Increased Conformational Flexibility of a Macrocycle–Receptor Complex Contributes to Reduced Dissociation Rates

Abstract Constraining a peptide in its bioactive conformation by macrocyclization represents a powerful strategy to design modulators of challenging biomolecular targets. This holds particularly true for the development of inhibitors of protein‐protein interactions which often involve interfaces lacking defined binding pockets. Such flat surfaces are demanding targets for traditional small molecules rendering macrocyclic peptides promising scaffolds for novel therapeutics. However, the contribution of peptide dynamics to binding kinetics is barely understood which impedes the design process. Herein, we report unexpected trends in the binding kinetics of two closely related macrocyclic peptides that bind their receptor protein with high affinity. Isothermal titration calorimetry, 19F NMR experiments and molecular dynamics simulations reveal that increased conformational flexibility of the macrocycle–receptor complex reduces dissociation rates and contributes to complex stability. This observation has impact on macrocycle design strategies that have so far mainly focused on the stabilization of bioactive ligand conformations.

Abstract: Constraining ap eptide in its bioactive conformation by macrocyclization represents ap owerful strategy to design modulators of challenging biomolecular targets. This holds particularly true for the development of inhibitors of protein-protein interactions which often involvei nterfacesl acking defined binding pockets. Such flat surfaces are demanding targets for traditional small molecules rendering macrocyclicp eptides promising scaffolds for novel therapeutics. However,t he contribution of peptide dynamics to binding kinetics is barely understood which impedest he design process. Herein, we reportu nexpected trends in the bindingk inetics of two closelyr elated macrocyclic peptides that bind their receptor protein with high affinity.I sothermal titration calorimetry, 19 FNMR experimentsa nd molecular dynamics simulations reveal that increased conformationalf lexibility of the macrocycle-receptor complex reduces dissociation rates and contributest oc omplex stability. This observation has impact on macrocycle design strategies that have so far mainly focusedo nt he stabilization of bioactive ligandc onformations.
Many therapeutically relevant bio-macromolecules cannot be targeted with traditionals mall molecule approaches. [1] Often these targets are characterized by relatively shallow surfacesa s they are frequently found at the interface of protein-protein interactions (PPI). [2] Due to their excellent surface recognition properties, large macrocyclic molecules are considered promising candidates to target protein areasi nvolved in PPIs. [3] This holds particularly true for peptide-derived macrocycles which display favorable binding properties being linked to their large surface areas as well as their uniques tructurala nd dynamic characteristics. [4] Despite their constrained structure,t hey exhibit considerable conformational freedom that allows for efficient samplingo fb ioactives tates. [3d, 5] In many cases, the effects of macrocyclization on the thermodynamicso fb inding are well investigated.O ften, favorable entropic profiles resulting from reduced flexibilitieso ft he free ligand translate into increased affinities. [3d, 6] However, the influenceo fm acrocyclization on the conformational dynamics of the bound state and the binding kinetics are less understood. Notably,the residence time of ab ound ligand (reciprocal of dissociation rate) is of particularinterest [7] as it strongly correlates with therapeutic efficacy. [8] Herein, we investigate two closely related peptide-derived macrocyclic ligands( MC18 and MC22)b inding the same receptorp rotein (Figure 1a)b ut showing distinct binding characteristics. Using isothermal titration calorimetry (ITC), 19 FNMR titration experimentsa nd molecular dynamics (MD) simulations,w es how that increasedc onformational flexibility of the macrocycle-receptor complexc ontributes to reducedd issociation rates and therebyt oh igher complex stability.
Previously,w er eported as et of macrocycles derived from a bacterialp eptideb inding epitope (L,g rey) composed of 11 aminoa cids (Figure 1a). [6a] Twom acrocyclic peptides show particularly high affinity for the receptorp rotein 14-3-3 isoform z (from now on called 14-3-3)a nd werep otent inhibitors of its interaction with Exoenzyme S. One of these peptidesc omprises am acrocycle containing 18 atoms( MC18,b lue) while the other one bears a2 2atom ring system( MC22,r ed). MC22 displays the highest affinity for the receptor.T he ligand-receptor complexes have been characterized via X-ray crystallography verifyingt he central groove of 14-3-3 as the binding site for all ligands ( Figure 1b). In addition, it was shown that macrocyclization indeed reduces flexibility in the free state of the ligand. Havinga ccess to thesew ell characterized ligand-receptor pairs, we were interestedi nt he impact of the different macrocyclization architectures on binding kinetics. For that purpose, we decided to use 19 FNMR spectroscopy which provides high sensitivity for the detection of biomolecular interactions. [9] To enable ligand-observed NMR experiments, the linear and both macrocyclic peptides were synthesized and N-terminallym odified with N-trifluoroacetyl glycine.
