Justin K. Murray

Exploration of Backbone Space in Foldamers Containing α- and β-Amino Acid Residues: Developing Protease-Resistant Oligomers that Bind Tightly to the BH3-Recognition Cleft of Bcl-xL

Protein–protein interactions play crucial roles in cell‐signaling events and are often implicated in human disease. Molecules that bind tightly to functional protein‐surface sites and show high stability to degradative enzymes could be valuable pharmacological tools for dissection of cell‐signaling networks and might ultimately lead to therapeutic agents. We recently described oligomers containing both α‐ and β‐amino acid residues that bind tightly to the BH3 recognition site of the anti‐apoptotic protein Bcl‐xL. The oligomers with highest affinity had a nine‐residue N‐terminal segment with a 1:1 α:β residue repeat and a six‐residue C‐terminal segment containing exclusively proteinogenic α‐residues. The N‐terminal portions of such (α/β+α)‐peptides are highly resistant to proteolysis, but the C‐terminal α‐segments are susceptible. This study emerged from efforts to modify the α‐segment in an (α/β+α)‐peptide in a way that would diminish proteolytic degradation but retain high affinity for Bcl‐xL. Some of the oligomers reported here could prove useful in certain biological applications, particularly those for which extended incubation in a biological milieu is required.

Engineering Potent and Selective Analogues of GpTx-1, a Tarantula Venom Peptide Antagonist of the NaV1.7 Sodium Channel

NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20× and 1000× over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp(29), Lys(31), and Phe(34) near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000× selectivity against NaV1.4 and NaV1.5.

Parallel synthesis of peptide libraries using microwave irradiation

The application of microwave irradiation to solid-phase peptide synthesis increases product purity and reduces reaction time. Parallel synthesis in 96-well polypropylene filter plates with microwave irradiation is an efficient method for the rapid generation of combinatorial peptide libraries in sufficient purity to assay the products directly for biological activity without HPLC purification. In this protocol, the solid-phase support is arrayed into each well of a 96-well plate, reagents are delivered using a multichannel pipette and a microwave reactor is used to complete peptide coupling reactions in 6 min and Fmoc-removal reactions in 4 min under temperature-controlled conditions. The microwave-assisted parallel peptide synthesis protocol has been used to generate a library of difficult hexa-β-peptides in 61% average initial purity (50% yield) and has been applied to the preparation of longer α- and β-peptides. Using this protocol, a library of 96 different hexapeptides can be synthesized in 24 h (excluding characterization).

Dipeptidyl-Quinolone Derivatives Inhibit Hypoxia Inducible Factor-1α Prolyl Hydroxylases-1, -2, and -3 with Altered Selectivity

Intracellular levels of the hypoxia-inducible transcription factor (HIF) are regulated under normoxic conditions by prolyl hydroxylases (PHD1, 2, and 3). Treatment of cells with PHD inhibitors stabilizes HIF-1α, eliciting an artificial hypoxic response that includes the transcription of genes involved in erythropoiesis, angiogenesis, and glycolysis. The different in vivo roles of the three PHD isoforms are not yet known, making a PHD-selective inhibitor useful as a biological tool. Although several chemical series of PHD inhibitors have been described, significant isoform selectivity has not been reported. Here we report the synthesis and activity of dipeptidyl analogues derived from a potent but non-selective quinolone scaffold. The compounds were prepared by Pd-catalyzed reductive carbonylation of the 6-iodoquinolone derivative to form the aldehyde directly, which was then attached to a solid support via reductive amination. Amino acids were coupled, and the resulting dipeptidyl-quinolone derivatives were screened, revealing retention of PHD inhibitory activity but an altered PHD1, 2, and 3 selectivity profile. The compounds were found to be ∼10-fold more potent against PHD1 and PHD3 than against PHD2, whereas the specific parent compound had shown no appreciable selectivity among the different PHD isoforms.

An antiangiogenic neurokinin-B/thromboxane A2 regulatory axis

Establishment of angiogenic circuits that orchestrate blood vessel development and remodeling requires an exquisite balance between the activities of pro- and antiangiogenic factors. However, the logic that permits complex signal integration by vascular endothelium is poorly understood. We demonstrate that a “neuropeptide,” neurokinin-B (NK-B), reversibly inhibits endothelial cell vascular network assembly and opposes angiogenesis in the chicken chorioallantoic membrane. Disruption of endogenous NK-B signaling promoted angiogenesis. Mechanistic analyses defined a multicomponent pathway in which NK-B signaling converges upon cellular processes essential for angiogenesis. NK-B−mediated ablation of Ca2+ oscillations and elevation of 3′–5′ cyclic adenosine monophosphate (cAMP) reduced cellular proliferation, migration, and vascular endothelial growth factor receptor expression and induced the antiangiogenic protein calreticulin. Whereas NK-B initiated certain responses, other activities required additional stimuli that increase cAMP. Although NK-B is a neurotransmitter/ neuromodulator and NK-B overexpression characterizes the pregnancy-associated disorder preeclampsia, NK-B had not been linked to vascular remodeling. These results establish a conserved mechanism in which NK-B instigates multiple activities that collectively oppose vascular remodeling.

