key: cord-0829352-h11bcb1e authors: McCall, J. Owen; Kadam, Sunil; Katz, Leonard title: A High Capacity Microbial Screen for Inhibitors of Human Rhinovirus Protease 3C date: 1994 journal: Biotechnology (N Y) DOI: 10.1038/nbt1094-1012 sha: 240d5cb8a1344e58ba5c10422027d0c9f788f923 doc_id: 829352 cord_uid: h11bcb1e We have developed a high capacity screen for compounds that inhibit the 3C protease of human rhinovirus–1b. The assay uses a recombinant strain of Escherichia coli expressing both the protease and a tetracycline resistance–conferring protein modified to contain the minimal protease cleavage site. Cultures growing in microtiter plates containing tetracycline are treated with potential inhibitors and simultaneously monitored for change in growth over time using an oxygen probe. Most of the cultures, not containing an inhibitor of the 3C protease, show reduced growth due to cleavage of the essential gene product; normal growth is seen only in the infrequent culture that contains an inhibitor. In the present example, we have used the tetA gene of plasmid pACYC184 as the modified gene. The system has been validated using inhibitors of protease 3C, and has been used to identify three new inhibitors of the enzyme, active in the micromolar range. A derivative of the 3C protease of HRV strain lb is expressed by E. coli. The genome ofHRV is a single molecule of positive strand RNA whose sequence consists of one long open reading frame encoding a polyprotein of 2,157 amino acids. Following synthesis, the polyprotein is processed to yield all the constituent enzymatic and structural pmteins of HRV. Most of the proteolytic processing sites are cleaved specifically by the 3C protease, whose activity is an absolute requirement for viral development 9 . RT-PCR was used to amplify the protease 3C coding region of HRV strain lb, along with the small upstream coding region 3B. The amplified DNA was cloned into the pUC18-based expression vector pKB130 (ref. IO) , placing expression of the cloned sequence under the control of the tightly regulated araBAD promoter (Fig. l) . This construction directs the expression of the 3B-3C portion of the polyprotein as a fusion to the eleven N-terminal amino-acyl residues of E. coli 11-galactosidase (/lG'). The resulting plasmid, pOM99, synthesized an arabi-1012 BIO/TECHNOLOGY VOL. 12 OCTOBER 1994 nose-inducible protein with a molecular weight of 25 kD as determined by SDS-PAGE (data not shown), consistent with the predicted molecular weight of 23 . 6 kD for unprocessed {3G' -3B-3C product. (The molecular weight of mature 3C protease is 20.5 kD.) The induced protein, whose identity was verified by N-terminal sequencing, constituted approximately 4% of tot.al cellular protein as determined by laser scanning densitometry of coomassie-stained SDS-PAGE gels. The material was found exclusively within the insoluble fraction of whole cell lysates (data not shown). The protease 3C derivative shows intracellular proteolytic activity. Though insoluble and apparently not capable of significant self-processing, the protease 3C derivative displayed specific proteolytic activity against a modified bacterial target protein supplied in vivo. The tetracycline resistance-conferring protein TetA, encoded by the plasmid pACYC184 (ref. 11) , was used to construct a target molecule for rhinovirus protease activity. It had been shown previously that small, in-frame insertion mutations of tetA can be introduced at the unique Sall site of this plasmid without significant loss of tetracycline resistance 12 • An oligonucleotide encoding the 10 amino acid sequence pOM99 (4165bp) FIGURE 1. Schematic diagram of plasmid pOM99, constructed for the expression of HRV protease 3C in f. coli. The 3B-3C coding region of the human rhlnovirus genome (strain 1 b) was amplified using RT-PCR, and inserted into the expression plasmid pKB13010 as an Sstl-Pstl fragment. The translation product contains an Initial eleven amino acids derived from the amino terminus of Lacz. Transcription initiates from the strong, tightly regulated araBAD promoter, which is controlled by the araC regulatory gene also on the plasmid. Expression is induced by the addition of arabinose to the growth medium. • FIGURE 2. LB agar plates containing 10 µg/ml tetracycline, streaked with strain OM86 (OH5a/pOM98, pOM99) and incubated overnight at 37°C. The plate on the left