Precision peptide theranostics: developing N- to C-terminus optimized theranostics targeting cholecystokinin-2 receptor.

Peptides are ideal for theranostic development as they afford rapid target accumulation, fast clearance from background tissue, and exhibit good tissue penetration. Previously, we developed a novel series of peptides that presented discreet folding propensity leading to an optimal candidate [68Ga]Ga-DOTA-GA1 ([D-Glu]6-Ala-Tyr-NMeGly-Trp-NMeNle-Asp-Nal-NH2) with 50 pM binding affinity against cholecystokinin-2 receptors (CCK2R). However, we were confronted with challenges of unfavorably high renal uptake. Methods: A structure activity relationship study was undertaken of the lead theranostic candidate. Prudent structural modifications were made to the peptide scaffold to evaluate the contributions of specific N-terminal residues to the overall biological activity. Optimal candidates were then evaluated in nude mice bearing transfected A431-CCK2 tumors, and their biodistribution was quantitated ex vivo. Results: We identified and confirmed that D-Glu3 to D-Ala3 substitution produced 2 optimal candidates, [68Ga]Ga-DOTA-GA12 and [68Ga]Ga-DOTA-GA13. These radiopeptides presented with high target/background ratios, enhanced tumor retention, excellent metabolic stability in plasma and mice organ homogenates, and a 4-fold reduction in renal uptake, significantly outperforming their non-alanine counterparts. Conclusions: Our study identified novel radiopharmaceutical candidates that target the CCK2R. Their high tumor uptake and reduced renal accumulation warrant clinical translation.

Development of Highly Potent Clinical Candidates for Theranostic Applications against Cholecystokinin-2 Receptor Positive Cancers.

Peptide receptor radionuclide therapy (PRRT) is a promising form of systemic radiation therapy designed to eradicate cancer. Cholecystokinin-2 receptor (CCK2R) is an important molecular target that is highly expressed in a range of cancers. This study describes the synthesis and in vivo characterization of a novel series of 177Lu-labeled peptides ([177Lu]Lu-2b-4b) in comparison with the reference CCK2R-targeting peptide CP04 ([177Lu]Lu-1b). [177Lu]Lu-1b-4b showed high chemical purity (HPLC ≥ 94%), low Log D7.4 (-4.09 to -4.55) with strong binding affinity to CCK2R (KD 0.097-1.61 nM), and relatively high protein binding (55.6-80.2%) and internalization (40-67%). Biodistribution studies of the novel 177Lu-labeled peptides in tumors (AR42J and A431-CCK2R) showed uptake one- to eight-fold greater than the reference compound CP04 at 1, 24, and 48 h. Rapid clearance and high tumor uptake and retention were established for [177Lu]Lu-2b-4b, making these compounds excellent candidates for theranostic applications against CCK2R-expressing tumors.

Cyclic Peptides as Drugs for Intracellular Targets: The Next Frontier in Peptide Therapeutic Development.

Developing macrocyclic peptides that can reach intracellular targets is a significant challenge. This review discusses the most recent strategies used to develop cell permeable cyclic peptides that maintain binding to their biological target inside the cell. Macrocyclic peptides are unique from small molecules because traditional calculated physical properties are unsuccessful for predicting cell membrane permeability. Peptide synthesis and experimental membrane permeability is the only strategy that effectively differentiates between cell permeable and cell impermeable molecules. Discussed are chemical strategies, including backbone N-methylation and stereochemical changes, which have produced molecular scaffolds with improved cell permeability. However, these improvements often come at the expense of biological activity as chemical modifications alter the peptide conformation, frequently impacting the compound's ability to bind to the target. Highlighted is the most promising approach, which involves side-chain alterations that improve cell permeability without impact binding events.

Polymer mediated transport of the Hsp90 inhibitor LB76, a polar cyclic peptide, produces an Hsp90 cellular phenotype.

LB76 is a cyclic peptide that shows great promise as a selective heat shock protein 90 (Hsp90) inhibitor. However despite strong binding to and inhibition of Hsp90 in cell lysate its polar structure prevents it from crossing the cell membrane. We have developed a pH responsive polymer nanoparticle that effectively encapsulates LB76 from solution without need for purification. The nanoparticle releases the molecule upon crossing the cell membrane. Treatment of human colon cancer HCT116 cells with nanoparticles laden with LB76 produces the typical phenotype associated with Hsp90 inhibition, providing evidence of a therapeutically active payload.

Delivering bioactive cyclic peptides that target Hsp90 as prodrugs.

