Chapter 2 Fragment Screen of T cell immunoreceptor with Ig and ITIM domains
2.2 Results
2.2.6 Reexamination of fragment binding to TIGIT WT
For screening purposes, we introduced the C69S mutation in order to avoid any non-specific covalent adducts that would produce false positive hits. Solvent-exposed C69 is a part of the AX6G lock motif, however, the side chain is directed away from the
binding interface so we rationalized this mutation would not drastically affect lock motif conformation (Figure 2-13A). It is reasonable to reconfirm fragment binding against TIGIT WT in order in order to eliminate any notion that this mutation affected TIGIT conformation or introduced a non-native binding site. Uniformly 15N-labeled TIGIT WT IgV domain was produced for rescreening the fragment hits found in our C69S screen. Interestingly, the SOFAST HMQC spectra of TIGIT WT revealed shift perturbations in peaks associated with fragment binding when compared to the C69S screening construct spectra (Figure 2-13B). Serine is spatially an acceptable analog for cysteine and usually a well-tolerated substitution when cysteine is solvent-exposed, but the polar hydroxyl sidechain of serine introduces a different chemical environment for its neighboring residues. This change in chemical environment is visualized in the comparison of the NMR spectra and suggests that the shifted resonances are localized closely to C69. Alternatively, the chemical shift perturbations could suggest a conformational change in the IgV domain, however, in this case the global similarity between the two spectra do not suggest a broader conformational change. The fact that shifted residues seen between the two protein construct spectra are shared with those seen in fragment binding would suggest that the fragment binding site is near or directly associated with the C69S lock motif residue mutation.
Figure 2-13. Examination of TIGIT C69S mutation.
(A) Structure of TIGIT-PVR interaction with the inset highlighting C69 side chain (sticks) directed away from interaction interface. (B) Overlay of SOFAST HMQC spectra of TIGIT C69S (blue) and TIGIT WT (red). Resonances that shift during fragment binding are highlighted with green circles.
We rescreened the best 10 fragment hits by VS against 15N-labeled TIGIT WT IgV and found that cluster 1, 3, and 7 hits with a VS of 4 or 3 when screened against the C69S protein construct did not bind to TIGIT WT (Figure 2-14A). This would suggest that the introduction of serine for cysteine artificially created a new fragment binding site for which the majority of fragments found in the C69S screen bind. The only fragment that still exhibited binding against TIGIT WT was the cluster 5 hit (5) that induced peak broadening upon binding. As previously discussed, this peak broadening could indicate intermediate exchange, protein aggregation, or changes in protein dynamics. If 5 is binding in the intermediate exchange regime with Kd less than 10 µM, it would be assumed that shifted peak intensities would return when the protein is saturated with ligand at concentrations several times higher than the Kd. The return of peak intensity was not seen at saturating levels of 5 suggesting that this fragment is not exhibiting intermediate exchange (Figure 2-14B). It is likely then that 5 is either inducing insoluble protein aggregation or changing the protein dynamics. To test this hypothesis, 5 was removed from the NMR sample by dialysis and re-collecting HMQC spectra on the dialyzed sample. The rescue of broadened signal intensities in the dialyzed sample suggests that peak broadening is
Figure 2-14. Binding of identified fragment hits against TIGIT WT.
(A) Fragment binding of 1 (800 µM, structure in inset) to TIGIT C69S (left) is not recapitulated with TIGIT WT (right). In the left spectra, peaks that shift between C69S and WT spectra as in Figure 2-13B are highlighted by green circles. (B) Titration of 5 (structure in inset) does not reveal peak intensity rescue at high concentrations. Zoom in right spectra is highlighted by black box in left spectra. (C) Peak broadening upon binding of 5 is shown in left spectra. Right spectra are collected on the same sample as in left after dialysis to remove 5 and shows the rescue of broadened peaks.
fragment specific and that the reversible binding of 5 induces a change in protein dynamics that leads to the loss of peak intensity (Figure 2-14C). The most acceptable explanation of the change in protein dynamics that leads to peak broadening is that 5 induces dimerization or soluble oligomerization of TIGIT and, thereby, decreases the molecular tumbling rate. This slower tumbling rate results in shorter T2 relaxation rates and a loss of signal over the experiment due to increased T2 relaxation.
The results of our focused rescreen of fragment hits against TIGIT WT suggest that the majority of fragment hits identified during initial screening are binding through the C69S mutation introduced in the screening construct. It is possible that the serine mutation introduces a site for hydrogen bond formation, both as a donor and acceptor, which fragments can interact with. The shared set of shift perturbations seen between fragment hits and TIGIT WT compared against apo TIGIT C69S further suggests that the mutated residue is involved in fragment interaction. Therefore, we have concluded that the majority of hits identified in our initial fragment screen are artifactual and specific to the C69S screening construct. This leads us to believe that TIGIT is much less
“druggable” than initially indicated by fragment screening. Rescreening the fragment library against TIGIT WT is not predicted to produce significantly different screening results as the AX6G lock motif is not considered to be conformationally impacted by the C69S mutation, particularly at the TIGIT-PVR interface “hot spot.” The lone reconfirmed hit, fragment 5, likely induces dimerization or oligomerization of the TIGIT IgV domain and has potential as a TIGIT inhibitor if the ligand-induced dimerization could be optimized to be strong enough to disrupt TIGIT interaction with PVR.