Phuong Nguyen Tran

We introduce a peptide prodrug activation strategy that employs the trivalent Lewis-acidic metal ions Ga3+, Fe3+, In3+, and Co3+, chelated by triazacyclononanediacetic acid monoamides. The metal chelates trigger rate-tunable, metal-mediated, autolytic amide bond cleavage (TMAC) adjacent to N-methylated and canonical amino acids. Our studies reveal that the four TMAC-compatible metal ions induce amide bond hydrolysis with divergent pH dependence and mechanistic pathways. We subsequently linked Ga3+, Fe3+, In3+, and Co3+ complexes to the N-terminus of a potent cytotoxic cargo, monomethyl auristatin E (MMAE), to produce the proof-of-concept prodrug PD1. In vitro toxicity studies establish IC50 values in HeLa and PC-3 cells that broadly correlate with complex cleavage rates; the metal ion-free PD1 conjugate, the Zn-PD1 complex, as well as the Ga3+, Fe3+, In3+, and Co3+ chelate hydrolysis products alone confer no toxicity. Attachment of a cancer-targeting peptide to the metal chelate, furnishing PD2, enables targeted delivery of the therapeutic payload in vivo, compatible with noninvasive nuclear imaging using positron emission tomography with 68Ga or single-photon computed tomography with 111In. The corresponding natGa-PD2 complex selectively suppressed tumor growth in vivo with minimal off-target toxicity. These studies establish metal identity as a general design parameter for programming amide bond cleavage and introduce TMAC as a versatile platform to engender peptide prodrugs with tunable release kinetics.

We introduce a solid‐phase synthesis platform for the rapid, modular, and scalable synthesis of the synthetic metallophore KO‐NH ₂ , which is readily recognized by bacterial siderophore transporters, but has optimized affinity for Ga ³⁺ over Fe ³⁺ . Four synthetic protochelin (PC) analogues, KK‐COOH, KO‐NH ₂ , KO‐COOH, and OK‐COOH were constructed. Growth recovery, radioactive uptake, and covalent photocrosslinker studies in Escherichia coli identified CirA and Fiu as the dominant outer membrane transport proteins capable of binding KO‐NH ₂ ‐linked, bifunctional constructs. Computational studies further supported that the structural framework of PC can be effectively mimicked by KO or OK peptides. Thermodynamic stability studies revealed an atypical inversion of thermodynamic stability trends, with logK _GaL of 39.9 and a logK _FeL of 36, demonstrating improved stability with Ga ³⁺ . Studies in naïve mice with ⁶⁸ Ga‐labeled derivatives identify the lead [ ⁶⁸ Ga][Ga(KO‐NH ₂ )] ³⁻ , which remained intact in vivo and demonstrated selective, enhanced uptake in an E. coli soft tissue model of infection (1.28 % ID/g, with a 3:1 ratio of infected to inflamed/normal muscle tissue). Linkage of KO‐NH ₂ to the antibiotic ampicillin produces an efficacious antibiotic, which supersedes the potency of the parent antibiotic ampicillin 4‐fold (MIC ₉₈ = 1.9 µM) effectively treats a soft tissue infection of ampicillin‐resistant E. coli .

We introduce a solid‐phase synthesis platform for the rapid, modular, and scalable synthesis of the synthetic metallophore KO‐NH ₂ , which is readily recognized by bacterial siderophore transporters, but has optimized affinity for Ga ³⁺ over Fe ³⁺ . Four synthetic protochelin (PC) analogues, KK‐COOH, KO‐NH ₂ , KO‐COOH, and OK‐COOH were constructed. Growth recovery, radioactive uptake, and covalent photocrosslinker studies in Escherichia coli identified CirA and Fiu as the dominant outer membrane transport proteins capable of binding KO‐NH ₂ ‐linked, bifunctional constructs. Computational studies further supported that the structural framework of PC can be effectively mimicked by KO or OK peptides. Thermodynamic stability studies revealed an atypical inversion of thermodynamic stability trends, with logK _GaL of 39.9 and a logK _FeL of 36, demonstrating improved stability with Ga ³⁺ . Studies in naïve mice with ⁶⁸ Ga‐labeled derivatives identify the lead [ ⁶⁸ Ga][Ga(KO‐NH ₂ )] ³⁻ , which remained intact in vivo and demonstrated selective, enhanced uptake in an E. coli soft tissue model of infection (1.28 % ID/g, with a 3:1 ratio of infected to inflamed/normal muscle tissue). Linkage of KO‐NH ₂ to the antibiotic ampicillin produces an efficacious antibiotic, which supersedes the potency of the parent antibiotic ampicillin 4‐fold (MIC ₉₈ = 1.9 µM) effectively treats a soft tissue infection of ampicillin‐resistant E. coli .

