Paralogue-selective degradation of the lysine acetyltransferase EP300.

The transcriptional coactivators EP300 and CREBBP are critical regulators of gene expression that share high sequence identity but exhibit non-redundant functions in basal and pathological contexts. Here, we report the development of a bifunctional small molecule, MC-1, capable of selectively degrading EP300 over CREBBP. Using a potent aminopyridine-based inhibitor of the EP300/CREBBP catalytic domain in combination with a VHL ligand, we demonstrate that MC-1 preferentially degrades EP300 in a proteasome-dependent manner. Mechanistic studies reveal that selective degradation cannot be predicted solely by target engagement or ternary complex formation, suggesting additional factors govern paralogue-specific degradation. MC-1 inhibits cell proliferation in a subset of cancer cell lines and provides a new tool to investigate the non-catalytic functions of EP300 and CREBBP. Our findings expand the repertoire of EP300/CREBBP-targeting chemical probes and offer insights into the determinants of selective degradation of highly homologous proteins.

Preparation of a Zirconium-89 Labeled Clickable DOTA Complex and Its Antibody Conjugate.

Desferrioxamine B (DFO) is the clinical standard chelator for preparing zirconium-89 labeled antibodies. In the current study, the stabilities of a zirconium-89 labeled panitumumab (PAN; Vectibix®) with three different chelators (DFO, DFO*, and DOTA) were compared. PAN is an anti-HER1/EGFR monoclonal antibody approved by the FDA for the treatment of HER1-expressing colorectal cancers and was used as the model antibody for this study. DFO/DFO* conjugates of PAN were directly radiolabeled with zirconium-89 at room temperature to produce [89Zr]Zr-DFO/DFO*-PAN conjugates following a well-established procedure. A zirconium-89 labeled DOTA-PAN conjugate was prepared by an indirect radiolabeling method. A cyclooctyne-linked DOTA chelator (BCN-DOTA-GA) was first radiolabeled with zirconium-89 at 90 °C under a two-step basic pH adjustment method followed by conjugation with PAN-tetrazene at 37 °C to produce a labeled conjugate, BCN-[89Zr]Zr-DOTA-GA-PAN. High reproducibility of the radiolabeling was observed via this two-step basic pH adjustment. The overall radiochemical yield was 40-50% (n = 12, decay uncorrected) with a radiochemical purity of >95% in 2 h synthesis time. All three conjugates were stable in whole human serum for up to 7 days at 37 °C. The kinetic inertness of the conjugates was assessed against the EDTA challenge. BCN-[89Zr]Zr-DOTA-GA-PAN exhibited excellent inertness followed by [89Zr]Zr-DFO*-PAN. [89Zr]Zr-DFO-PAN displayed the lowest level of inertness.

A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins.

Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins.

Structural Relationships to Efficacy for Prazole-Derived Antivirals.

Here, an in vitro characterization of a family of prazole derivatives that covalently bind to the C73 site on Tsg101 and assay their ability to inhibit viral particle production is presented. Structurally, increased steric bulk on the 4-pyridyl of the prazole expands the prazole site on the UEV domain toward the ß-hairpin in the Ub-binding site and is coupled to increased inhibition of virus-like particle production in HIV-1. Increased bulk also increased toxicity, which is alleviated by increasing flexibility. Further, the formation of a novel secondary Tsg101 adduct for several of the tested compounds and the commercial drug lansoprazole. The secondary adduct involved the loss of the 4-pyridyl substituent to form an irreversible species, with implications for increasing the half-life of the active species or its specificity toward Tsg101 UEV. It is also determined that sulfide derivatives display effective viral inhibition, presumably through cellular sulfoxidation, allowing for delayed conversion within the cellular environment, and identify SARS-COV-2 as a target of prazole inhibition. These results open multiple avenues for the design of prazole derivatives for antiviral applications.

PIM1 targeted degradation prevents the emergence of chemoresistance in prostate cancer.

PIM kinases have important pro-tumorigenic roles and mediate several oncogenic traits, including cell proliferation, survival, and chemotherapeutic resistance. As a result, multiple PIM inhibitors have been pursued as investigational new drugs in cancer; however, response to PIM inhibitors in solid tumors has fallen short of expectations. We found that inhibition of PIM kinase activity stabilizes protein levels of all three PIM isoforms (PIM1/2/3), and this can promote resistance to PIM inhibitors and chemotherapy. To overcome this effect, we designed PIM proteolysis targeting chimeras (PROTACs) to target PIM for degradation. PIM PROTACs effectively downmodulated PIM levels through the ubiquitin-proteasome pathway. Importantly, degradation of PIM kinases was more potent than inhibition of catalytic activity at inducing apoptosis in prostate cancer cell line models. In conclusion, we provide evidence of the advantages of degrading PIM kinases versus inhibiting their catalytic activity to target the oncogenic functions of PIM kinases.

hRpn13 shapes the proteome and transcriptome through epigenetic factors HDAC8, PADI4, and transcription factor NF-κB p50.