contains arablnose, the plate on the right does not. --~c m, EVLFQGPVYR was ligated into the Sall site, resulting in the production of a TetA protein containing a protease 3C cleavage site (TetA'c). The amino acid sequence inserted does not exactly correspond to any known rhinoviral cleavage site, but rather is one that has been shown to be efficiently cleaved in vitro using synthetic oligopeptides 13 • 14 • The modified pACYCl84, designated pOM98, was introduced into the strain carrying the protease 3C expression plasmid pOM99. On plates containing 10 µglml tetracycline, overnight growth of this strain carrying the two plasmids was indistinguishable from that on plates lacking the antibiotic. However, when the growth medium was supplemented with arabinose to induce the synthesis of the protease 3C, growth was severely curtailed (Fig. 2) . When pOM98 was replaced with pACYC184 in the same host, the addition of arabinose to the growth medium had no effect on growth (see below), indicating that inhibition of growth in the presence of arabinose was dependent upon the presence of the protease 3C target sequence within the TetA protein. To verify that the protease 3C derivative actually cleaved the modified TetA protein within the bacterial cell, "S-methioninelabeled cultures were prepared in both the presence and absence of arabinose. Whole-cell protein extracts were subjected to SOS-PAGE and autoradiography (Fig. 3) . Growth in the absence of arabinose (lane 1) produced two prominent bands, corresponding to the pOM98-coded Cml (chloramphenicol acetyltransferase) and TetAJC proteins. When arabinose was included in the growth medium, the TetA 3 c band was no longer observed (lane 2), while the Cml band remained. The resulting TetA 3 c cleavage products were not visible, probably due to the rapid degradation of nonfunctional proteins often seen in E. coli' 5 • When the protease 3C gene was absent from the expression vector, arabinose induction failed to cause the disappearance of the band. The expression strain lacking pOM98 or pACYC184 fails to produce the bands marked "Cml" and "TetA/TetK" in Figure 3 (data not shown). A high throughput screen for inhibitors. To enable large numbers of cultures to be handled and processed efficiently, cells were grown in 96 well microtiter plates, enabling the semiautomation of manipulations and full automation of data collection. Although protease induction clearly sensitizes cells to tetracycline during overnight growth on agar plates, it was found that the difference in growth rate between induced and noninduced broth cultures was too small to be differentiated by monitoring the change in culture optical density over the shorter time periods (60-90 min.) desirable in a high-throughput assay (Fig. 4) . However, since growth under the conditions used quickly becomes oxygen-limited, growth rate was determined by measuring the change in fluorescence emission of an oxygenquenched ruthenium complex' 6 applied to the floor of the microtiter wells. As oxygen in the medium is depleted by growing cells, fluorescence increases to a maximum level determined by the concentration of the indicator. The addition of arabinose caused protease induction and reduced cellular oxygen uptake due to the intracellular accumulation of inhibitory levels of tetracycline. Since the microtiter trays are incubated unsealed, a reduction in oxygen uptake allows air to diffuse into the growth medium and quench the fluorescence probe causing the gradual decrease in fluorescence (closed triangles, Fig. 4 ). The growth (respiration) of uninduced strain OM86 (DH5adpOM98, pOM99) caused an increase in fluorescence by rapidly depleting available oxygen during the first hour of growth (open squares, Fig. 5 ). In the presence of arabinose, however, the growth rate was decreased, reflected by a much slower depletion of oxygen from the culture medium (closed symbols, Fig. 5 ). Under these conditions, the protease 3C synthesized is apparently able to cleave the tetracycline resistance protein, resulting in inhibition of growth by the antibiotic. Addition of the target oligopeptide (EVLFQGPVY) to the growth medium reversed the growth inhibition , presumably by competing with the target protein for proteolysis (triangles, F ig. 5). A control peptide of similar size (eight residues) had no effect on growth (data not shown). Oligopeptide added at