Metabotropic glutamate receptors (mGluRs) are important drug targets because of their
involvement in several neurological diseases. Among mGluRs, mGlu5 is a particularly high-profile target because its positive
or negative allosteric modulation can potentially treat schizophrenia or anxiety and chronic pain, respectively. Here, we
computationally and experimentally probe the functional binding of a novel photoswitchable mGlu5 NAM, termed
alloswitch-1, which loses its NAM functionality under violet light. We show alloswitch-1 binds deep in the allosteric
pocket in a similar fashion to mavoglurant, the co-crystallized NAM in the mGlu5 transmembrane domain crystal structure.
Alloswitch-1, like NAM 2-Methyl-6-(phenylethynyl)pyridine (MPEP), is significantly affected by P655M mutation deep in the
allosteric pocket, eradicating its functionality. In MD simulations, we show alloswitch-1 and MPEP stabilize the co-crystallized
water molecule located at the bottom of the allosteric site that is seemingly characteristic of the inactive receptor state.
Furthermore, both NAMs form H-bonds with S809 on helix 7, which may constitute an important stabilizing interaction for
NAM-induced mGlu5 inactivation. Alloswitch-1, through isomerization of its amide group from trans to cis is able to form an
additional interaction with N747 on helix 5. This may be an important interaction for amide-containing mGlu5 NAMs, helping to
stabilize their binding in a potentially unusual cis-amide state. Simulated conformational switching of alloswitch-1 in silico
suggests photoisomerization of its azo group from trans to cis may be possible within the allosteric pocket. However,
photoexcited alloswitch-1 binds in an unstable fashion, breaking H-bonds with the protein and destabilizing the co-crystallized
water molecule. This suggests photoswitching may have destabilizing effects on mGlu5 binding and functionality.