Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1

Abstract

We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to pre-inhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties

The Lock is the Key: Development of Novel Drugs through Receptor Based Combinatorial Chemistry

“…156 Measuring Akt1 kinase activity under varying substrate and triligand concentrations eliminated the possibility of a competitive mode of Akt1 inhibition by the triligand with respect to ATP and peptide substrates. 150 This confirmed that the triligand binds to a location away from the active site of the kinase and that inhibition occurs via an allosteric mechanism. Finally, the anchor, biligand, and triligand were tested for the ability to recognize Akt from the ovarian cancer cell line OVCAR3 in immunoprecipitation (IP) experiments.…”

“…150 Akt1 kinase is responsible for signal transduction from the plasma membrane to downstream effector molecules that control cell growth, apoptosis, and translation. 151 To ensure the development of an allosteric site inhibitor, Millward et al carried out an initial screen against a large OBOC peptide library on a kinase preinhibited with an ATP-competitive inhibitor, Ac7.…”

“…151 To ensure the development of an allosteric site inhibitor, Millward et al carried out an initial screen against a large OBOC peptide library on a kinase preinhibited with an ATP-competitive inhibitor, Ac7. 150 One of the Nterminal azido-amino acid-containing peptides generated in the initial screen showed almost 95% inhibition of the Akt1 kinase in the absence and presence of the conjugated small molecule inhibitor and was therefore employed as an anchor for biligand development (Fig. 18).…”

“…The authors observed IP of the protein that likely corresponds to the GSK3β kinase by the triligand, and to a lesser degree, by the anchor and the biligand. 150 The underlying rationale for GSK3 binding to ligands is yet to be explained. However, IP experiments confirm the increase in capture efficiency of ligands, particularly in stimulated cells, as they are being translated from anchor to triligand with their affinity and selectivity criteria increased.…”

Exploitation of the Ornithine Effect Enhances Characterization of Stapled and Cyclic Peptides

“…However, peptides that are protected from degradation via cyclization or stapling have been shown to exhibit high potency and low toxicity, resulting in more promising candidates for drug administration than their linear counterparts [3][4][5][6][7]. A myriad of nonlinear peptides are found naturally in plants, fungi, and bacteria as well as synthetic ones produced in the laboratory [10][11][12][13][14][15][16][17][18]. Valinomycin, for example, is a cyclic dodecadepsipeptide produced by Streptomyces fulvissimus with potent antibacterial properties while also acting as a potassium-selective ionophore [19,20].…”