The HTS assay based on relieving the growth inhibition associated with the in vitro activation of T3S secretion in Y. pestis
described above has proved to be both effective and convenient. The rate of false-positive results was low, in the sense that the screening of 70,966 compounds yield only 431 positive hits (0.6%), among which strong positive hits were observed at a rate of 0.01%. Z
′ values (43
), a statistical measure of assay quality, between 0.5 and 1 are generally regarded as excellent for HTS methods. In our HTS assay, the Z
′ value for random assay plates averaged 0.7 (31
). The assay clearly yields bona fide inhibitors. Addition of the test compounds to the bacterial suspension in the appropriate medium is the only preincubation manipulation required, and luminometry is the only postincubation step. The use of a luminescent Y. pestis
derivative allows a reliable means of detection of modest growth at low bacterial densities and avoids interference by common artifacts (e.g., bubbles) that afflict direct measurements of growth by determination of the OD. This method is strongly biased against compounds that inhibit bacterial growth, an effect that would yield false-positive results by some other approaches. It also selects for compounds that act on the bacteria rather than on target cells, as the latter are not present in the assay. The chief disadvantage of this approach is that it may fail to detect some classes of inhibitors because they do not relieve growth inhibition. For example, compounds that interfere at the level of formation of the connection of the secretion apparatus with target cells would probably not be detected.
Several other techniques have been used to screen for T3S inhibitors. These include enzyme-linked immunosorbent assay-based detection of proteins secreted from EPEC (19
), inhibition of sheep erythrocyte lysis by EPEC (1
), and inhibition of induction of a yopE
fusion in Y. pseudotuberculosis
. The erythrocyte lysis assay probably depends on pore formation by T3S translocon proteins (EspB and EpsD in EPEC, which are paralogues of Yersinia
YopB and YopD, respectively), while the yopE
fusion assay depends on the observation that the induction of T3S in the yersiniae is accompanied by substantial increases in the levels of transcription of effector genes, including yopE
). Each of these approaches has yielded some potential inhibitors. The yopE
fusion method has been used most extensively, because, like our method, it is well suited to HTS. Initial experiments by the use of this method yielded 4 lead compounds from the screening of 9,400 compounds from the ChemBridge DiverSet F library. Initial structure-activity relationship studies (13
) have been performed with two of these compounds, a salicylaldehyde compound (previously designated INP0007 [20
]) and a 2-arylsulfonylamino-benzanilide, by using activity assays based on whole bacteria, because the targets of these compounds are not established. The activities of INP0007 and selected analogs against both Salmonella enterica
serovar Typhimurium have been tested in vitro and in a tissue culture model (20
), and the activities of these compounds against Chlamydia trachomatis
have been tested in tissue culture (2
). The results of these studies are consistent with the inhibition of T3S in these species.
The inhibitory compounds identified in our assay are a diverse group and in all cases are distinct from those discussed above. One interesting feature of the inhibition data is the differential action of the inhibitors on the secretion of different Yop species. Only compound 2 acted effectively against all three Yop species examined, while each of the other compounds inhibited YopH secretion more effectively than they inhibited the secretion of YopD and YopM. Moreover, the relative activities against the secretion of YopD and YopM also varied among the compounds. These differences suggest that the compounds differ in their mechanisms of action. The abilities of compounds 1 and 3, but not those of compounds 2 and 4, to inhibit the secretion of Tir by EPEC provides additional evidence that the mechanisms of action are diverse. Like INP0007 and related compounds, the novel scaffolds of compounds 1 and 3 clearly indicate that they have spectra of activity that extend beyond the yersiniae. This confirms the utility of the Yersinia system as a general platform for identifying T3S inhibitors.
Compound 2 was remarkable in comparisons with the other T3S inhibitors described here and elsewhere for its low IC50 and its ability to cause very complete inhibition. While the toxicity of this compound for mammalian cells is unfortunate, studies of its mechanism of action against T3SSs may prove useful in the development of improved inhibitors. It is also possible that modifications of this compound may sufficiently reduce its toxicity to provide a useful therapeutic index.
One puzzling observation is that INP0007 inhibited the growth of both Y. pestis
and Y. pseudotuberculosis
, with stronger inhibition against the former being observed. This conflicts with the results of Kauppi et al. (22
). We cannot explain this inconsistency and can only suggest that it may be due to differences in the growth medium used or may be due to the presence of a minor but potent growth inhibitor in our commercial preparation. Minor contaminants were observed by mass spectroscopy (see Fig. S1 in the supplemental material), but the effect of this compound on bacterial growth is an issue that should be revisited.