Titanium-45 is a positron-emitting radiometal with decay characteristics attractive for clinical positron emission tomography (PET) imaging (t1/2 = 3.08 h, β+ = 84.8%, β+ = .439 MeV). We report methods for producing 45Ti at commercial scale via 13 MeV proton irradiation of spot-welded natSc foil cyclotron targets that are thick to 13 MeV protons and robust to beam currents up to 100 µA, producing tens of GBq (>1 Ci) of 45Ti. Titanium-45 is extracted from bulk natSc via ZR resin and recovered in 0.1 M oxalic acid with radiochemical yields of 72.7 ± 0.6% (n = 3) and 78 ± 3% (n = 3) from 0.25 and 0.5 mm thick natSc targets, respectively. Separation conditions are optimized for rapid and facile radiopharmaceutical incorporation by coordination using siderophore chelators which have previously demonstrated compatibility with 45Ti. Purified 45Ti contains <1 µg natFe and achieves apparent molar activity (AMA) of 29 - 440 GBq/µmol (0.8 – 11.9 Ci/µmol) at EoB for productions ranging from 35 – 122 GBq (0.096 – 3300 Ci). AMAs were determined utilizing the catechol based chelator tren-2,3-dihydroxy-benzamide (TREN-CAM), forming [45Ti][Ti(TREN-CAM)]2-. We observed an increase of AMA in correlation with production size and a decrease of AMA in correlation with increased Sc target mass, indicating that stable contaminants in the target material likely limit currently achievable AMA. The methods described herein establish and validate a robust 45Ti isotope production framework which enables commercial scale production and clinical translation of 45Ti PET agents.

Positron emission tomography (PET) is an emerging tool under clinical investigation for the detection of bacterial infections. The ability to identify the site of infection and the pathogen's potential susceptibility to subsequent treatment approaches can streamline treatment and prevent misuse of antibiotics. Previous studies identified TREN‐CAM as a promising scaffold for radiometal labeling using the positron‐emitting radionuclide ⁶⁸ Ga. Here, we investigate the suitability of ^67/68 Ga‐labeled TREN‐CAM and enterobactin for the noninvasive imaging of an Escherichia coli infection. In vitro uptake experiments with [ ⁶⁷ Ga]Ga ^III ‐TREN‐CAM demonstrate superior uptake in E. coli K12 and Pseudomonas aeruginosa PAO1 when compared with [ ⁶⁷ Ga]Ga ^III ‐DFO, a compound currently under clinical investigation for infection imaging. Selectivity experiments indicate that [ ⁶⁷ Ga]Ga ^III ‐TREN‐CAM is recognized by the active transmembrane transport machinery for ^nat Fe ^III ‐Ent. Using a soft tissue E. coli infection model, we demonstrate that [ ⁶⁸ Ga]Ga ^III ‐TREN‐CAM selectively accumulates in infected tissue. Notably, [ ⁶⁸ Ga]Ga ^III ‐TREN‐CAM provides better infection‐to‐inflammation contrast, accelerated blood clearance, and lower off‐target accumulation compared to [ ⁶⁸ Ga]Ga ^III ‐Ent and [ ⁶⁸ Ga]Ga ^III ‐citrate. The potential of [ ⁶⁸ Ga]Ga ^III ‐TREN‐CAM to serve as a tool to monitor the therapeutic efficacy of cefiderocol is also evaluated, offering insight into its utility in guiding antibiotic treatment.

Positron emission tomography (PET) is an emerging tool under clinical investigation for the detection of bacterial infections. The ability to identify the site of infection and the pathogen's potential susceptibility to subsequent treatment approaches can streamline treatment and prevent misuse of antibiotics. Previous studies identified TREN‐CAM as a promising scaffold for radiometal labeling using the positron‐emitting radionuclide ⁶⁸ Ga. Here, we investigate the suitability of ^67/68 Ga‐labeled TREN‐CAM and enterobactin for the noninvasive imaging of an Escherichia coli infection. In vitro uptake experiments with [ ⁶⁷ Ga]Ga ^III ‐TREN‐CAM demonstrate superior uptake in E. coli K12 and Pseudomonas aeruginosa PAO1 when compared with [ ⁶⁷ Ga]Ga ^III ‐DFO, a compound currently under clinical investigation for infection imaging. Selectivity experiments indicate that [ ⁶⁷ Ga]Ga ^III ‐TREN‐CAM is recognized by the active transmembrane transport machinery for ^nat Fe ^III ‐Ent. Using a soft tissue E. coli infection model, we demonstrate that [ ⁶⁸ Ga]Ga ^III ‐TREN‐CAM selectively accumulates in infected tissue. Notably, [ ⁶⁸ Ga]Ga ^III ‐TREN‐CAM provides better infection‐to‐inflammation contrast, accelerated blood clearance, and lower off‐target accumulation compared to [ ⁶⁸ Ga]Ga ^III ‐Ent and [ ⁶⁸ Ga]Ga ^III ‐citrate. The potential of [ ⁶⁸ Ga]Ga ^III ‐TREN‐CAM to serve as a tool to monitor the therapeutic efficacy of cefiderocol is also evaluated, offering insight into its utility in guiding antibiotic treatment.