Engineering Highly Potent and Selective Microproteins against Nav1.7 Sodium Channel for Treatment of Pain

Abstract

The prominent role of voltage-gated sodium channel 1.7 (Nav1.7) in nociception was revealed by remarkable human clinical and genetic evidence. Development of potent and subtypeselective inhibitors of this ion channel is crucial for obtaining therapeutically useful analgesic compounds. Microproteins isolated from animal venoms have been identified as promising therapeutic leads for ion channels, because they naturally evolved to be potent ion channel blockers. Here, we report the engineering of highly potent and selective inhibitors of the Nav1.7 channel based on tarantula ceratotoxin-1 (CcoTx1). We utilized a combination of directed evolution, saturation mutagenesis, chemical modification, and rational drug design to obtain higher potency and selectivity to the Nav1.7 channel. The resulting microproteins are highly potent (IC 50 to Nav1.7 of 2.5 nM) and selective. We achieved 80-and 20-fold selectivity over the closely related Nav1.2 and Nav1.6 channels, respectively, and the IC 50 on skeletal (Nav1.4) and cardiac (Nav1.5) sodium channels is above 3000 nM. The lead molecules have the potential for future clinical development as novel therapeutics in the treatment of pain.

Aspartic Acid Isomerization Characterized by High Definition Mass Spectrometry Significantly Alters the Bioactivity of a Novel Toxin from Poecilotheria

“…With over 220,000 species of venomous animals, there is a seemingly unlimited number of possible candidates for investigation [73]. As an example, spider venoms are particularly attractive with an estimated 18 million peptides from their venoms [74]. With this voluminous accumulation of data, and an even larger repository of yet uncharacterized molecules, it was a reasonable discourse for researchers to invest in their therapeutic potential.…”

From identification to functional characterization of cyriotoxin‐1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei

“…Due to these similarities, CyrTx-1a may share the same binding site on TTX-sensitive Na V channels as the one determined by mutational analysis and in silico docking for HnTx-IV, HwTx-IV, and GpTx-1a (Cai et al, 2015;Minassian et al, 2013;Murray et al, 2016). Indeed, positively charged amino acids Lys 25 , His 26 , Lys 27 , and Lys 30 , surrounded by hydrophobic Phe 5 and Trp 28 clustered on one toxin face may be involved in interactions with negatively charged Glu 753 , Glu 811 , Asp 816 , and Glu 818 or aliphatic residues (Met 750 ) located in S1-S2 and S3-S4 loops of DII domain of TTX-sensitive Na V channels (Klint et al, 2014;Li et al, 2004;Liu et al, 2012;Xiao et al, 2008;Xiao, Blumenthal, Jackson, Liang, & Cummins, 2010 From this point of view and compared to toxins belonging to the NaSpTx family 1 such as HnTx-I, HnTx-III, Hd1a, HnTx-IV, ProTx-III, Cm1a, GpTx-1, and HwTx-IV previously reported to interact with this subtype, CyrTx-1a is thus among the most efficient peptides (Cardoso et al, 2015;Klint et al, 2014;Klint et al, 2015;Liu et al, 2012;Liu et al, 2013;Murray et al, 2015;Murray et al, 2016;Shcherbatko et al, 2016;Xiao et al, 2008). In addition to hNa V 1.7 channels, CyrTx-1a was also shown to be highly potent to block the TTX-S hNa V 1.1, 1.2, 1.3, and 1.6 channels with the following increasing order for mean IC 50 values (between approximately 75 and 300 nM):…”

“…From this point of view and compared to toxins belonging to the NaSpTx family 1 such as HnTx-I, HnTx-III, Hd1a, HnTx-IV, ProTx-III, Cm1a, GpTx-1, and HwTx-IV previously reported to interact with this subtype, CyrTx-1a is thus among the most efficient peptides (Cardoso et al, 2015;Klint et al, 2014;Klint et al, 2015;Liu et al, 2012;Liu et al, 2013;Murray et al, 2015;Murray et al, 2016;Shcherbatko et al, 2016;Xiao et al, 2008). In addition to hNa V 1.7 channels, CyrTx-1a was also shown to be highly potent to block the TTX-S hNa V 1.1, 1.2, 1.3, and 1.6 channels with the following increasing order for mean IC 50 values (between approximately 75 and 300 nM):…”

“…This is likely to be mainly due to the presence of Asn 23 instead of Ser 23 in the HnTx-I sequence that excludes any Na V 1.7 channel activity (Klint, Chin, & Mobli, 2015), while a high potency for Na V 1.7 channels associated with a good selectivity against Na V 1.5 and Na V 1.4 channels is due to the conservation of highly functional residues (Murray et al, 2015;Xiao et al, 2008). Indeed, CyrTx-1a possesses several highly conserved and crucial amino acids, known to govern the Na V channel activity, such as the Phe 5 , Pro 11 , Leu 20 , Ser 23 , His 26 , and more importantly the Trp 28 and Lys 30 residues (Minassian et al, 2013;Murray et al, 2016;Shcherbatko et al, 2016). In addition, its sequence includes a hydrophobic patch (Gly 4 , Gly 6 , and Val 31 ) that has been described to reinforce the inhibitory potency of ICK toxins at Na V 1.7 channels (Agwa, Huang, Craik, Henriques, & Schroeder, 2017).…”

Using automated patch clamp electrophysiology platforms in pain‐related ion channel research: insights from industry and academia

“…IonFlux, QPatch, SyncroPatch, PE384 and Qube) or low, fixed well volumes (IonWorks) allows expensive, low‐quantity compounds and molecules (e.g. peptide toxin fractions; see Klint et al , ; Shcherbatko et al , ; Deuis et al , ) to be tested in limited, small‐volume applications. Micro‐fluidic channels also provide fast external solution exchange rates (complete solution exchange between 10 and 50 ms), sufficiently fast to allow for recordings from rapidly desensitizing ligand‐gated ion channels (e.g.…”