Competitive ABPP

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Scheme. 18 – Synthesis of 4-Oxo-β-Lactam compounds 119 and 120. A. Synthesis of a 4-Oxo-β-Lactam with N-benzyl substituent, 119, from N-benzyl 4-(aminomethyl)benzoate; B. Synthesis of a 4-Oxo-β-Lactam with a free

carboxylic acid, 120, by hydrogenation of 120.

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Figure 45 – Library of 4-Oxo-β-Lactams.

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Figure 46 – Example of a competitive ABPP experiment for selected para-substituted 4-Oxo-β-Lactam compounds (117, 121, 122, 123, 124, 125) compounds in U937 whole cell lysates against FP-Rhodamine (1 µM). The region

between 75 and 150 kDa is reproduced on the right with higher contrast.

Figure 47 – Example of a competitive ABPP experiments for selected para-substituted 4-Oxo-β-Lactam compounds (132, 133, 134, 135, 136, 137) compounds in U937 whole cell lysates against FP-Rhodamine (1 µM). The region

between 75 and 150 kDa is reproduced on the right with higher contrast.

107 for the entire 4-Oxo-β-Lactam library are presented in the attachments section. The analysis of the gel showed selective blocking of some targets of the FP probe.

These gels offered a preliminary glimpse into the reactivity of our compounds, showing a surprisingly selective profile of inhibition, in some cases also with good potency. The para-substituted compounds showed very efficient labeling of a protein at 25 kDa (compounds 117, 121-125), which could potentially be HNE or PR3. Some of the meta-substituted compounds also inhibited this band, but generally with less potency (compounds 132-137). The area between 75 kDa and 150 kDa, where most DPP family members fall was difficult to analyze due to the lower abundance of some proteins but seemed to suggest there was more efficient inhibition of these enzymes by the meta-substituted compounds, with compounds 132 and 135 showing inhibition of a band at 100 kDa, which could potentially be either DPP8 or DPP9. Overall, the results seemed to be hindered by the lack of resolution in the ~30 kDa section, where a lot of proteins overlap.

For these reasons, the experiments were repeated using fractioned cell lysates, with separated soluble and membrane fractions of proteins, which usually provide better resolution.

The inhibitors were incubated with membrane and soluble lysate fractions of U937 cells for 30 minutes at 3 different concentrations, 0.1 µM, 1 µM and 10 µM. After 30 minutes 1 µM of FP-Rhodamine was added. 30 minutes later the reaction was quenched with loading buffer and the mixture was analyzed by SDS-PAGE. Compound reactions were compared against a control sample pre-incubated with DMSO for 30 minutes and then FP-Rhodamine. The gel of fractioned lysates offered a much more readable profile of protein labeling than before. The gels for selected compounds are presented in Figure 48.

The full results for the entire 4-Oxo-β-Lactam library are presented in the attachments.

The results suggested that the compounds are potent and selective inhibitors of a selected group of serine hydrolases. A band at approximately 25 kDa in the membrane fraction (potentially HNE) was strongly inhibited by several compounds (for example compounds 117, 121 and 124), even at low concentrations of 100 nM. Bands at approximately 100 kDa in the soluble fraction (likely to be DPP8, DPP9 and/or FAP) seemed to also be strongly inhibited by some compounds (for example 132, 135 and 139). Bands lower than 25 kDa in the soluble fraction also showed some inhibition (probable LYPLA1 and LYPLA2) for most tested compounds.

Other bands which were harder to identify with confidence also show inhibition when compared with the DMSO control, suggesting additional enzymes are being hit by the

108 compounds. These identifications were suggested by a combination of previous knowledge of the expected targets of 4-Oxo-β-Lactams and known labeling patterns of FP-probes.

A significant number of bands increased in labeling intensity when compared to the DMSO control. This can be explained by inhibition of serine proteases, which led to an increase in their substrates, suggesting the increasing bands could potentially be proteins that are usually cleaved by serine hydrolases that were inhibited by the 4-Oxo-β-Lactams.

Overall the results suggested that para-substituted compounds result in a remarkably potent inhibition of the band at 25 kDa in the membrane fraction, while the meta-substituted compounds seemed to have a much higher potency against the bands around 100 kDa in the soluble fraction, with some compounds, like 135, showing significant inhibition at 100 nM and seemingly complete inhibition at the two higher concentrations.

This suggested potential SAR dynamics involving positioning of the substituents in the aromatic ring attached to the 4-Oxo-β-Lactam core. This had previously been observed in work of our group with HNE inhibition, where compounds with substituents at the para-position showed higher potency.²⁷⁰

Ortho-substituted compounds showed almost no blocking of relevant FP-labeled bands and were not presented here, with only some minor bands being partially inhibited at the highest concentration of 4-Oxo-β-Lactam compound (gels for these compounds are presented in the attachments). Compounds 119 and 120 showed inhibition of some bands, but no unique features that warranted further study.

The data presented above was obtained when the compounds were tested in U937 whole cell lysates. To evaluate the capacity of selected compounds to cross the cell membrane and inhibit serine hydrolases we performed a competitive ABPP assay in whole U937 cells. Briefly, U937 cells were diluted to 1 million cells/mL in serum-free media and incubated with selected compounds of the 4-Oxo-β-Lactam library at 1 and 10 µM of concentration. DMSO controls were also performed. The mixtures were incubated at 37°C for one hour. The reactions were stopped by centrifugation, removal of media and PBS wash, followed by resuspension and lysis in PBS. Protein content was normalized to 1 mg/mL and the samples were labeled with FP-Rhodamine for 30 minutes. The results are shown in Figure 49.

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Figure 48 – Competitive ABPP gels with soluble and membrane fractions of U937 cells for selected compounds.

Disappearance of a band when compared with DMSO control indicates that band is a target of the compound. Clear inhibition of some targets is observed. Analysis of the molecular weights seems to suggest that the compounds are

inhibitors of DPP8 and/or DPP9 (soluble fraction, ~100 kDa), PREP (soluble fraction, ~75 kDa) and HNE (membrane fraction, ~25 kDa).

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Figure 49 – Competitive ABPP in U937 whole cells using selected compounds from the 4-Oxo-β-Lactam library. The full gels are shown in A. A cropped version with higher contrast to emphasize lower abundance proteins is shown in

B.

The gel profiles suggested that 4-Oxo-β-Lactam compounds were generally able to cross the cell membrane. This was suggested by the different FP labeling profiles observed when compared with the DMSO control for nearly all compounds at different points in the gels. Some particular bands that were identified in the whole cell lysate assays seem

111 to also be inhibited in these assays, including the band at approximately 25 kDa for compounds 117, 121 and 126, for example. Interestingly, multiple bands that are not visible in the DMSO-treated sample are being strongly stained by FP in compound-treated samples, particularly, bands above 150 kDa, which are visible in samples treated with compounds 117, 122, 123, 126, 128 and 135. Additional bands present this pattern across the gel. This phenomenon might be the result of inhibition of serine proteases, with the proteins corresponding to bands of increased labeling being potential substrates of these enzymes. Overall, a significant change in the profile of serine hydrolase labeling by FP was observed, suggesting that 4-Oxo-β-Lactams efficiently cross the cell membrane, in sufficient concentrations to cause changes in enzymatic behavior that are visible by SDS-PAGE and highlighting that these compounds can potentially be used in assays with whole cells.