Molecular glues for manipulating enzymes: trypsin inhibition by benzamidine-conjugated molecular glues

The inhibitory effect of benzamidine as blocker on the protease activity of trypsin is enhanced by covalent conjugation with bioadhesive molecular glue.


Introduction
If the behaviours of enzymes are manipulated by noncovalent interactions, 1-4 one may possibly alter their functions and eventually control related biological events. In this context, one ambitious goal might be to noncovalently operate enzymes such that they perform different functions from their original tasks. In early studies, for instance, amphiphilic molecules have been utilized to introduce enzymes to non-aqueous media in order to expand the range of substrates. 5 Nevertheless, from a pharmacological viewpoint, noncovalent enhancement or attenuation of certain enzymatic activities 6-9 is a highly important and challenging subject. As a proof-of-concept study, we developed a benzamidine (BA) derivative appended with a particular bioadhesive polymer, i.e., molecular glue (Glue n -BA, Fig. 1), which bears at its side-chain termini multiple guanidinium ion (Gu + ) pendants that can be salt-bridged with oxyanionic groups on target protein surfaces. BA is known to inhibit the protease activity of trypsin by blocking its substrate-binding site (Fig. 2a). 10 In proximity to this binding site, trypsin has oxyanionic regions 11 (blue-coloured) that allow the glue moiety of Glue n -BA to adhere (Fig. 2a). Hence, we envisioned that Glue n -BA could inhibit the protease activity of trypsin much more than a BA derivative without the glue moiety such as TEG-BA ( Fig. 1), if the adhesion of the glue moiety (Glue n ) does not hamper the appropriate BA positioning toward the active site (Fig. 2b). Fig. 1 Schematic structures of bioadhesive molecular glues Glue n -R (n ¼ 10 and 29) and m Glue n -R (n ¼ 27) conjugated with benzamidine (R ¼ BA) as a trypsin inhibitor, and those of the reference molecular glue Glue n -Ph without an inhibitory terminus and TEGylated benzamidine (TEG-BA) without the glue moiety.
We have developed a series of dendritic molecular glues that bear multiple guanidinium ion (Gu + ) pendants in the periphery of their water-soluble dendritic scaffolds. [12][13][14][15][16] Such dendritic molecular glues tightly adhere to proteins, 12-14 phospholipid membranes 15 and clay nanosheets 16 in aqueous media via the formation of multiple salt bridges between their Gu + pendants and oxyanionic groups located on those targets. Most interesting along the line of this study was the observation that the photomechanical motion of an azobenzene-cored molecular glue can be transmitted to a phospholipid vesicular membrane via salt-bridge interactions and can consequently modulate its transmembrane ion permeation. 15 This nding motivated us to extend the scope of the present study more to bio-related applications, i.e., noncovalent manipulation of enzymes. Recently, we conrmed that non-dendritic, linear polymers bearing side-chain Gu + pendants 17 are readily accessible alternatives to our prototype dendritic molecular glues. Hence, in the present study, we designed linear Glue n -BA with short (n ¼ 10) and long (n ¼ 29) glue moieties (Fig. 1). In addition to TEG-BA as a reference, we also prepared m Glue n -BA (n ¼ 27, Fig. 1) carrying 27 Gu + units along the polymer main chain. As highlighted in this article, Glue 10 -BA inhibited the trypsin activity much more than TEG-BA ( Fig. 1) without the glue moiety, whereas m Glue 27 -BA ( Fig. 1) was inferior to TEG-BA despite the fact that it carries 27 Gu + units and has a higher affinity than TEG-BA for trypsin.

