Stephen V. Fiacco

Serum Stable Natural Peptides Designed by mRNA Display

Peptides constructed with the 20 natural amino acids are generally considered to have little therapeutic potential because they are unstable in the presence of proteases and peptidases. However, proteolysis cleavage can be idiosyncratic, and it is possible that natural analogues of functional sequences exist that are highly resistant to cleavage. Here, we explored this idea in the context of peptides that bind to the signaling protein Gαi1. To do this, we used a two-step in vitro selection process to simultaneously select for protease resistance while retaining function–first by degrading the starting library with protease (chymotrypsin), followed by positive selection for binding via mRNA display. Starting from a pool of functional sequences, these experiments revealed peptides with 100–400 fold increases in protease resistance compared to the parental library. Surprisingly, selection for chymotrypsin resistance also resulted in similarly improved stability in human serum (~100 fold). Mechanistically, the decreases in cleavage results from both a lower rate of cleavage (kcat) and a weaker interaction with the protease (Km). Overall, our results demonstrate that the hydrolytic stability of functional, natural peptide sequences can be improved by two orders of magnitude simply by optimizing the primary sequence.

N‐Methyl Scanning Mutagenesis Generates Protease‐Resistant G Protein Ligands with Improved Affinity and Selectivity

ment of the a-carbon side chain to the amide nitrogen atom often disrupts peptide function. We were interested in strategies that enhance proteolytic stability while preserving the function of the underlying peptide. Toward that end, we have explored the effect of inserting single N-methyl amino acids into functional peptides, a method we refer to as N-methyl scanning mutagenesis. Our hope was to find one or more substitutions that enhance protease resistance while retaining binding affinity. We chose the G-protein-binding core-motif peptide DKLYWWEFL, which binds the heterotrimeric inhibitory G protein Gai1*GDP with good affinity (Kd=200 nm), as our model system. [4] Our strategy was to systematically construct nine variants of this peptide bearing a single N-methyl analogue of the natural residue at each position. One challenge we faced was that previous synthetic methods for incorporating N-methyl residues involved long multistep reactions per coupling or A in low yields. [5,6] The synthetic difficulty is the result of coupling the secondary amine of an N-methyl peptide. [7] This coupling must go to near completion, in a short period of time to avoid diketopiperazine formation. [6] To couple Nmethyl residues we followed a strategy similar to Giralt and co-workers, [6] but used the additive pair O-(7-azabenzotriazol-1yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU)/ 1-hydroxy-7-azabenzotriazole (HOAt). Coupling each N-methyl amino acid goes to near completion in 20 min in one step with no preactivation for all residues we tested. As a result, we were able to construct the N-methyl peptides reliably with a final yield of 20‐25% (Table 1).

Directed Evolution of Scanning Unnatural-Protease-Resistant (SUPR) Peptides for in Vivo Applications.

Peptides typically have poor biostabilities, and natural sequences cannot easily be converted into drug‐like molecules without extensive medicinal chemistry. We have adapted mRNA display to drive the evolution of highly stable cyclic peptides while preserving target affinity. To do this, we incorporated an unnatural amino acid in an mRNA display library that was subjected to proteolysis prior to selection for function. The resulting “SUPR (scanning unnatural protease resistant) peptide” showed ≈500‐fold improvement in serum stability (t 1/2 =160 h) and up to 3700‐fold improvement in protease resistance versus the parent sequence. We extended this approach by carrying out SUPR peptide selections against Her2‐positive cells in culture. The resulting SUPR4 peptide showed low‐nanomolar affinity toward Her2, excellent specificity, and selective tumor uptake in vivo. These results argue that this is a general method to design potent and stable peptides for in vivo imaging and therapy.

Automated, Resin-Based Method to Enhance the Specific Activity of Fluorine-18 Clicked PET Radiotracers

Radiolabeling of substrates with 2-[18F]fluoroethylazide exploits the rapid kinetics, chemical selectivity, and mild conditions of the copper-catalyzed azide-alkyne cycloaddition reaction. While this methodology has proven to result in near-quantitative labeling of alkyne-tagged precursors, the relatively small size of the fluoroethylazide group makes separation of the 18F-labeled radiotracer and the unreacted precursor challenging, particularly with precursors >500 Da (e.g., peptides). We have developed an inexpensive azide-functionalized resin to rapidly remove unreacted alkyne precursor following the fluoroethylazide labeling reaction and integrated it into a fully automated radiosynthesis platform. We have carried out 2-[18F]fluoroethylazide labeling of four different alkynes ranging from <300 Da to >1700 Da and found that >98% of the unreacted alkyne was removed in less than 20 min at room temperature to afford the final radiotracers at >99% radiochemical purity with specific activities up to >200 GBq/μmol. We have applied this technique to label a novel cyclic peptide previously evolved to bind the Her2 receptor with high affinity, and demonstrated tumor-specific uptake and low nonspecific background by PET/CT. This resin-based methodology is automated, rapid, mild, and general allowing peptide-based fluorine-18 radiotracers to be obtained with clinically relevant specific activities without chromatographic separation and with only a minimal increase in total synthesis time.

Abstract P5-01-10: In vitro and in vivo imaging of Her2 with cyclic peptides derived from directed evolution

The implementation of personalized medicine for Her2-positive breast cancer treatment (Trastuzumab and Pertuzumab) has made a dramatic effect on overall patient outcome. Unfortunately, there are currently no FDA-approved imaging agents to monitor Her2-positive breast cancer leaving physicians to rely on invasive biopsies and anatomical imaging to monitor treatment with Her2-targeted therapeutics. There is also increasing evidence that women initially diagnosed with Her2-negative breast can present with Her2-positive disease upon recurrence. Noninvasive, whole-body visualization of Her2 would identify Her2-recurrent disease and serve as a powerful tool to monitor the effectiveness of new and emerging Her2-targeted therapeutics (e.g. TDM-1). While many Her2-antibody based imaging agents have been used in preclinical applications, their long circulating half-lives, high liver uptake, and poor synthetic accessibility pose a challenges for clinical translation. Here we describe the use of highly stable cyclic peptides derived from biological display as imaging agents for Her2-positive breast cancer. These peptides have antibody-like high affinity ( Citation Format: Lindsay E Kelderhouse, Amanda Hardy, Yong Pan, Patrea Rhea, Argentina Ornelas, Seth Gammon, Peiying Yang, Stephen Fiacco, Steven W Millward. In vitro and in vivo imaging of Her2 with cyclic peptides derived from directed evolution [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-01-10.