The most challenging issue facing peptide drug development is producing a molecule with optimal physical properties while maintaining target binding affinity. Masking peptides with protecting groups that can be removed inside the cell, produces a cell-permeable peptide, which theoretically can maintain its biological activity. Described are series of prodrugs masked using: (a) O-alkyl, (b) N-alkyl, and (c) acetyl groups, and their binding affinity for Hsp90. Alkyl moieties increased compound permeability, Papp, from 3.3 to 5.6, however alkyls could not be removed by liver microsomes or in-vivo and their presence decreased target binding affinity (IC50 of ≥10 µM). Thus, unlike small molecules, peptide masking groups cannot be predictably removed; their removal is related to the 3-D conformation. O-acetyl groups were cleaved but are labile, increasing challenges during synthesis. Utilising acetyl groups coupled with mono-methylated amines may decrease the polarity of a peptide, while maintaining binding affinity.

Protein-protein inhibitor designed de novo to target the MEEVD region on the C-terminus of Hsp90 and block co-chaperone activity.

Protein-protein interactions control all cellular functions. Presented is the first de novo designed protein-protein inhibitor that targets the C-terminus of heat shock protein 90 (Hsp90) and blocks co-chaperones from binding. Compound LB76, which was created from an Hsp90 co-chaperone, selectively pulls down Hsp90 from cell lysates, binds to Hsp90's C-terminal domain, and blocks the interactions between Hsp90 and TPR-containing co-chaperones. Through these interactions, LB76 inhibits the protein-folding function of Hsp90. Blocking these protein-protein interactions between Hsp90 and C-terminal co-chaperones regulate the cell's entire protein-folding machinery.

Synthesis and Structure-Activity Relationships of Inhibitors That Target the C-Terminal MEEVD on Heat Shock Protein 90.

Herein, we describe the synthesis and structure-activity relationships of cyclic peptides designed to target heat shock protein 90 (Hsp90). Generating 19 compounds and evaluating their binding affinity reveals that increasing electrostatic interactions allows the compounds to bind more effectively with Hsp90 compared to the lead structure. Exchanging specific residues for lysine improves binding affinity for Hsp90, indicating some residues are not critical for interacting with the target, whereas others are essential. Replacing l- for d-amino acids produced compounds with decreased binding affinity compared to the parent structure, confirming the importance of conformation and identifying key residues most important for binding. Thus, a specific conformation and electrostatic interactions are required in order for these inhibitors to bind to Hsp90.

RITA Mimics: Synthesis and Mechanistic Evaluation of Asymmetric Linked Trithiazoles.

The established cytotoxic agent RITA contains a thiophene-furan-thiophene backbone and two terminal alcohol groups. Herein we investigate the effect of using thiazoles as the backbone in RITA-like molecules and modifying the terminal groups of these trithiazoles, thereby generating 41 unique structures. Incorporating side chains with varied steric bulk allowed us to investigate how size and a stereocenter impacted biological activity. Subjecting compounds to growth inhibition assays on HCT-116 cells showed that the most potent compounds 7d, 7e, and 7h had GI50 values of 4.4, 4.4, and 3.4 µM, respectively, versus RITA (GI50 of 800 nM). Analysis of these compounds in apoptosis assays proved that 7d, 7e, and 7h were as effective as RITA at inducing apoptosis. Evaluating the impact of 7h on proteins targeted by RITA (p53, c-Myc, and Mcl-1) indicated that it acts via a different mechanism of action to that of RITA. RITA suppressed Mcl-1 protein via p53, whereas compound 7h suppressed Mcl-1 expression via an alternative mechanism independent of p53.

Thioimidazoline based compounds reverse glucocorticoid resistance in human acute lymphoblastic leukemia xenografts.

Glucocorticoids form a critical component of chemotherapy regimens for pediatric acute lymphoblastic leukemia (ALL) and the initial response to glucocorticoid therapy is a major prognostic factor, where resistance is predictive of poor outcome. A high-throughput screen identified four thioimidazoline-containing compounds that reversed dexamethasone resistance in an ALL xenograft derived from a chemoresistant pediatric ALL. The lead compound (1) was synergistic when used in combination with the glucocorticoids, dexamethasone or prednisolone. Synergy was observed in a range of dexamethasone-resistant xenografts representative of B-cell precursor ALL (BCP-ALL) and T-cell ALL. We describe here the synthesis of twenty compounds and biological evaluation of thirty two molecules that explore the structure-activity relationships (SAR) of this novel class of glucocorticoid sensitizing compounds. SAR analysis has identified that the most effective dexamethasone sensitizers contain a thioimidazoline acetamide substructure with a large hydrophobic moiety on the acetamide.