The dissociation process is generally considered to be dominated by the properties of the ligand-receptor complex. [8,14] The 19 FNMR spectra provide information about the flexibility of the boundl igand since decreases in linewidth (n 0.5 )c orrelate with increased flexibility (Figure 2a). [15] Interestingly,t his analysis indicates higherflexibility for the bound state of macrocycle MC22 (n 0.5 = 12.4 Hz) compared to MC18 (n 0.5 = 33.7 Hz) and linear ligand L (n 0.5 = 33.5 Hz). While this providesa ni nitial insight into the dynamic properties of these ligand-receptor complexes,i ti sn ot cleari ft his is merely al ocal effect observed for the fluorine label.
To dissect the different contributions to the properties of the bound state, we employed MD simulations for all three ligand-receptor complexes. [16] The corresponding crystal structures (PDB ID 4n7g, 4n7y,4 n84) served as as tarting point for these calculations. Ligands werep repared using Maestro [17] and considered as they had been used in ITC and 19 FNMR experiments (includingt heir N-terminalt rifluoroacetyl glycine). MD simulations were performed for 1.5 msp er complex taking snapshotse very 20 ps.S napshots were clusteredu sing a2 cutoff for the minimum distance between clusters.T he cluster probability distributionss erved to calculate conformational entropies (S conf )w hich represent am easuref or the conformational flexibility of peptidesa nd proteins. [18] Comparing conformational entropies of thesel igand-receptor complexes with their dissociation rates reveals ac lear correlation (Figure 3a)i ndicating that increased flexibilityo ft he complexi sl inked with reduced k off values. Ac loser look at the conformational entropies reveals that the complex of MC22 (S conf = 7.5 kcal mol À1 )s hows increased flexibility compared to the complex of L and MC18 (S conf = 6.6 and 6.4 kcal mol À1 ,F igure 3b)w hich is consistent with the above mentioned narrower NMR linewidth (n 0.5 )f or the MC22-receptor complex. Interestingly,d ifferences in the overall flexibilitya ppear to originate mainly from the ligands (Figure 3b).
Considering the relatively high structural similarity of MC18 and MC22,w ew ere interested to locate the regions responsible for the different flexibilities in the two receptor-bound macrocycles. For that purpose, the root mean square fluctuations (RMSF) were calculated for all main chain atoms andf or the carbon atoms within the ligand crosslink in MC18 (blue) and MC22 (red, Figure 4a,b). Based on these RMSF values, the two macrocyclesd isplay similar flexibilities within the peptide core sequence (X 1 LDX 2 ,F igure 4a,X = crosslinking amino acids), but differ in their terminal regionsa nd the crosslink itself (Figure 4a,b). Here, MC22 showsconsiderably higherflexibility than MC18 mainly contributing to overall differences in conformational entropies of the bound state. Ac loser look at both bound macrocycles including ac olor coding for RMSF valuesi llustrates these differences in flexibility (Figure 4c,d,i ndicatingl ow (white) to high (orange) flexibility). Both peptides exhibit highest flexibility for their very terminal amino acids which is in line with previously reported crystal structures showingl ess defined electron density in these regions (PDB ID:4 N7Y and4 N84). [6a] Notably,b oth termini in MC18 exhibit lower flexibility than corresponding areasi nMC22.I na ddition, the crosslink in MC18 appearst ob ec onsiderably more rigid than in MC22.T his behavior can be explained by the observation that the crosslink in MC18 reaches further into the hydrophobic groove of 14-3-3 ( Figure S10 and S11) which may constrain its conformational freedom.I mportantly,t hese MD findings are in line with the decreased NMR linewidthf or the Nterminal fluorine label in MC22 (Figure 2a).
Ta ken together,t hese results provide mechanistic insight into the contribution of peptide flexibility to receptor binding using ITC and 19 FNMR experiments combined with MD simulations. Although both macrocycles exhibit similart hermodynamic profiles, 19 FNMR reveals intriguingd ifferences in binding kinetics. Strikingly,r educed dissociation rates (and thereby increased affinity) correlate with increased conformationalf lexibilitieso ft he ligand-receptor complex.T his observation has implications for the design of high affinity peptides and macrocycles which so far focused on the stabilization of ab ioactive conformation in the free state. Our findings suggest complementing this strategy with ac onsideration of the bound state aiming for increased flexibility.H owever,w ec annot conclude general design principles based on these initial findings. Taken into consideration that in some cases crosslink incorporation was also reported to result in al oss of entropic contributions to binding, [19] any endeavor towards affinity maturation of macrocyclic ligands is highly advised to involveathorough biophysical characterization of receptor binding. Even though, such integrated optimization strategiesa re time and resource demanding, they may provide the possibility to obtain ligands with both increased affinity and prolonged residence time, of which the latter is an important pharmacological parameter towards high drug efficacy.I naddition, our findings highlight the potentialo fl oop-like peptide epitopes as startingp oint for macrocyclic ligands as they exhibit reducedi ntramolecular hy-drogen bond stabilization when compared to repetitive secondary elements such as a-helix and b-sheet.I mportantly, loop-like epitopes are underrepresented in current stabilization approaches that predominantly focusona-helices.