PADLOC: A Powerful Tool to Assign Disulfide Bond Connectivities in Peptides and Proteins by NMR Spectroscopy

The determination of the disulfide bond connectivity in a peptide or protein represents a significant challenge. It is notoriously difficult to use NMR spectroscopy to assign disulfide connectivities because NMR spectra lack direct evidence for disulfide bonds. These bonds are typically inferred from three-dimensional structure calculations, which can result in ambiguous disulfide assignment. Here, we present a new NMR based methodology, in which the disulfide connectivity is obtained by applying Bayesian rules of inference to the local topology of cysteine residues. We illustrate how this approach successfully predicts the disulfide connectivity in proteins for which crystal structures are available in the protein data bank (PDB). We also demonstrate how this methodology is used with experimental NMR data for peptides with complex disulfide topologies, including hepcidin, Kalata-B1, and μ-Conotoxin KIIIA. In the case of μ-Conotoxin KIIIA, the PADLOC connectivity (1-15,2-9,4-16) differs from previously published results; additional evidence is presented demonstrating unequivocally that this newly proposed connectivity is correct.

Single Residue Substitutions That Confer Voltage-Gated Sodium Ion Channel Subtype Selectivity in the NaV1.7 Inhibitory Peptide GpTx-1

There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit NaV1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with NaV1.7 and related NaV subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on NaV1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a NaV1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work.

Sustained inhibition of the NaV1.7 sodium channel by engineered dimers of the domain II binding peptide GpTx-1.

Many efforts are underway to develop selective inhibitors of the voltage-gated sodium channel NaV1.7 as new analgesics. Thus far, however, in vitro selectivity has proved difficult for small molecules, and peptides generally lack appropriate pharmacokinetic properties. We previously identified the NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity via structure-guided analoging. To further understand GpTx-1 binding to NaV1.7, we have mapped the binding site to transmembrane segments 1-4 of the second pseudosubunit internal repeat (commonly referred to as Site 4) using NaV1.5/NaV1.7 chimeric protein constructs. We also report that select GpTx-1 amino acid residues apparently not contacting NaV1.7 can be derivatized with a hydrophilic polymer without adversely affecting peptide potency. Homodimerization of GpTx-1 with a bifunctional polyethylene glycol (PEG) linker resulted in a compound with increased potency and a significantly reduced off-rate, demonstrating the ability to modulate the function and properties of GpTx-1 by linking to additional molecules.

Exploration of structure--activity relationships among foldamer ligands for a specific protein binding site via parallel and split-and-mix library synthesis.

We describe the use of parallel and split-and-mix library synthesis strategies for exploration of structure-activity relationships among peptidic foldamer ligands for the BH3-recognition cleft of the anti-apoptotic protein Bcl-xL. This effort began with a chimeric (alpha/beta+alpha)-peptide oligomer (composed of an alpha/beta-peptide segment and an alpha-peptide segment) that we previously identified to bind tightly to the target cleft on Bcl-xL. The side chains that interact with Bcl-xL were varied in a 1000-member one-bead-one-compound library. Fluorescence polarization (FP) screening identified four new analogues with binding affinities similar to that of the lead compound but no analogues with enhanced affinity. These results suggested that significant improvements in affinity were unlikely in this series. We then used library synthesis to examine backbone variations in the C-terminal alpha-peptide segment of the lead compound. These studies provided an opportunity for direct comparison of parallel and split-and-mix synthesis formats for foldamer libraries with respect to synthetic variability and assay sensitivity. We found that compounds from both the parallel and one-bead-one-compound libraries could be reliably screened in a competition FP assay without purification of library members. Our findings should facilitate the use of combinatorial library synthesis for exploration of foldamers as inhibitors of protein-protein interactions.

Engineering Antibody Reactivity for Efficient Derivatization to Generate NaV1.7 Inhibitory GpTx-1 Peptide–Antibody Conjugates

The voltage-gated sodium channel NaV1.7 is a genetically validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain; however functional NaV1.7 targeting antibodies are not known. In this report, we describe NaV1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the NaV1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of 13 potential cysteine conjugation sites in the antibody scaffold was tuned using a model alkylating agent. Subsequent reactions with the peptide identified cysteine locations with the highest conversion to desired conjugates, which blocked NaV1.7 currents in whole cell electrophysiology. Variations in attachment site, linker, and peptide loading established design parameters for potency optimization. Antibody conjugation led to in vivo half-life extension by 130-fold relative to a nonconjugated GpTx-1 peptide and differential biodistribution to nerve fibers in wild-type but not NaV1.7 knockout mice. This study describes the optimization and application of antibody derivatization technology to functionally inhibit NaV1.7 in engineered and neuronal cells.