Ted J. Gauthier

Monitoring a Nuclear Factor-κB Signature of Drug Resistance in Multiple Myeloma

The emergence of acquired drug resistance results from multiple compensatory mechanisms acting to prevent cell death. Simultaneous monitoring of proteins involved in drug resistance is a major challenge for both elucidation of the underlying biology and development of candidate biomarkers for assessment of personalized cancer therapy. Here, we have utilized an integrated analytical platform based on SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring mass spectrometry, a versatile and powerful tool for targeted quantification of proteins in complex matrices, to evaluate a well-characterized model system of melphalan resistance in multiple myeloma (MM). Quantitative assays were developed to measure protein expression related to signaling events and biological processes relevant to melphalan resistance in multiple myeloma, specifically: nuclear factor-κB subunits, members of the Bcl-2 family of apoptosis-regulating proteins, and Fanconi Anemia DNA repair components. SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring methods were developed for quantification of these selected target proteins in amounts of material compatible with direct translation to clinical specimens (i.e. less than 50,000 cells). As proof of principle, both relative and absolute quantification were performed on cell line models of MM to compare protein expression before and after drug treatment in naïve cells and in drug resistant cells; these liquid chromatography-multiple reaction monitoring results are compared with existing literature and Western blots. The initial stage of a systems biology platform for examining drug resistance in MM has been implemented in cell line models and has been translated to MM cells isolated from a patient. The ultimate application of this platform could assist in clinical decision-making for individualized patient treatment. Although these specific assays have been developed to monitor MM, these techniques are expected to have broad applicability in cancer and other types of disease.

Constrained cyclic peptides as immunomodulatory inhibitors of the CD2:CD58 protein-protein interaction

The interaction between the cell-cell adhesion proteins CD2 and CD58 plays a crucial role in lymphocyte recruitment to inflammatory sites, and inhibitors of this interaction have potential as immunomodulatory drugs in autoimmune diseases. Peptides from the CD2 adhesion domain were designed to inhibit CD2:CD58 interactions. To improve the stability of the peptides, β-sheet epitopes from the CD2 region implicated in CD58 recognition were grafted into the cyclic peptide frameworks of sunflower trypsin inhibitor and rhesus theta defensin. The designed multicyclic peptides were evaluated for their ability to modulate cell-cell interactions in three different cell adhesion assays, with one candidate, SFTI-a, showing potent activity in the nanomolar range (IC50: 51 nM). This peptide also suppresses the immune responses in T cells obtained from mice that exhibit the autoimmune disease rheumatoid arthritis. SFTI-a was resistant to thermal denaturation, as judged by circular dichroism spectroscopy and mass spectrometry, and had a half-life of ∼24 h in human serum. Binding of this peptide to CD58 was predicted by molecular docking studies and experimentally confirmed by surface plasmon resonance experiments. Our results suggest that cyclic peptides from natural sources are promising scaffolds for modulating protein-protein interactions that are typically difficult to target with small-molecule compounds.

Structure–activity relationships of peptidomimetics that inhibit PPI of HER2‐HER3

Human epidermal growth factor receptor-2 (HER2) is a tyrosine kinase family protein receptor that is known to undergo heterodimerization with other members of the family of epidermal growth factor receptors (EGFR) for cell signaling. Overexpression of HER2 and deregulation of signaling has implications in breast, ovarian, and lung cancers. We have designed several peptidomimetics to block the HER2-mediated dimerization, resulting in antiproliferative activity for cancer cells. In this work, we have investigated the structure-activity relationships of peptidomimetic analogs of Compound 5. Compound 5 was conformationally constrained by N- and C-terminal modification and cyclization as well as by substitution with d-amino acids at the N-and C-termini. Among the compounds studied in this work, a peptidomimetic Compound 21 with d-amino acid substitution and its N- and C-termini capped with acetyl and amide functional groups and a reversed sequence compared to that of Compound 5 exhibited better antiproliferative activity in HER2-overexpressed breast, ovarian, and lung cancer cell lines. Compound 21 was further evaluated for its protein-protein interaction (PPI) inhibition ability using enzyme fragment complementation assay, proximity ligation assay, and Western blot analysis. Results suggested that Compound 21 is able to block HER2:HER3 interaction and inhibit phosphorylation of the kinase domain of HER2. The mode of binding of Compound 21 to HER2 protein was modeled using a docking method. Compound 21 seems to bind to domain IV of HER2 near the PPI site of EGFR:HER2, and HER:HER3 and inhibit PPI.