Results and discussion
Glue n -BA was synthesized using a "click" reaction 18-23 between TEG-BA and a three-armed monomer containing Gu + , azide and alkyne moieties. The reaction mixture was subjected to preparative size exclusion chromatography to allow fractionation of Glue 10 -BA and Glue 29 -BA. m Glue n -BA (average n ¼ 27, Fig. 1) was synthesized in a similar fashion by polymerizing a Gu + -containing linear monomer with terminal azide and alkyne groups. The average molecular weights of Glue n -BA and m Glue n -BA were estimated by 1 H NMR spectroscopy and static light scattering (SLS) analysis (Table S1 †).
We rst investigated the effect of conjugation of molecular glues to BA on the binding affinity for trypsin. Trypsin is known to alter its conformation upon interaction with metal ions, 24 polymers 25 and proteins, 26 resulting in circular dichroism (CD) spectral changes. Upon mixing with Glue 10 -BA, trypsin also changed its CD spectrum. As shown in Fig. S10c, † the CD intensity of trypsin (5 mM) at 237 nm decreased upon titration with Glue 10 -BA (0-7 mM) at 25 C in Tris-HCl buffer (50 mM Tris-HCl, 10 mM CaCl 2 , pH 8.0). According to the reported method, 27,28 we estimated the association constant (K assoc ) of Glue 10 -BA with trypsin to be 5.5 Â 10 5 M À1 by tting the fractions of bound trypsin (Fig. 3, red) to the Hill equation. 27,28 As expected, Glue 29 -BA bearing a larger number (29) of Gu + pendants exhibited a signicantly higher K assoc value of 3.2 Â 10 6 M À1 (Fig. 3, brown and S10b †), reecting an important role of multivalency. In sharp contrast, when TEG-BA without the glue moiety was used in the titration, the CD spectral change of trypsin was too small to detect unless the concentration range of TEG-BA for the titration was extended to 200 mM (Fig. 3, blue and S10a †). Accordingly, the K assoc value was estimated to be 1.6 Â 10 4 M À1 , which is 35-and 200-fold lower than those observed for Glue 10 -BA and Glue 29 -BA, respectively. We also found that Glue 10 -Ph without BA (Fig. 1) binds to trypsin (Fig. S9 †) with a K assoc value (2.8 Â 10 5 M À1 ; Fig. 3, green and S11a †) that is comparable to that of Glue 10 -BA, indicating that the glue moiety predominantly contributes to the binding affinity of Glue 10 -BA. Notably, the K assoc value of m Glue 27 -BA containing 27 Gu + units along the main chain (8.2 Â 10 4 M À1 ; Fig. 3, purple and S11b †) was 40-fold lower than that of Glue 29 -BA possessing an almost comparable number of Gu + pendants, and even 6.7-fold lower than that of Glue 10 -BA. As previously reported, 12 the poor binding behaviour of m Glue 27 -BA is most likely due to a presumably small conformational exibility of its in-chain Fig. 2 (a) Schematic illustration of the trypsin-catalyzed hydrolysis of N-p-tosyl-L-arginine methyl ester (TAME) to N-p-tosyl-L-arginine, exhibiting its characteristic absorption at 247 nm. 10,11 Benzamidine (BA) derivatives are known to bind to the substrate-binding site of trypsin and inhibit its enzymatic activity. 10 Trypsin has oxyanionic regions on its surface (blue-coloured) in proximity to the substrate-binding site, which allow the glue moiety of Glue n -BA to adhere. (b) Schematic representation of how the inhibitory effect of the blocking unit BA on the protease activity of trypsin is enhanced by conjugation with molecular glue.
Gu + units compared with that of the Gu + units at the side-chain termini in Glue n -BA.
Trypsin hydrolyses peptide linkages at the carboxyl side of lysine and arginine residues. 29 As already described in Fig. 2a, this protease activity is inhibited by BA. 10 Considering the exceptionally high affinity of Glue 29 -BA for trypsin, we expected that this BA-appended molecular glue might be the best inhibitor among those listed in Fig. 1. However, as observed by dynamic light scattering (DLS; Fig. S12 †), trypsin/Glue 29 -BA, in contrast with other complexes such as trypsin/Glue 10 -BA and trypsin/Glue 10 -Ph, tends to form large aggregates (>200 nm), most likely due to the formation of physical crosslinks between its excessively long glue moiety and trypsin. Therefore, we conducted inhibitory assay experiments using Glue 10 -BA and m Glue 27 -BA, along with Glue 10 -Ph and TEG-BA as references, but did not use Glue 29 -BA. Nevertheless, we found that upon conjugation with Glue 10 , BA's inhibitory effect was considerably enhanced. As shown in Fig. 4 (black), when N-p-tosyl-L-arginine methyl ester (TAME, 1 mM) as a substrate was mixed with trypsin (20 nM) at 25 C in Tris-HCl buffer (50 mM Tris-HCl, 10 mM CaCl 2 , pH 8.0), TAME was hydrolysed to N-p-tosyl-Larginine (Fig. 2a), exhibiting an increase in its characteristic absorption at 247 nm. 10 However, when 10 mM of Glue 10 -BA was added to the reaction system, the hydrolysis of TAME was considerably decelerated (Fig. 4, red). Although TEG-BA did not exhibit detectable inhibition at 10 mM (Fig. 4, blue), Glue 10 -BA explicitly inhibited the trypsin activity even at 2.5 mM (Fig. 4,  orange). As shown in Fig. 5, the hydrolytic activity of trypsin was evaluated using the pseudo-rst order reaction kinetics, and normalized to that of untreated trypsin (20 nM). The sigmoidal prole, obtained for the case with TEG-BA in Fig. 5 (blue), allowed estimation of the half-maximal inhibitory concentration (IC 50 ) of TEG-BA as 79 mM. Notably, Glue 10 -BA exhibited a 13-fold greater inhibitory effect (IC 50 ¼ 6.2 mM; Fig. 5, red) than TEG-BA. In sharp contrast, when Glue 10 -Ph was used in place of Glue 10 -BA, no inhibition of the trypsin activity was observed (Fig. 5, green) even when [Glue 10 -Ph] was higher than 20 mM. Hence, the adhesion of the glue moiety does not hamper the enzymatic activity of trypsin, but primarily contributes to the stabilization of the BA/trypsin complex. As previously described, the binding affinity of m Glue 27 -BA for trypsin is only 15% of that of Glue 10 -BA, but still 5-fold higher than that of TEG-BA. However, m Glue 27 -BA exhibited a lower inhibitory effect than TEG-BA under identical conditions, and minimally inhibited the hydrolytic activity of trypsin (Fig. 5, purple). We presume that the poor conformational exibility of the in-chain Gu + units in the glue moiety hinders the ability of the conjugated BA terminus to properly block the substrate-binding site of trypsin. To rationalize the concept of blocker-appended molecular glues for pharmacological applications, this issue should be taken into consideration.