The anti-cancer target hRpn13 is a proteasome substrate receptor. However, hRpn13-targeting molecules do not impair its interaction with proteasomes or ubiquitin, suggesting other critical cellular activities. We find that hRpn13 depletion causes correlated proteomic and transcriptomic changes, with pronounced effects in myeloma cells for cytoskeletal and immune response proteins and bone-marrow-specific arginine deiminase PADI4. Moreover, a PROTAC against hRpn13 co-depletes PADI4, histone deacetylase HDAC8, and DNA methyltransferase MGMT. PADI4 binds and citrullinates hRpn13 and proteasomes, and proteasomes from PADI4-inhibited myeloma cells exhibit reduced peptidase activity. When off proteasomes, hRpn13 can bind HDAC8, and this interaction inhibits HDAC8 activity. Further linking hRpn13 to transcription, its loss reduces nuclear factor κB (NF-κB) transcription factor p50, which proteasomes generate by cleaving its precursor protein. NF-κB inhibition depletes hRpn13 interactors PADI4 and HDAC8. Altogether, we find that hRpn13 acts dually in protein degradation and expression and that proteasome constituency and, in turn, regulation varies by cell type.

Perfluorinated Iridium Catalyst for Signal Amplification by Reversible Exchange Provides Metal-Free Aqueous Hyperpolarized [1-13C]-Pyruvate.

Hyperpolarized (HP) carbon-13 [13C] enables the specific investigation of dynamic metabolic and physiologic processes via in vivo MRI-based molecular imaging. As the leading HP metabolic agent, [1-13C]pyruvate plays a pivotal role due to its rapid tissue uptake and central role in cellular energetics. Dissolution dynamic nuclear polarization (d-DNP) is considered the gold standard method for the production of HP metabolic probes; however, development of a faster, less expensive technique could accelerate the translation of metabolic imaging via HP MRI to routine clinical use. Signal Amplification by Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) achieves rapid hyperpolarization by using parahydrogen (p-H2) as the source of nuclear spin order. Currently, SABRE is clinically limited due to the toxicity of the iridium catalyst, which is crucial to the SABRE process. To mitigate Ir contamination, we introduce a novel iteration of the SABRE catalyst, incorporating bis(polyfluoroalkylated) imidazolium salts. This novel perfluorinated SABRE catalyst retained polarization properties while exhibiting an enhanced hydrophobicity. This modification allows the easy removal of the perfluorinated SABRE catalyst from HP [1-13C]-pyruvate after polarization in an aqueous solution, using the ReD-SABRE protocol. The residual Ir content after removal was measured via ICP-MS at 177 ppb, which is the lowest reported to date for pyruvate and is sufficiently safe for use in clinical investigations. Further improvement is anticipated once automated processes for delivery and recovery are initiated. SABRE-SHEATH using the perfluorinated SABRE catalyst can become an attractive low-cost alternative to d-DNP to prepare biocompatible HP [1-13C]-pyruvate formulations for in vivo applications in next-generation molecular imaging modalities.

Dietary fish oil enriched in very-long-chain polyunsaturated fatty acid reduces cardiometabolic risk factors and improves retinal function.

Very-long-chain polyunsaturated fatty acids (VLCPUFAs; C24-38) constitute a unique class of PUFA that have important biological roles, but the lack of a suitable dietary source has limited research in this field. We produced an n-3 C24-28-rich VLCPUFA-oil concentrated from fish oil to study its bioavailability and physiological functions in C57BL/6J mice. The serum and retinal C24:5 levels increased significantly compared to control after a single-dose gavage, and VLCPUFAs were incorporated into the liver, brain, and eyes after 8-week supplementation. Dietary VLCPUFAs resulted in favorable cardiometabolic changes, and improved electroretinography responses and visual performance. VLCPUFA supplementation changed the expression of genes involved in PPAR signaling pathways. Further in vitro studies demonstrated that the VLCPUFA-oil and chemically synthesized C24:5 are potent agonists for PPARs. The multiple potential beneficial effects of fish oil-derived VLCPUFAs on cardiometabolic risk and eye health in mice support future efforts to develop VLCPUFA-oil into a supplemental therapy.

