment failure due to the corrosive liquid and precipitation of the peptide or protein. Consequently, these problems lower the quality of the assay, result in statistically unreliable data, and prohibit the use of these assays in high throughput screening processes. Furthermore, these conventional methods require numerous steps making the overall assay lengthy, and are an impediment to rapid drug discovery.
An object of this invention is to provide a rapid and reliable assay for identifying and analyzing compounds that modulate the activity of dual substrate enzymes.
This invention provides an assay for identifying and measuring the activity of compounds that interact with dual substrate enzymes. The assay utilizes a resin to capture and bind to one of the two substrates that interact with the dual substrate enzyme. Typical resins are in the form of beads, which can be charged. A typical substrate for the FabD enzyme is called Acyl Carrier Protein (ACP). The capture of ACP by a resin such as charged beads eliminates the need for trichloroacetic acid precipitation. The virtual unlimited surface area provided by such resins overcomes the limited binding capacity of standard microplate wells that are required by conventional assays. Use of these resins permits the use of a low-specific activity radiolabeled form of one of the substrates in the assay. Furthermore, the use of resins eliminates the need for modifying enzymatic substrates for capture, for example by synthesizing a biotinylated tag on the substrate. The present assay also uses scintillation proximity assay resins for measuring radioactivity, thus eliminating the need for liquid scintillation cocktails used in the prior art assays, which are comprised of toxic organic substances that are difficult to work with. The co-dispensing of resins (i.e., the capture resin and the scintillation resin) in this invention reduces the number of steps that must be performed according to prior art assays. The wide variety of commercially available resins that can be used in the present assay offers universal applications for substrate capture and scintillation counting. The utility of this invention is demonstrated by the measurement of enzyme activity and the activity of both known enzyme inhibitors, as well as potential inhibitors from chemical libraries.
This invention is a method for identifying an inhibitor of a dual substrate enzyme; wherein a first substrate is a macromolecule that is enzymatically modified in the presence of the dual substrate enzyme to accept the radiolabeled portion of a second substrate, said second substrate bearing a radiolabeled portion, such that some or all of the radiolabeled portion of the radiolabeled substrate is transferred to the macromolecule substrate to form a radiolabeled macromolecule, said method comprising:
a. adding a capture resin to a buffered mixture of an enzyme, a non-radiolabeled first substrate, a radiolabeled second substrate, allowing the enzyme to catalyze transfer of the radiolabeled portion of the radiolabeled second substrate to the non-radiolabeled first substrate, in the presence or absence of a test compound;
b. removing unreacted radiolabeled second substrate;
c. adding a scintillant resin to the enzyme-radiolabeled first substrate mixture; and
d. measuring the amount of radiolabeled first substrate reacted in the presence of a test compound by scintillation counting, measuring the amount of radiolabeled first substrate reacted in the absence of a test compound by scintillation counting, and comparing the two measurements.
The assay provided by this invention includes a method wherein the first substrate is a macromolecule selected from a peptide or protein.
In a preferred embodiment, the first substrate is an Acyl Carrier Protein (ACP).
In another embodiment, the enzyme is selected from a fatty acid biosynthesis enzyme, a phosphate transfer enzyme, or a protein kinase enzyme.
Another embodiment of the invention includes use of a scintillation proximity assay resin (SPA) as the scintillant used for measuring the radiolabeled first substrate.
In a preferred embodiment of this invention, the unreacted radiolabeled second substrate is removed by filtration.
Another aspect of the invention is a method wherein the filtration of radiolabeled second substrate is carried out using an automated filtration and washing apparatus.
In another embodiment of the invention, the first substrate may be radiolabeled, with the second substrate being nonradiolabeled, and wherein the enzyme is an agent that catalyzes the transfer of the radiolabeled portion of said first substrate to said second substrate.