Conclusions
Through a comparative inhibition study on the protease activity of trypsin using Glue n -BA, m Glue n -BA and TEG-BA (Fig. 1) as potential trypsin inhibitors, we demonstrated that an active-site   blocker such as BA efficiently inhibits the trypsin activity when its conjugated glue moiety (Glue n ) can hold the blocker stably onto the active site through adhesion to a proximal oxyanionic region (Fig. 2b). Of particular interest is the obviously smaller inhibitory effect of m Glue 27 -BA compared to TEG-BA, despite the fact that m Glue 27 -BA has a 5-fold higher affinity than TEG-BA for trypsin. The incorporation of a mechanism to respond to biological or physical stimuli for controlling the operation of the blocker unit is an interesting subject worthy of further investigation.

Trypsin activity assay
To a Tris-HCl buffer (50 mM Tris-HCl, 10 mM CaCl 2 , pH 8.0) solution of trypsin (20 nM) was added a Tris-HCl buffer solution of TEG-BA, and the mixture was incubated at 25 C for 1 min. Then, to the resultant solution was added a Tris-HCl buffer solution of N-p-tosyl-L-arginine methyl ester hydrochloride (TAME, nal concentration 1 mM; Fig. 2a), and the absorption intensity at 247 nm (ref. 10) was traced over a period of 1 min. The trypsin activity was determined using pseudo-rst order reaction kinetics and normalized to that of untreated trypsin. The trypsin activities in the presence of Glue n -BA, m Glue n -BA and Glue n -Ph were likewise evaluated.