O and 90 minutes produced a burst of growth followed by a decline during subsequent 30 minute periods. Thus the peptide can readily compete with the TetA 3 c protein allowing growth in tetracycline-containing medium. As the intracellular pool of the target peptide diminishes, however, • hydrolysis ofTetA 3 c increases and the cells again become sensitive to tetracycline. It has been found that peptides larger than about six amino acids are generally not efficiently taken up by wild-type E. coli 11 • However, our data strongly suggest that the nine amino acid target peptide is entering the cells. The size limitation on uptake is believed to be a function of outer membrane pore size since the hydrodynamic volume of the peptide, rather than the number of amino-acyl residues, appears to be the critical fac-tor11. It is possible that the particular sequence of the target peptide allows compact folding or that very little needs to enter the cell to be effective. Alternatively, the TetA protein may be assisting in the entry of the peptide. Tetracycline resistance is known to be associated with increased uptake of aminoglycosides, compounds of greater molecular weight than the target peptide 18 . Novel inhibitors of protease 3C were identified using the fluorescence-based as.say. More than 20,000 natural product extracts or purified compounds were examined using the highthroughput format described above. Two active compounds, shown in Figure 6 , were recovered from microbial extracts. The phytotoxin radicinin 19 [I) was purified from a microbial extract identified using this assay, as was citrinin hydrate [2), a novel compound similar to the microbial toxin citrinin 20 • A protease 3C inhibitor was also identified from the group of pure compounds screened. Kalafungin [3), a polyketide antibiotic 2 1. 22 , was selected for testing due to its resemblance to thysanone, a natural product previously shown to be an inhibitor of protease 3C 23 • These three compounds were also found to inhibit the activity of purified protease 3C in an in vitro assay based on the hydrolysis of the target peptide (manuscript in preparation). The IC50 for these three substances were: radicinin, 500 µM; citrinin hydrate, 280 µM; kalafungin, 3.3 µM. A detailed description of these compounds will be published elsewhere. We also tested these compounds, as well as TPCK (N-tosyl-L-phenylalanine chloromethylketone, a nonspecific thiol protease inhibitor2 4 ) and the target peptide by spotting solutions on LB plates containing both arabinose and tetracycline that had been seeded with strain OM86 (DH5n/pOM98, pOM99). All five compounds exhibited activity, seen as zones of growth surrounding each spot following overnight incubation (data not shown). Kalafungin, radicinin, and citrinin hydrate, all produced large zones of protection, probably due at least in part to their hydrophilic character and corresponding diffusibility. The target peptide, a larger hydrophobic molecule with limited diffusibility, produced a smaller zone. To verify that exposure to these compounds has a direct effect upon the intracellular levels of TetA ,c, autoradiographs were prepared as before, using arabinose-induced cultures grown in the presence of three of the inhibitors. In Figure 3 , lanes 3, 4 and 5 represent cells treated with kalafungin, radicinin and citrinin hydrate respectively. All show various levels ofuncleaved TetA'c protein, indicating substantial protection from 3C cleavage. In searching for enzyme-specific inhibitors, the initial screening is often done using an in vitro assay comprised of the purified enzyme and substrate, in this case the viral protease and a synthetic peptide target; the more laborious in vivo (infected cell) assay is usually reserved for verifying activities identified using the in vitro assay. In vitro assays may have limitations. First, a sufficient supply of soluble, active, purified enzyme must be available. Second, nonspecific inhibition of the protease can become a problem, especially in complex mixtures such as natural product extracts. Finally, large or hydrophilic molecules 1014 BIO/TECHNOLOGY VOL. 12 OCTOBER 1994 ' 8.5 !C ... .,., that appear as active in an in vitro screen often cannot traverse a cell membrane and are, therefore, not therapeutically useful. The screen described here is an attempt to overcome these limitations by using a bacterial