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Kaposi’s sarcoma—associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS), the most common cancer in AIDS patients. All herpesviruses express a conserved dimeric serine protease that is required for generating infectious virions, and is therefore of pharmaceutical interest. Given the past challenges of developing drug-like active-site inhibitors to this class of proteases, small-molecules targeting allosteric sites are of great value. In light of evidence supporting a strong structural linkage between the dimer interface and the protease active-site, we have focused our efforts on the dimer interface for identifying dimer disrupting inhibitors. Here, we describe a high throughput screening approach for identifying small molecule dimerization inhibitors of KSHV protease. The helical mimetic, small molecule library used, as well as general strategies for selecting compound libraries for this application will also be discussed. This methodology can be applicable to other systems where an alpha helical moiety plays a dominant role at the interaction site of interest, and in vitro assays to monitor function are in place.
Herpesviruses make up one of the most prevalent viral families including eight human types that cause a variety of devastating illnesses. The standard course of treatment for common herpesviral infections is a class of broad—acting viral DNA replication inhibitors, which exhibit undesirable toxicity, poor oral bioavailability, and in some cases inadequate efficacy. Efforts by pharmaceutical companies to target the active-site of the essential dimeric serine protease of human herpesviruses (HHV) have yet to yield a drug-like candidate(1–6). Given the evidence supporting a conformational linkage between protease dimerization and activation, we have focused our efforts on identifying molecules that target the dimer interface(7–12). In the case of KSHV protease (KSHV Pr) the dimer interface covers approximately 2500 Å2, and includes the α-helix 5 of each monomer as the major constituent (Figure 1)(13). In vitro studies with KSHV Pr and other HHV proteases have shown the dimer interface to be very sensitive to genetic perturbation, where single point mutations often lead to a loss of dimerization and activity(11). Furthermore the dimerization affinity is weak; with a reported KD of 1.7µM for KSHV Pr(10). These characteristics define the dimer interface as a suitable candidate for dimerization inhibitors. We first tested this approach by inserting the key interfacial α—helix 5 residues on the internally—stabilized α—helix of a mini—protein(14). The resulting macromolecule disrupted the KSHV Pr dimer and inhibited enzyme activity, proving that targeting the dimer interface is a viable route for identifying novel inhibitors. In order to identify small molecule dimer disruptors of KSHV Pr, a workflow was developed that begins with high throughput screening (focus of this chapter), and continues with experiments that assess dimerization, mode of binding, and broad specificity against other HHV proteases (Figure 2) (15,16). Since two α-helices are the major component of the KSHV Pr dimer interface, screened a library of helical mimetic small molecules. This library was comprised of roughly 200 compounds that were originally developed by computational design to disrupt the interacting α—helix of p53 tumor suppressor protein with oncoprotein MDM2(17). Screening was performed in a 96—well plate format using a fluorogenic activity assay. The substrate used is an optimized hexa—peptide attached to 7—amino—4—carbamoylmethyl coumarin (ACC), where cleavage at the scissile bond releases the ACC group resulting in increased fluorescence(7,12). We have successfully used this approach to identify the small molecule inhibitor DD2 (Figure 3), which binds a novel allosteric pocket at the dimer interface of KSHV Pr, and traps it in an inactive monomeric state(16). Here we describe the important considerations in library selection as well as the detailed steps in the high throughput fluorogenic screening assay.
Recombinant KSHV Pr is expressed in Escherichia coli and purified as reported previously(16). 80 µM aliquots of purified protease are flash frozen in storage buffer (same as assay buffer described in section 3.2) and stored at −20 °C. The total amount of protein required will vary depending on the assay and the number of plates being screened.
The protease substrate is an optimized hexa—peptide with the fluorogenic reporter group 7— amino-4—carbamoyl—methylcoumarin (ACC), which allows for monitoring enzyme activity spectroscopically. The peptide sequence is Ac—Pro—Val—Tyr—tBug—Gln—Ala—ACC with an observed KM of 8.5 ± 0.8 µM for KSHV Pr. Substrate is synthesized using standard FMOC chemistry and purified as reported previously(7,12). Substrate stocks of 10 mM in 100% DMSO are stored at −20 °C. The total amount of substrate required will vary depending on the assay and the number of plates being screened. (Ac = acetyl group, tBug = t-butyl glycine).
For general considerations on library selection see Note 1. Compound libraries are generally provided in ready-to-use format, dissolved in 100% DMSO and plated in either 96—well or 384—well plates. An additional dilution step with 100% DMSO or plate reformatting may be necessary depending on the screening assay conditions. In order to avoid screening artifacts resulting from compound aggregation or precipitation, a final screening concentration between 10—30 µM is recommended. Our compound library was in a ready-to-use format at a concentration of 1mM in a 96—well plate. Compound libraries are stored at −20 °C.
