Apropiación y representación del territorio desde el ejercicio cotidiano

As discussed, the purpose of the study at this stage was to find potent inhibitors that can act as precursors to aid the identification of the target protease(s). The inhibitors presented in Table 3. 1 were tested in vivo using the assay described in section 2.1.1, but before discussing the results it is worth reiterating some of the limitations of the chosen assay technique that should be taken into consideration during interpretation. Firstly, the growth rate of C. difficile is rather inconsistent despite the application of some technical improvements (discussed in section 2.1.1). Secondly, because the compounds do not directly inhibit growth, the observed effect may appear relatively insignificant as the samples were normalised by OD. However the behaviour of the bacterium in the presence of the inhibitor could be affected in a subtle way. Secondly, the small inaccuracy in the stock concentration may affect the relative potencies when comparing different inhibitors. Nevertheless, a given compound may be reliably assayed and compared at various concentrations from the same stock solution. Thirdly, a Western blot is not suitable for precise quantitative analysis in the absence of a control band since the intensity of a particular band on the blot varies from one membrane to the next and also depends on the length of the exposure time during imaging. Furthermore, this intensity is judged by eye, which further reduces the accuracy. Despite this, it is usually reliable to compare bands on the same membrane, and a negative and positive control on each membrane acts as a useful reference. As mentioned in the assay section 2.1.1, the band for full length SlpA appears when the target protease is inhibited since there is no longer full cleavage of SlpA by the protease. The extent to which this cleavage is inhibited correlates well with the full

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length SlpA band intensity, because the better the inhibitor inactivated the target protease the less it cleaves SlpA and therefore the more intense the band becomes. Thus, within the limitations noted above, this band can be exploited as a semi-quantitative scale for a lower limit of inhibitor potency, where the stronger the band the more potent the inhibitor.

Initially, the inhibitor EP-LR-NH2 was compared side-by-side with the natural product itself (E-64) at two different concentrations (50 µM and 250 µM). The anti-LMW Western blot of this comparison is shown (Figure 3. 5, includes a negative control lane in which no inhibitors were added). It is readily noticeable that EP-LR-NH2, which retains the key stereochemical and functional features of the natural product, is probably somewhat more potent than E- 64 since the full length SlpA band is clearly visible at 50 µM in EP-LR-NH2 but not present in E-64. This was very encouraging as it implies that to some extent the considerations taken and assumptions made during inhibitor design were justified and with optimisation could potentially lead to more potent inhibitors. The fact that all the LMW-SLP bands are equal indicates that the same amount of sample was loaded on each lane and suggests the results can be interpreted reliably. Comparing inhibition at 250 µM, there is no significant difference between EP-LR-NH2 and E-64. It was decided to introduce a concentration of 100 µM in order to improve the interpretation of results when comparing different inhibitors on Western blot.

Figure 3. 5: Activity of EP-LR-NH2 vs. E-64, as measured by Western blot using anti-LMW SLP. C. difficile 630 cultures were treated with each compound at 50 µM and 250 µM.

E-64

EP-LR-NH

SlpA

LMW-SLP

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The importance of the warhead was next investigated. EtEP-OH, Ac-LR-NH2, EtEP-LR-NH2, (R,R)EP-LR-NH2 were tested alongside two positive controls (EP-LR-NH2 and E-64) and the negative control, with all compounds at 100 µM (Figure 3. 6). Figure 3. 6 demonstrates that both positive controls possess similar activity at 100 µM which is in line with previous observations. It is also evident that the substitution of the N-terminal trans-(S,S)-epoxysuccinyl warhead with an acetyl group or a trans-(R,R)-epoxysuccinyl warhead leads to a complete loss of activity. This validates the hypothesis that the epoxide warhead is not only critical for inhibition but its binding to the protease is also highly stereoselective. Furthermore, the warhead on its own without any appended specificity element was determined to be inactive, illustrating that both parts, the warhead and the specificity element, are essential for achieving inhibition.