cell-based design. The strategy of this screen centers upon the rescue of a growing culture from the lethal activity of protease 3C against a (conditionally) essential bacterial protein. This strategy was first described by Block and Grafstrom, who constructed a prototype screen based on the protease of HIV-I but did not use it to actually screen compounds". We found the prototype screen to be unworkable in practice, mainly due to the high toxicity of HIV-I protease to the host cell (unpublished results). The screen presented here uses a viral protease with a much more restricted specificity and contains modifications to improve clonal stability and throughput. The assay is based upon strain OM86 (DH5a/pOM98, pOM99) which expresses HRV-1 b protease 3C under control of the araBAD promoter, and constitutively expresses a mcxlified TotA protein containing a protease 3C cleavage site (TetAK). The addition of arabinose to the growth medium induces expression of the 3C protease, which results in the inactivation of cellular TetA 3 c protein, rendering the cells sensitive to tetracycline. Adding a protease inhibitor to the growth medium will reestablish tetracycline resistance and pennit growth if ( l) the inhibitor has sufficient activity against protease 3C that TetA:,c cleavage will be blocked, (2) a sufficient amount can traverse the cytoplasmic membrane, and (3) it is not itself toxic to the cell. Cells growing in the presence of tetracycline therefore die following the addition of arabinose, unless rescued by the presence of an inhibitor of the 3C protease. In addition to the obvious need for a protease inhibitor to have a high level of activity against its target, it must also possess many other characteristics to be therapeutically useful. Chief among these are the ability to reach the target molecule within the cell and a lack of cellular toxicity. Since bacterial and human cells share many features, we reason that any candidate compound that is either unable to traverse a cell membrane, or shows initial toxicity, should be eliminated in a first screen. Although porin mutants of E. coli are available wherein the cell membrane presents less of a barrier than in wild-type cells, such strains were less favored, along with the naturally more penneable Gram positive species, due to an excessive rate of positive sample presentation (data not shown). The araB operator/promoter was chosen to control 3C protease expression due to its very low basal level of transcription in the noninduced state 25 • We have found that this can be an important consideration as overexpression of some viral proteases is extremely toxic to bacterial cells (unpublished observations), and even sublethal baseline expression of such proteins could lead to clonal instability. Bacterial strain, plasmids and media. All work employed £ . coli strain DHSc. [F· q,80dlacZaM !5 endAJ recAl hsdRJ7(r,-m• ') sup£44 thi-1 >,-gyrA96 re/Al a(lacZ}kargf)Ul69] (Bethesda Research Labs, Gaithersberg, MD). In all instances cells were grown using Luria-Bertani (LB) broth or plates26, el Proteases and their inhibitors: today and tomorrow Conference rcport-Elastasc inhibitors for the treatment of emphysema-Approaches to synthesis and biological evaluation The role of proteases in growth, invasion. and spread of cancer cells Candida proteinases and candidosis Participation of the proteolysis system in realization of influenza virus virulence and development of the infectious process: Antiviral effect of pmtease inhibitors Enzyme inhibitors in medicine Isolation ofrhinoviruses and coronaviruses from 38 colds in adults ls a rhinovirus vaccine possible? 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The sy nthesis of a tetrapeptide Spectroscopic characterization of complexes of ruthenium (II) and iridium (Ill) with 4,4' -diphenyl,2 .2' ,bipyridine and 4,7-diphenyl-1,10-phenanthroline The interdction of 1.-ardbinose and o-fucose with ArdC protein We thank Sally Darwin for the amino-terminal protein sequence determination, Ken Idler for nucleotide sequence determination, Jyoti Patel for oligonucleotide synthesis, Carole Carter for growing the virus, Ronald Rasmussen for synthesis of the fluorescent substrate, Warren Kati for the IC,., determinations, Shaun Tennant and Jill Hochlowslci for chemical isolations , and Jennifer Poddig for assistance in assay development and screening.