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    • For molecules with potential for

    2022-07-04

    For molecules with potential for therapeutic use, high potency is desired, preferably in the nM or lower concentration range. However, the glycine receptor specific peptides identified with the New England Biolabs Ph.D. libraries, both in this study (Fig. 3) and by Tipps et al. (2010), acted at low μM concentrations. This could be due to the pentavalent display of these libraries. Multivalent display can lead to an avidity effect where phage displaying multiple copies of a particular peptide, each binding to the target, increase the apparent affinities of the multivalent phage-peptide fusions, compared to when peptides are tested alone. This phenomenon has been well documented in the phage display literature (Lowman, 1997). In order to overcome this, one can utilize a monovalent display system, such a phagemid display system, when performing panning procedures (Lowman, 1997). Alternatively, peptide potency could be increased through affinity maturation by using the sequence of the most promising peptides from initial screens, such as D7.2-122 or D7.2-123 in this case, as a scaffold for the creation of a new phage library in which all displayed peptides would be some variation of the originally-identified sequences. We are investigating both possible methods to identify peptides that act with greater potency at the glycine receptor. The variety of amino Tolcapone receptor sequences seen in the 40 identified peptides (Table 1) reflects the large and diverse nature of the pentameric glycine receptor as it is likely that such a large molecular structure presents many potential binding sites for the various peptides contained within the library. Further, it should be noted that phage display can only identify peptides capable of interacting with a particular target, and does not provide information about any possible functional effect of the peptides at that target. Therefore, we narrowed our consensus search to only the eight peptides that enhanced EC5–10 receptor currents ≥20% and revealed several possible amino acid consensus sequences that may contribute to their abilities to positively modulate glycine receptor function (Table 2). The most prevalent consensus was the sequence PxxS, found in four of eight peptides, where the proline residue is located at either positions 3 or 4 and the serine is located at the C-terminal positions 6 or 7. Further, this consensus is expanded within the top two enhancing peptides, D7.2-122 and D7.2-123, to TxPxxS. These two peptides also both contained TTxxxxx, which may also be responsible for their robust abilities to enhance glycine receptor function. Additionally, these two peptides share the sequence TxP with D7.2-118, which just missed the cutoff for ≥20% receptor enhancement (19.1% potentiation of EC5–10 glycine response). In addition to these sequences, we found that three of eight allosteric enhancers contained the sequence TxT, and two contained xxxQxPx. Of the 17 of 25 characterized peptides that did not enhance receptor function ≥20%, TxT was found in three (D7.1-104, D7.1-112, and D7.2-015), QxP was found in one (D7.2-120) and PxxS was found in one other (D7.1-110). It may be that these three consensus sequences allow for interaction with the glycine receptor, and enhancing activity relies on the presence of other specific sequences within the peptide; alternatively, other amino acids within these peptides may interfere with the glycine receptor-enhancing activity that these consensus sequences contribute. Further, it is possible that these peptides could enhance receptor activity at higher concentrations, but are less potent than the eight that did. Zinc is a biphasic modulator of the glycine receptor, capable of enhancing receptor function at concentrations below approximately 10µM, and inhibiting it at higher concentrations (Bloomenthal et al., 1994, Harvey et al., 1999, Laube et al., 1995). It is clear that some peptides, such as D7.2-123−B, contain sufficient contaminating zinc to affect glycine receptor function. However, ICP-MS measures total zinc, and there is no way to tell how much of this value reflects free zinc concentrations acting at the glycine receptor. Further, the relationship between the degree of receptor modulation observed and zinc concentration of the various peptides indicates that peptide effects cannot solely be due to contaminating zinc (Fig. 8). For example, peptides that range in activity between −24.19% to 46.60% change in EC5–10 glycine responses contain between 12.38−15.36nM zinc, a very small range that could not account for the degree of variance observed in the peptide effects. Further, D7.2-123−B had very similar effects at 30 and 100μM concentrations, despite there being over a 3-fold difference in zinc concentration (108.68 vs. 362.38nM zinc) in those two peptide preparations. Glycine receptor potentiation by zinc typically peaks around 1 μM zinc (Miller et al., 2005), with significantly different effects between 100 and 300nM zinc. Therefore, the similarity in effects between the different concentrations of D7.2-123−B must be due to some interaction between both zinc and peptide effects at the glycine receptor.