Finally, no significant difference was identified in the extent of inhibition at this concentration between the free acid and the ester form of the epoxide warhead. It is difficult to speculate on which of the analogues are more likely to accomplish superior cell penetration or reach the target protease more easily and thus it is challenging to differentiate between them at this stage with regards to their relative activities. Another speculation is that the ethyl ester compound might be hydrolysed to the acid form in the culture and therefore it possesses similar potency. Further investigation to answer these intriguing questions fell outside the principal focus of this research and hence this further work will be carried out in subsequent studies.

Figure 3. 6: Activity of inhibitors, as measured by Western blot of S-layer fractions using anti-LMW SLP. C.

difficile 630 cultures were treated with compound at 100 µM. The warhead was modified and a specificity element was kept as LR-NH2, as present in the parent precursor.

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The effects of the variation of the specificity element on inhibition were examined, and the warhead was kept in its acid form (as seen in E-64) for all inhibitors to ease directly comparison (Figure 3. 7). The data suggests that increasing the peptide length to mimic P2 up to P5 of the substrate for strain 630 enhanced inhibition (EP-KTEL-NH2 was more active than EP-KT-NH2). However, EP-K-NH2 and EP-KTE-NH2 were found to be unexpectedly inactive at the concentration tested. The activity of EP-KT-NH2 and inactivity of EP-K-NH2 might imply that at least two amino acids are required for binding, but it is difficult to suggest why EP-KTE-NH2 was inactive whilst EP-KTEL-NH2 was active. Maybe the glutamic acid is unfavoured in the binding, but this unfavourableness is compensated by adding a leucine in the chain. On the other hand, EP-KA-NH2, EP-KTT-NH2 and EP-YT-NH2 (refer to strain 959 and 167) all show some level of activity. EP-YT-NH2 is the most potent compound from the entire series as measured by Western blot. The variation of the AA1 leads to highly intriguing results as can be seen for EP-KT-NH2, EP-YT-NH2, EP-LT-NH2 and EP-KT-NH2 in Figure 3. 7. These inhibitors carry the same AA2 residue, threonine, but a different AA1 residue. An alanine at AA1 position causes a consistent loss of activity, a phenomenon further confirmed by EP-ATT-NH2. The activity increases dramatically from leucine to tyrosine. EP-KT-NH2 and EP-KA-NH2, where the AA1 residue is the same but the AA2 amino acid is different, are observed to have similar activities. Furthermore, if threonine is shifted to the P2 position (as in EP-T-NH2), inhibition is lost.

Figure 3. 7: Activity of inhibitors, as measured by Western blot of S-layer fractions using anti-LMW SLP. C.

difficile 630 cultures were treated either with compound at 250 µM or 1% DMSO. The specificity elements were varied and the warhead was kept as EP, as present in E-64.

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Regarding the inhibitors designed through inspiration from the structure of E-64 (i.e. AA1= leucine: EP-LT-NH2, EP-LTT-NH2, EP-LTTL-NH2), only moderate activities were observed compared to EP-KTEL-NH2. The addition of a spacer in the middle of the peptidic sequence of the parent inhibitor did not affect its activity. Unfortunately EP-L-NH2 was not readily accessible using the routes described above, so a direct comparison with the related EP-R- NH2 was not possible.

Accumulating the knowledge gained from the comparison between EP-AA1XX-NH2 and EP- XXAA2-NH2 (where XX is the varied amino acid), the inactivity of EP-T-NH2 and the increased potency of EP-R-NH2 is compelling evidence that the AA1 position plays the decisive role in contribution towards binding whereas variation in the AA2 position does not affect the activity within the range of compounds explored here. Extending the length of the peptidic chain from AA3 to AA4 does not gain substantial additional activity. On the contrary, the potency of EP-R-NH2 indicates that even one amino acid in combination with the warhead is enough to support a relatively effective inhibition.

Looking at the unanticipated inactivity of EP-K-NH2 and EP-KTE-NH2, this could be associated with the limitation of the assay discussed above. The stock concentration of EP-K-NH2 and EP-KTE-NH2 might be much lower than determined by mass, consequently their real concentration in this particular experiment could be significantly below 100 µM which may suggest why no activity was detected.