Tools and tactics to define specificity of metabolic chemical reporters.

Metabolic chemical reporters (MCRs) provide easily accessible means to study glycans in their native environments. However, because monosaccharide precursors are shared by many glycosylation pathways, selective incorporation has been difficult to attain. Here, a strategy for defining the selectivity and enzymatic incorporation of an MCR is presented. Performing ß-elimination to interrogate O-linked sugars and using commercially available glycosidases and glycosyltransferase inhibitors, we probed the specificity of widely used azide (Ac4GalNAz) and alkyne (Ac4GalNAlk and Ac4GlcNAlk) sugar derivatives. Following the outlined strategy, we provide a semiquantitative assessment of the specific and non-specific incorporation of this bioorthogonal sugar (Ac4GalNAz) into numerous N- and O-linked glycosylation pathways. This approach should be generally applicable to other MCRs to define the extent of incorporation into the various glycan species.

Monitoring response to a clinically relevant IDH inhibitor in glioma-Hyperpolarized 13C magnetic resonance spectroscopy approaches.

Background: Mutant isocitrate dehydrogenase (IDHmut) catalyzes 2-hydroxyglutarate (2HG) production and is considered a therapeutic target for IDHmut tumors. However, response is mostly associated with inhibition of tumor growth. Response assessment via anatomic imaging is therefore challenging. Our goal was to directly detect IDHmut inhibition using a new hyperpolarized (HP) 13C magnetic resonance spectroscopy-based approach to noninvasively assess α-ketoglutarate (αKG) metabolism to 2HG and glutamate. Methods: We studied IDHmut-expressing normal human astrocyte (NHAIDH1mut) cells and rats with BT257 tumors, and assessed response to the IDHmut inhibitor BAY-1436032 (n ≥ 4). We developed a new 13C Echo Planar Spectroscopic Imaging sequence with an optimized RF pulse to monitor the fate of HP [1-13C]αKG and [5-12C,1-13C]αKG with a 2.5 × 2.5 × 8 mm3 spatial resolution. Results: Cell studies confirmed that BAY-1436032-treatment leads to a drop in HP 2HG and an increase in HP glutamate detectable with both HP substrates. Data using HP [5-12C,1-13C]αKG also demonstrated that its conversion to 2HG is detectable without the proximal 1.1% natural abundance [5-13C]αKG signal. In vivo studies showed that glutamate is produced in normal brains but no 2HG is detectable. In tumor-bearing rats, we detected the production of both 2HG and glutamate, and BAY-1436032-treatment led to a drop in 2HG and an increase in glutamate. Using HP [5-12C,1-13C]αKG we detected metabolism with an signal-to-noise ratio of 23 for 2HG and 17 for glutamate. Conclusions: Our findings point to the clinical potential of HP αKG, which recently received FDA investigational new drug approval for research, for noninvasive localized imaging of IDHmut status.

Engineering CD276/B7-H3-targeted antibody-drug conjugates with enhanced cancer-eradicating capability.

CD276/B7-H3 represents a promising target for cancer therapy based on widespread overexpression in both cancer cells and tumor-associated stroma. In previous preclinical studies, CD276 antibody-drug conjugates (ADCs) exploiting a talirine-type pyrrolobenzodiazepine (PBD) payload showed potent activity against various solid tumors but with a narrow therapeutic index and dosing regimen higher than that tolerated in clinical trials using other antibody-talirine conjugates. Here, we describe the development of a modified talirine PBD-based fully human CD276 ADC, called m276-SL-PBD, that is cross-species (human/mouse) reactive and can eradicate large 500-1,000-mm3 triple-negative breast cancer xenografts at doses 10- to 40-fold lower than the maximum tolerated dose. By combining CD276 targeting with judicious genetic and chemical ADC engineering, improved ADC purification, and payload sensitivity screening, these studies demonstrate that the therapeutic index of ADCs can be substantially increased, providing an advanced ADC development platform for potent and selective targeting of multiple solid tumor types.

Orthogonal Enzyme-Substrate Design Strategy for Discovery of Human Protein Palmitoyltransferase Substrates.

Protein palmitoylation, with more than 5000 substrates, is the most prevalent form of protein lipidation. Palmitoylated proteins participate in almost all areas of cellular physiology and have been linked to several human diseases. Twenty-three zDHHC enzymes catalyze protein palmitoylation with extensive overlap among the substrates of each zDHHC member. Currently, there is no global strategy to delineate the physiological substrates of individual zDHHC enzymes without perturbing the natural cellular pool. Here, we outline a general approach to accomplish this on the basis of synthetic orthogonal substrates that are only compatible with engineered zDHHC enzymes. We demonstrate the utility of this strategy by validating known substrates and use it to identify novel substrates of two human zDHHC enzymes. Finally, we employ this method to discover and explore conserved palmitoylation in a family of host restriction factors against pathogenic viruses, including SARS-CoV-2.