310-Helix stabilization via side-chain salt bridges1

Decapeptides, Sb-10 (H-Api-Aib-Aib-Glu-Aib-Aib-Glu-Aib-Aib-Api-NH2) and Ipi-10 (H-Api-Aib-Aib-Lys-Aib-Aib-Lys-Aib-Aib-Api-NH2), where Aib is α-aminoisobutyric acid and Api is 4-aminopiperidine-4-carboxylic acid, are designed to be amphipathic as 310-helices; Sb-10 is the first example of a 310-helical peptide stabilized by side-chain salt bridging or ion pairing.

The structures of N-(t-butoxycarbonyl)-3-methylbenz[f]indole and N-benzenesulfonyl-4-bromo-3-methylbenz[f]indole

N-(t-butoxycarbonyl)-3-methylbenz[f]indole,1, C18H19NO2, crystallizes in orthorhombic space groupPna21 witha=6.0338(2),b=24.839(4),c=10.2481(7) Å,V=1535.9(3) Å3,Z=4. The structure was refined toR=0.051 for 2007 observed reflections. The benz[f]indole ring system is nearly planar, exhibiting mean deviation of 0.048 Å. The ester plane of thet-butoxycarbonyl substituent is nearly coplanar with the benz[f]indole ring system, forming a C−N−C=O torsion angle with magnitude 10.6(5)0.N-benzenesulfonyl-4-bromo-3-methyl-benz[f]indole,2, C19H14BrNO2S crystallizes in triclinic space groupP−1 witha=8.1400(5),b=10.0587(8),c=10.8863(7) Å, α=89.927(7), β=110.495(5), γ=96.846(6)0,V=828.2(3) Å3,Z=2. The structure was refined toR=0.050 for 2653 observed reflections. The benz[f]indole ring system is nearly planar, with mean deviation 0.016 Å, and forms a dihedral angle of 80.93(9)0 with the phenyl plane.

Design of cyclic and d-amino acids containing peptidomimetics for inhibition of protein-protein interactions of HER2-HER3

HER2 receptors are surface proteins belonging to the epidermal growth factor family of receptors. Their numbers are elevated in breast, lung, and ovarian cancers. HER2‐positive cancers are aggressive, have higher mortality rate, and have a poor prognosis. We have designed peptidomimetics that bind to HER2 and block the HER2‐mediated dimerization of epidermal growth factor family of receptors. Among these, a symmetrical cyclic peptidomimetic (compound 18) exhibited antiproliferative activity in HER2‐overexpressing lung cancer cell lines with IC50 values in the nanomolar concentration range. To improve the stability of the peptidomimetic, d‐amino acids were introduced into the peptidomimetic, and several analogs of compound 18 were designed. Among the analogs of compound 18, compound 32, a cyclic, d‐amino acid‐containing peptidomimetic, was found to have an IC50 value in the nanomolar range in HER2‐overexpressing cancer cell lines. The antiproliferative activity of compound 32 was also measured by using a 3D cell culture model that mimics the in vivo conditions. The binding of compound 32 to the HER2 protein was studied by surface plasmon resonance. In vitro stability studies indicated that compound 32 was stable in serum for 48 hours and intact peptide was detectable in vivo for 12 hours. Results from our studies indicated that 1 of the d‐amino acid analogs of 18, compound 32, binds to the HER2 extracellular domain, inhibiting the phosphorylation of kinase of HER2.

