These studies describe what we believe to be the first reported small molecules mimicking a specific neurotrophin domain, capable of functioning as ligands to selectively and potently activate the TrkB receptor.
We initially theorized that using loop subdomain pharmacophore modeling and virtual screening, the a priori likelihood of identifying compounds functioning through TrkB would be substantially greater than that for compounds identified in conventional high-throughput screening for neurotrophic activity (which would identify compounds functioning through any mechanism). As in our previous studies on p75NTR
), we found that this method provides a manageable group of compounds for biologic testing, with a high rate of recovery (5 activated of 7 tested) of the desired activity relative to that which would be obtained using high-throughput screening (<1%, refs. 43
), and similar to that of other virtual screens beginning with more structural information (45
). Multiple lines of experimental evidence suggest that these small molecules function as TrkB ligands, including: specific activation of TrkB and two primary downstream signaling intermediates; inhibition of activity by the kinase inhibitor K252a and a monoclonal antibody directed to the ECD of TrkB; blocking of compound binding to the ECD of TrkB by BDNF; compound effects on TrkB-expressing, but not TrkA- or TrkC-expressing, NIH-3T3 cells; and inhibition of BDNF binding to TrkB, with no inhibition of neurotrophin binding to TrkA, TrkC, or p75NTR
, in addition to a negative Cerep screen. While the present studies failed to detect interaction of LM22A-4 with p75NTR
or prevention of cell death through p75NTR
, future studies will address whether such ligands might nevertheless affect its complex intracellular adaptor/signaling milieu. The lack of effect of BDNF antibodies, compound inhibition of BDNF maximal activity, and LM22A-4 induction of greater neurite outgrowth than maximal BDNF suggest that the LM22A compounds do not act as BDNF secretagogues. Thus, we suggest that the identification, within this small group of tested compounds, of chemically diverse compounds sharing the common mechanism of TrkB activation supports the validity of the model employed in these studies.
Recent studies show that small molecules may bind with relatively high affinity to protein surfaces through “hot spots,” small groups of residues that contribute significant energy to the protein-protein interaction of interest. Small molecule binding can disrupt protein-protein interactions and inhibit the functions they mediate (46
). Moreover, the small molecule may induce conformational changes in the binding domain that differ from those associated with binding of the natural ligand (48
). Given the findings of small molecule–mediated TrkB agonism presented here, and our previous findings with p75NTR
), we suggest that hot-spot binding and induction of conformational changes in the receptor may allow these small molecules to act as activating ligands, though there may be differences from the natural ligand with respect to the coupling and kinetics of the induced signaling. Further structural studies of compound-receptor complexes will be needed to confirm this hypothesis.
Of note, other natural ligands of TrkB may also induce signaling quantitatively and qualitatively different from that induced by BDNF. Mutation of the SHC-binding site of TrkB in mice caused loss of NT-4–dependent, but not BDNF-dependent, neurons in vivo and decreased survival of sensory neurons in culture and ERK activation in response to NT-4 relative to BDNF (51
). In another study, c-fos promoter activity in cortical neurons was found to be more responsive to exogenous NT-4 than BDNF (52
). Such differential activation of downstream signaling by ligands may reflect and/or play an active role in partial agonism and may therefore be significant in the effects of LM22A compounds on endogenous BDNF actions. Moreover, the effects of differential activation by small molecule ligands may vary with respect to other native TrkB ligands (NT-4, NT-3) and lead to unexpected inhibition or synergy. Further studies comparing signaling activation kinetics and pathways between the compounds and protein ligands, including coexposure, will be needed to evaluate these possibilities.
Additionally, it would be reasonable to expect that the methodology presented here would also yield pure BDNF/TrkB antagonists, though we have not specifically assayed for these as of yet. Unfortunately, the small numbers of compounds and lack of available close structural analogs make detailed structure-activity relationship analysis infeasible at this point; this will be addressed in future studies employing larger libraries and/or synthesis of directed analogs.
The lack of rotational symmetry of LM22A-1 and -3 and the small size of the compounds suggest that they are functionally monovalent (i.e., do not interact with more than one receptor monomer). This apparent monovalency raises interesting questions regarding mechanisms of TrkB activation by the compounds. Previous studies have characterized synthetic peptides modeled on loop regions of BDNF, with the goal of using active peptides to derive active non-peptide small molecules that might function as BDNF mimetics (24
). In each of these studies, peptides with neurotrophic activity were found; however, the ability to activate TrkB was either not described (24
) or not detected (54
). Hence whether such peptides can function as ligands that activate TrkB remains to be determined. Unlike TrkA and TrkC, for which there is evidence that “monovalent” cyclic peptidomimetics can exhibit small degrees of agonist activity (55
), for TrkB only “dimeric” peptides have as yet been found to demonstrate trophic activity. This finding is consistent with the classical model in which the BDNF dimer is thought to induce dimerization leading to activation (autophosphorylation) of TrkB. If the compounds act in a monomeric fashion, one possible mechanism by which this activation might occur is that the compound binds to a TrkB monomer, inducing a conformational change that promotes dimerization. Also, if, as has been proposed for TrkA, the unliganded receptor monomer is in equilibrium with dimeric or higher multimeric states (57
), then the compounds might stabilize and/or promote phosphorylation of complexes, without inducing dimerization. It is unlikely that each of the 4 compounds characterized here is capable of forming high-affinity, bivalent dimers in solution, which then function as dimeric ligands. Co-crystallization and other future kinetic and structural studies will address the questions of how the mechanisms of activation and binding of the small molecules to TrkB compare with those of BDNF.
The ability of the prototype compound LM22A-4 to trigger activation of TrkB and its downstream signaling intermediates in the hippocampus and striatum is consistent with the observed effects on motor learning after TBI, though the anatomic substrates and mechanisms of that effect will require further investigation.
These findings, along with the compound’s ability to improve cell survival in several in vitro neurodegenerative disease models, open new opportunities for the further development of derivatives that may have therapeutic effects in a large number of acute and chronic neurological and neuropsychiatric disorders. The prospect that small molecule TrkB ligands could have the BDNF-like effects of promoting neurogenesis and synaptic function suggests further novel therapeutic applications of such compounds. The finding that BDNF interaction with p75NTR
promotes mechanisms underlying pain (23
) raises the interesting possibility that small molecules activating TrkB without mimicking BDNF-p75NTR
interactions might reduce the likelihood of pain as a side effect occurring in BDNF-based therapeutics. In addition, the partial agonist profile of the compounds described here may support their use under circumstances where excessive TrkB signaling is undesirable.
The methods and compounds described here provide a basis for drug discovery in other receptor systems and for designing and characterizing novel non-peptide TrkB ligands optimized for blood brain barrier penetration and other pharmacological features.