Positive and negative controls are an important measure of assay performance and should ideally be included in every row (or column) of the assay plate. In assays, where timing is of particular importance, such as a protease assay, these controls allow for accurate calculation of % inhibition in each row (or column), as well as comparison of data across all rows (or columns). DMSO serves as the negative control and is used in the place of test compound. An ideal positive control may be a known active compound, such as a protease inhibitor in our case. The total amount of control compound required will vary depending on the assay and the number of plates being screened. At the time we performed our screen potent reversible KSHV Pr inhibitor did not exist, therefore substrate alone, which mimics the absence of protease activity, served as the positive control.
Prior to performing a high-throughput screen, it is critical to evaluate the quality of the screening assay by calculating a Z’—factor (see Note 2). High-throughput screening is typically performed in small assay volumes, therefore small fluctuations in liquid handling may affect the final readout. For this reason working with professionally calibrated pipettes is highly recommended. The following is a step—by—step protocol for manually performing a screen in one 96—well plate. The ability to screen multiple plates at once depends on the assay conditions and access to liquid handlers.
This work was supported by NIH grants T32 GMO7810, AIO67423 (C.S.C.), P50 GM 082250 and by the American Lebanese and Syrian Associated Charities and St Jude Children’s Research Hospital (R.K.G.).
1KSHV Pr activity is regulated by a dimerization-driven conformational switch. The two α- helices located at the dimer interface became our rationale for screening a helical mimetic small-molecule library for inhibitors. α-helices make up the largest class of protein secondary structure and are commonly found at the interface of protein-protein interactions(18,19). Therefore significant effort has been aimed at developing non-peptide small molecule mimetics of α-helices. The key characteristic of such molecules is a rigid scaffold with functionalities presented in the same orientation as the i, i + 3 or i + 4, and i + 7 residue positions on an α—helix. The chemotypes of commonly reported scaffolds include biphenyls, allenes, alkylidene cycloalkanes, spiranes, benzylideneacetophenones, trisubstituted imidazole, indanes, polycyclic ethers, benzodiazepines and teraryl units(18). We had access to a small focused library of helical mimetic small molecules synthesized to disrupt the interaction of p53 and MDM2, which also includes an α-helix(17). The scaffolds were made of two or three aryl rings connected by amide bonds, with side chains in the i, i + 4, and i + 7 positions. It is worth noting that commercial helical mimetic libraries, as well as general protein-protein interaction libraries, are now available for purchase through companies like BioFocus. In the absence of suitable small molecule test candidates, peptidic alternatives such as hydrocarbon-stapled helical peptides can also serve as a starting point(20–22).
2The Z’—factor is the measure of assay quality and is vital to identifying true hits in a high throughput screen(23). The value is typically calculated from the positive and negative control experiments, which in our screen correspond to the “low” and “high” signals respectively. The resulting numerical values range between 0 and 1, where numbers closest to 1 are favorable. The method of calculating the Z’—factor has been reported previously. Z’—factor optimization is achieved by varying the assay conditions such that the separation between the “low” and “high” signal is increased. We optimized our KSHV Pr assay by increasing the assay incubation temperature, the reaction incubation time, and including centrifugations steps.
3DMSO exhibits an inhibitory affect in most assays, therefore its final concentration should be kept to a minimum. However, library compounds and protease substrates are generally dissolved in 100% DMSO stocks, and often require some DMSO to remain dissolved in aqueous buffer. For this reason it is important to determine the DMSO tolerance of screening assays. This is done by performing the basic assay in the presence of increasing concentration of DMSO until an inhibitory condition is reached. The DMSO tolerance for the KSHV Pr assay is 5% (v/v).
4Some compounds form aggregates that interact with proteins in a non-specific manner(24,25). Aggregate forming compounds may result in a false-positive inhibitory effect when screened as part of large libraries. Including a small amount of detergent, such as 0.01% Triton X—100 (v/v), will eliminate aggregation-based hits in most cases(25). In the case of assays like the KSHV Pr assay that don’t tolerate detergents, including 1mg/ml BSA eliminates non-specific interactions. Ideally detergents are included in the initial screen. However, since the library we screened was small, we counter screened our hits in the presence of BSA.
5Some compounds may exhibit fluorescent properties in the wavelengths monitored by the assay. For example a compound that absorbs light in the same region as our ACC substrate, may interfere with the final assay readout. Therefore, it’s important to monitor the abortions and emission properties of all screening hits at the wavelengths used to make read measurements.
6A true inhibitor has a sigmoidal dose response cure with a hill slop value that is around 1. The concentration of inhibitor that achieves 50% inhibition is the IC50. Dose response curves are obtained by repeating the screening assay with increasing concentrations of compound, ranging from 0 to 100 µM. Generally a 3-fold dilution of compound is used (100 µM, 33.3 µM, 11.1 µM, etc.). Percent inhibition values (see Methods 3.11) are then plotted as a function of inhibitor concentration.
7Since the goal of the screen is to identify dimer disruptors, secondary assays that monitor dimerization must be available. We used two independent but complementary methods to determine weather our hit DD2 was a dimer disruptor(7,16). The first was an FPLC assay and the second was a 2D—NMR approach using protease containing a reporter at the dimer interface. Both methods were carried out as described previously.