CPProtectides: Rapid uptake of well-folded β-hairpin peptides with enhanced resistance to intracellular degradation

Cell penetrating peptides (CPPs) have emerged as powerful tools for delivering bioactive cargoes, such as biosensors or drugs to intact cells. One limitation of CPPs is their rapid degradation by intracellular proteases. β‐hairpin “protectides” have previously been demonstrated to be long‐lived under cytosolic conditions due to their secondary structure. The goal of this work was to demonstrate that arginine‐rich β‐hairpin peptides function as both protectides and as CPPs. Peptides exhibiting a β‐hairpin motif were found to be rapidly internalized into cells with their uptake efficiency dependent on the number of arginine residues in the sequence. Cellular internalization of the β‐hairpin peptides was compared to unstructured, scrambled sequences and to commercially available, arginine‐rich CPPs. The unstructured peptides displayed greater uptake kinetics compared to the structured β‐hairpin sequences; however, intracellular stability studies revealed that the β‐hairpin peptides exhibited superior stability under cytosolic conditions with a 16‐fold increase in peptide half‐life. This study identifies a new class of long‐lived CPPs that can overcome the stability limitations of peptide‐based reporters or bioactive delivery mechanisms in intact cells.

Design of novel lipidated peptidomimetic conjugates for targeting EGFR heterodimerization in HER2 + cancer

The human epidermal growth factor receptor (EGFR) family is known to be involved in cell signaling pathways. The extracellular domain of EGFR consists of four domains, of which domain II and domain IV are known to be involved in the dimerization process. Overexpression of these receptors is known to play a significant role in heterodimerization of these receptors leading to the development of cancer. We have designed peptidomimetic molecules to inhibit the EGFR heterodimerization interaction that have shown antiproliferative activity and specificity for HER2-positive cancer cell lines. Among these, a peptidomimetic, compound 5, exhibited antiproliferative activity at low nanomolar concentrations in HER2-overexpressing cancer cell lines. To improve the stability of this peptidomimetic, we have designed and synthesized a novel conjugate of peptidomimetic compound 5 with a lipid, stearic acid. The antiproliferative activity of this conjugate was evaluated in HER2-positive cancer cell lines. Results suggested that the conjugate exhibited selective antiproliferative activity in HER2-overexpressing breast and lung cancer cell lines and was able to block HER2:HER3 heterodimerization. Also, the conjugate showed improved stability with a half-life of 5 h in human serum compared to the half-life of 2 h for parent compound 5. The binding affinity of the conjugate to HER2 protein was evaluated by SPR analysis, and the mode of binding of the lipid conjugate to domain IV of HER2 protein was demonstrated by docking analysis. Thus, this novel lipid conjugate can be used to target HER2-overexpressing cancers.

Generating Isoform-Specific Antibodies : Lessons from Nucleocytoplasmic Glycoprotein Skp1

Antibodies that discriminate protein isoforms differing by modifications at specific amino acids have revolutionized studies of their functions. Skp1 is a novel nucleocytoplasmic glycoprotein that is hydroxylated at proline-143 and then O-glycosylated by a pentasaccharide attached via a GlcNAcα1, 4(trans)- hydroxyproline linkage. Skp1 isoform-specific antibodies were successfully obtained by immunizing mice or rabbits with KLH-coupled synthetic peptides bearing either unmodified Pro, 4(trans)-hydroxyproline, or D-GlcNAcα1,4 (trans)-hydroxyproline, and screening with corresponding BSA-conjugates or by Western blotting toward a panel of Skp1 isoforms. Antibodies specific for Skp1 or HO-Skp1 were not found in exhaustive murine trials, yet monospecific polyclonal antibodies were readily achieved in rabbits without crossadsorption. In all cases, antibodies were specific at the protein but not the peptide level, which suggests that conformation comprises part of the basis for recognition and which should be considered when developing screening strategies.

Selective Bioactivity of Synthetic Amphipathic Peptides Against Murine Macrophages Infected with Brucella abortus

The efficacy of current antibiotics is declining at an alarming rate due to the proliferation of multi-drug resistant bacteria [1]. Synthetic antimicrobial peptides are currently being investigated as potential agents to address the increased need of antibiotics with different mechanism of action [2]. A series of short, synthetic, amphipathic peptides, incorporating high levels of Cα,Cα-disubstituted glycines (Table 1) have been prepared to determine their suitability as antimicrobial agents against the intracellular pathogen Brucella abortus. Though exhibiting low direct bioactivity against B. abortus, the peptides show a remarkable selectivity towards killing Murine peritoneal macrophages that have been infected with the pathogen in vitro. In all instances, the a-helical permutation isomers of the peptides exhibited higher selectivity and overall activity than the 310-helical permutations. These results are being applied towards the development of therapeutic applications of synthetic peptides against intracellular pathogens of significant current interest, such as M. tuberculosis.