Methionine sulfoxide reductases and cholesterol transporter STARD3 constitute an efficient system for detoxification of cholesterol hydroperoxides.

Methionine sulfoxide reductases (MSRs) are key enzymes in the cellular oxidative defense system. Reactive oxygen species oxidize methionine residues to methionine sulfoxide, and the methionine sulfoxide reductases catalyze their reduction back to methionine. We previously identified the cholesterol transport protein STARD3 as an in vivo binding partner of MSRA (methionine sulfoxide reductase A), an enzyme that reduces methionine-S-sulfoxide back to methionine. We hypothesized that STARD3 would also bind the cytotoxic cholesterol hydroperoxides and that its two methionine residues, Met307 and Met427, could be oxidized, thus detoxifying cholesterol hydroperoxide. We now show that in addition to binding MSRA, STARD3 binds all three MSRB (methionine sulfoxide reductase B), enzymes that reduce methionine-R-sulfoxide back to methionine. Using pure 5, 6, and 7 positional isomers of cholesterol hydroperoxide, we found that both Met307 and Met427 on STARD3 are oxidized by 6α-hydroperoxy-3ß-hydroxycholest-4-ene (cholesterol-6α-hydroperoxide) and 7α-hydroperoxy-3ß-hydroxycholest-5-ene (cholesterol-7α-hydroperoxide). MSRs reduce the methionine sulfoxide back to methionine, restoring the ability of STARD3 to bind cholesterol. Thus, the cyclic oxidation and reduction of methionine residues in STARD3 provides a catalytically efficient mechanism to detoxify cholesterol hydroperoxide during cholesterol transport, protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

Synthesis and Evaluation of Fluorine-18-Labeled L-Rhamnose Derivatives.

The use of radiolabeled glucose for PET imaging resulted in the most commonly used tracer in the clinic, 2-deoxy-2-[18F]fluoroglucose (FDG). More recently, other radiolabeled sugars have been reported for various applications, including imaging tumors and infections. Therefore, in this study, we developed a series of fluorine-18-labeled L-rhamnose derivatives as potential PET tracers of various fungal and bacterial strains. Acetyl-protected triflate precursors of rhamnose were prepared and radiolabeled with fluorine-18 followed by hydrolysis to produce L-deoxy [18F]fluororhamnose. The overall radiochemical yield was 7-27% in a 90 min synthesis time with a radiochemical purity of 95%. In vivo biodistribution of the ligands using PET imaging showed that 2-deoxy-2-[18F]fluoro-L-rhamnose is stable for at least up to 60 min in mice and eliminated via renal clearance. The tracer also exhibited minimal tissue or skeletal uptake in healthy mice resulting in a low background signal.

The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity.

Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases.

Site-Specifically Conjugated Single-Domain Antibody Successfully Identifies Glypican-3-Expressing Liver Cancer by Immuno-PET.

Primary liver cancer is the third leading cause of cancer-related deaths, and its incidence and mortality are increasing worldwide. Hepatocellular carcinoma (HCC) accounts for 80% of primary liver cancer cases. Glypican-3 (GPC3) is a heparan sulfate proteoglycan that histopathologically defines HCC and represents an attractive tumor-selective marker for radiopharmaceutical imaging and therapy for this disease. Single-domain antibodies are a promising scaffold for imaging because of their favorable pharmacokinetic properties, good tumor penetration, and renal clearance. Although conventional lysine-directed bioconjugation can be used to yield conjugates for radiolabeling full-length antibodies, this stochastic approach risks negatively affecting target binding of the smaller single-domain antibodies. To address this challenge, site-specific approaches have been explored. Here, we used conventional and sortase-based site-specific conjugation methods to engineer GPC3-specific human single-domain antibody (HN3) PET probes. Methods: Bifunctional deferoxamine (DFO) isothiocyanate was used to synthesize native HN3 (nHN3)-DFO. Site-specifically modified HN3 (ssHN3)-DFO was engineered using sortase-mediated conjugation of triglycine-DFO chelator and HN3 containing an LPETG C-terminal tag. Both conjugates were radiolabeled with 89Zr, and their binding affinity in vitro and target engagement of GPC3-positive (GPC3+) tumors in vivo were determined. Results: Both 89Zr-ssHN3 and 89Zr-nHN3 displayed nanomolar affinity for GPC3 in vitro. Biodistribution and PET/CT image analysis in mice bearing isogenic A431 and A431-GPC3+ xenografts, as well as in HepG2 liver cancer xenografts, showed that both conjugates specifically identify GPC3+ tumors. 89Zr-ssHN3 exhibited more favorable biodistribution and pharmacokinetic properties, including higher tumor uptake and lower liver accumulation. Comparative PET/CT studies on mice imaged with both 18F-FDG and 89Zr-ssHN3 showed more consistent tumor accumulation for the single-domain antibody conjugate, further establishing its potential for PET imaging. Conclusion: 89Zr-ssHN3 showed clear advantages in tumor uptake and tumor-to-liver signal ratio over the conventionally modified 89Zr-nHN3 in xenograft models. Our results establish the potential of HN3-based single-domain antibody probes for GPC3-directed PET imaging of liver cancers.

