Auxiliary Proteins are the Predominant Determinants of Differential Efficacy of Clinical Candidates Acting as AMPA Receptor Positive Allosteric Modulators

Abstract

Positive allosteric modulators (PAMs) of AMPA receptors boost cognitive performance in preclinical and clinical studies. Their therapeutic window is narrow, however, and clinical application will likely only occur if greater discrimination in activity is achieved. Toward that end, we compared the modulatory activity of two PAMs recently considered as clinical candidates, LY451395 (mibampator) and PF-04958242/BIIB104, on recombinant and native AMPA receptors (AMPARs). We found that the principle molecular determinant that shaped modulatory activity of both PAMs on deactivation (recombinant) and decay (synaptic) of AMPARs was the auxiliary protein incorporated into the receptor complexes. AMPARs containing the stargazin/g2 transmembrane AMPAR regulatory protein (TARP) were slowed to a .10-fold degree by both PAMs as compared with those incorporating g8 TARP. Neither subunit composition nor flip/flop splice variation had substantive effect. Similarly, stargazin/g2-containing mossy fiber EPSCs in cerebellar granule neurons were slowed to a ∼5-fold greater degree than EPSCs in hippocampal CA1 pyramidal cell neurons, which express the g8 TARP. LY451395 exhibited greater efficacy than BIIB104 at both synapses. These studies provide insight into the receptor constituents that determine efficacy of sulfonamide PAMs. We conclude that compounds that discriminate between AMPARs complexed with distinct TARPs, and particularly those with lower stargazin/g2 efficacy such as BIIB104, could act as viable procognitive therapeutics. SIGNIFICANCE STATEMENTPositive allosteric modulators (PAMs) of AMPA receptors enhance cognitive function in a variety of preclinical models. A clearer understanding of the critical determinants of PAM activity could yield critical insight into pathways to maximize their therapeutic index. Here we show that auxiliary proteins for AMPARs play a major, but thus far underappreciated, role in shaping recombinant and neuronal AMPAR modulation by two clinical candidate PAMs. These data will inform both clinical outcomes as well as future rational development of new modulators.

Structure, Function, and Pharmacology of Glutamate Receptor Ion Channels

“…NA, not applicable; GluN1/2D receptors show no desensitization in the continued presence of agonist. , 15 Traynelis and Wahl (1997), 16 Heckmann et al (1996), 17 Bowie (2002, Bowie and Lange (2002), 18 Schiffer et al (1997), 19 Varney et al (1996, # 5-6 (0.27-0.46) 2,6,8,10,11 " 1.4-3.3 2,8,10,15,18,28,31 " 3.9-7.4 2,4,8,10,28,31 # 55-170 .5-7.7 15,19,20,28,31 " 5.9-12 19,20,23,28,31 # 65-150 15,19 " 0.047-0.17 ,24,27,30 " 10-45 2,22,[24][25][26]30 13-22 22,30 " 0.07-0.27 2,8,22,[24][25][26]30 (2006), Dawe et al (2016) and Ishii et al (2020) for data on the effects of auxiliary subunits on different AMPA receptor splice isoforms. GluA2Q indicates cDNA for the unedited GluA2 with Gln at the Q/R/N site.…”

“…A recent medicinal chemistry campaign yielded a high-affinity series of benzothiadiazines, including some with nanomolar EC 50 values for potentiation of AMPA receptor signaling (Goffin et al, 2018). The biarylpropylsulfonamides include PEPA (flop-specific) and the high-affinity clinical candidate LY-451395 (or mibampator) (flip-specific) (Shepherd et al, 2002;Ishii et al, 2020). The tetrahydrofuran ether PF-04958242 (also known as BIIB104) (Table 7) completely occludes the desensitization state in flip-containing receptors, with less dependence on the flip/flop isoform for deactivation (Shaffer et al, 2015;Ishii et al, 2020).…”

“…The biarylpropylsulfonamides include PEPA (flop-specific) and the high-affinity clinical candidate LY-451395 (or mibampator) (flip-specific) (Shepherd et al, 2002;Ishii et al, 2020). The tetrahydrofuran ether PF-04958242 (also known as BIIB104) (Table 7) completely occludes the desensitization state in flip-containing receptors, with less dependence on the flip/flop isoform for deactivation (Shaffer et al, 2015;Ishii et al, 2020). A comparative study with BIIB104 and LY-451395 revealed that TARP auxiliary subunits shape the effects on deactivation of AMPA receptors and eliminate most differential effects of flip/flop splicing (Ishii et al, 2020).…”

“…The tetrahydrofuran ether PF-04958242 (also known as BIIB104) (Table 7) completely occludes the desensitization state in flip-containing receptors, with less dependence on the flip/flop isoform for deactivation (Shaffer et al, 2015;Ishii et al, 2020). A comparative study with BIIB104 and LY-451395 revealed that TARP auxiliary subunits shape the effects on deactivation of AMPA receptors and eliminate most differential effects of flip/flop splicing (Ishii et al, 2020). These data predict that the potentiating activity of PAMs could show regiospecificity at synapses that follows the expression pattern of auxiliary subunits (Tomita et al, 2006) (Fig.…”

Characterizing the binding and function of TARP γ8-selective AMPA receptor modulators

“…Malfunction of these receptors is associated with a variety of neurological and psychiatric disorders, rendering them a strategic drug target ( 3 ). AMPAR-targeting therapeutics that have advanced into clinical trials are either positive allosteric modulators that improve cognition ( 4 – 6 ) or negative allosteric modulators (NAMs) that have been trialed in epilepsy treatment ( 7 ). Nevertheless, because both modulator types target sequence-conserved receptor segments, the ligand-binding domain in the case of positive allosteric modulators ( 5 ) and the channel gate region for NAMs ( 8 , 9 ) (see Fig.…”