RO-275

RO-275 is a primary carboxamide derivative with potent HCN1 cation channel inhibitory activity (submicromolar concentrations), as shown in in vitro electrophysiology experiments. RO-275 is highly selective over HCN isoforms 2-4 and other relevant ion channels (hERG, Nav1.4). Moreover, the carboxamide derivative showed a low binding affinity for metabotropic glutamate receptor 5 (mGluR5), which is known to be targeted by structurally related compounds. Selectivity over other voltage- and ligand-gated cation channels expressed in neurons was not yet determined. Inhibition was directly assessed in overexpression systems only against human and rat HCN1 channels, but not on the mouse homologue, a rodent species widely used in neuroscience experiments. Inhibitory activity in nanomolar range was also observed against physiologically relevant variants, such as hHCN1/HCN2 heterotetramer and hHCN1 co-expressed with channel chaperone TRIP8b., but the effect on other heteromeric configurations (e.g., HCN1/HCN4) was not yet explored. Interestingly, RO-275 seems to have a different binding mode to HCN1 in comparison with unselective HCN channels inhibitors, due to the absence of use- and voltage-dependent inhibition, and also due to a delayed onset of the inhibitory activity. However, the exact binding site and interacting residues are currently unknown. In rat CA1 pyramidal neurons (ex vivo), RO-275 significantly, but only partially reduced the sag response at 1 µM concentration. Furthermore, RO-275 did not significantly enhance artificial excitatory postsynaptic potential (aEPSP) summation in these neurons, while the effect on HCN1 KO rodent brain slices was not assessed for this particular compound. Nonetheless. RO-275 showed no effect on spontaneously beating atrial cardiomyocytes at tested concentrations, due to its selectivity profile. The distinct pharmacological profile of RO-275 makes it suitable for investigating the physiological and pathological roles of HCN1, but further studies are needed to establish the pharmacological parameters on mouse HCN1, while localised dendritic patch clamp and high-resolution imaging are warranted to further investigate the effects on voltage sag and EPSP summation. Due to its favourable physicochemical and pharmacokinetic parameters, RO-275 can be suitable for both intraperitoneal (i.p.) and oral administration. At a 30 mg/kg dose (i.p.), RO-275 was efficient in improving working memory in a translationally relevant touchscreen task (TUNL) in rats with cognitive dysfunction induced with a NMDAR antagonist. Moreover, the same dose was shown to have no effect on heart rate in a rat telemetry assay, Further safety pharmacology studies are needed to investigate the long-term effects of RO-275 administration on motor learning and other cognitive functions. Toxicity studies are also warranted. Nonetheless, at present, RO-275 is the best available compound to interrogate the role of HCN1 in physiology and to investigate the outcome of selective pharmacological inhibition of HCN1 in animal models of CNS disorders, such as epilepsy, schizophrenia, autism etc.