December 2024
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Introduction A body of work in our laboratory suggests that neuroprotective drugs should be tested against increasingly stringent models of cerebral ischemia in order to identify the drug(s) and dose providing the highest therapeutic index (‘last man standing’). It is within this context that we have evaluated peptides, specifically arginine (R)-rich cell-penetrating peptides (CPPs), which have recently emerged as promising neuroprotective candidates. Conjugation of the well-known TAT (GRKKRRQRRRPQ) CPP with various peptide cargoes has provided neuroprotection against stroke-like insults in vitro and in vivo [1]. Surprisingly, arginine-rich CPPs on their own (i.e., without cargo) were reported to provide better neuroprotection than TAT-NR2B9c against stroke-like insults, particularly CPPs containing more R residues [2,3]. Maximal neuroprotection was achieved with R18 (currently in a phase II stroke clinical trial). The mechanism of neuroprotection was attributed primarily to neuronal entry of oligoarginine peptides causing concomitant withdrawal of NMDA receptors from the plasma membrane, in turn reducing excitotoxic Ca2+ influx [4]. Methods We now show that poly-arginine peptides are actually quite limited in the degree of neuroprotection conferred. Specifically, neurotoxic Ca2+ influx is no longer suppressed if the in vitro excitotoxic insult is prolonged or intense. Moreover, R18 alone caused synaptotoxicity at relatively higher doses, judged using multi-electrode arrays. Results Thus, the therapeutic index of current CPPs may be too limited: TAT-conjugated peptides may display insufficient efficacy, while the higher doses of long-chain oligoarginines required to improve neuroprotection approaches a neurotoxic threshold. We now identify a class of alkyl-polyarginine peptides which improves against these limitations. Compared to R18, acylated-R9 peptides provided more potent neuroprotection against prolonged/intense in vitro insults at equimolar doses, which was below the synaptotoxic threshold relative to R18. Testing acyl chain lengths from C4 to C14 in R9 peptides yielded the optimal therapeutic index was produced by C10-R9 or C12-R9. R9 alone caused neuronal plasma membrane hyperpolarization. Plasma membrane hyperpolarization enhances cell penetration by CPPs, but also inactivates NMDA receptors. Conclusion These findings do not support a mechanism requiring reduced cell surface NMDA receptors, but instead suggest a plasma membrane hyperpolarization based mechanism resulting in a superior therapeutic index provided by acyl-R9’s.