Comparison of Three Transcytotic Pathways for Distribution to Brain Metastases of Breast Cancer.

Advances in drug treatments for brain metastases of breast cancer have improved progression-free survival but new, more efficacious strategies are needed. Most chemotherapeutic drugs infiltrate brain metastases by moving between brain capillary endothelial cells, paracellular distribution, resulting in heterogeneous distribution, lower than that of systemic metastases. Herein, we tested three well-known transcytotic pathways through brain capillary endothelial cells as potential avenues for drug access: transferrin receptor (TfR) peptide, low-density lipoprotein receptor 1 (LRP1) peptide, albumin. Each was far-red labeled, injected into two hematogenous models of brain metastases, circulated for two different times, and their uptake quantified in metastases and uninvolved (nonmetastatic) brain. Surprisingly, all three pathways demonstrated distinct distribution patterns in vivo. Two were suboptimal: TfR distributed to uninvolved brain but poorly in metastases, while LRP1 was poorly distributed. Albumin distributed to virtually all metastases in both model systems, significantly greater than in uninvolved brain (P < 0.0001). Further experiments revealed that albumin entered both macrometastases and micrometastases, the targets of treatment and prevention translational strategies. Albumin uptake into brain metastases was not correlated with the uptake of a paracellular probe (biocytin). We identified a novel mechanism of albumin endocytosis through the endothelia of brain metastases consistent with clathrin-independent endocytosis (CIE), involving the neonatal Fc receptor, galectin-3, and glycosphingolipids. Components of the CIE process were found on metastatic endothelial cells in human craniotomies. The data suggest a reconsideration of albumin as a translational mechanism for improved drug delivery to brain metastases and possibly other central nervous system (CNS) cancers.In conclusion, drug therapy for brain metastasis needs improvement. We surveyed three transcytotic pathways as potential delivery systems in brain-tropic models and found that albumin has optimal properties. Albumin used a novel endocytic mechanism.

225Ac-MACROPATATE: A Novel α-Particle Peptide Receptor Radionuclide Therapy for Neuroendocrine Tumors.

Neuroendocrine tumors (NETs) express somatostatin receptors (SSTRs) 2 and 5. Modified variants of somatostatin, the cognate ligand for SSTR2 and SSTR5, are used in treatment for metastatic and locoregional disease. Peptide receptor radionuclide therapy with 177Lu-DOTATATE (DOTA-octreotate), a ß-particle-emitting somatostatin derivative, has demonstrated survival benefit in patients with SSTR-positive NETs. Despite excellent results, a subset of patients has tumors that are resistant to treatment, and alternative agents are needed. Targeted α-particle therapy has been shown to kill tumors that are resistant to targeted ß-particle therapy, suggesting that targeted α-particle therapy may offer a promising treatment option for patients with 177Lu-DOTATATE-resistant disease. Although DOTATATE can chelate the clinically relevant α-particle-emitting radionuclide 225Ac, the labeling reaction requires high temperatures, and the resulting radioconjugate has suboptimal stability. Methods: We designed and synthesized MACROPATATE (MACROPA-octreotate), a novel radioconjugate capable of chelating 225Ac at room temperature, and assessed its in vitro and in vivo performance. Results: MACROPATATE demonstrated comparable affinity to DOTATATE (dissociation constant, 21 nM) in U2-OS-SSTR2, a SSTR2-positive transfected cell line. 225Ac-MACROPATATE demonstrated superior serum stability at 37°C over time compared with 225Ac-DOTATATE. Biodistribution studies demonstrated higher tumor uptake of 225Ac-MACROPATATE than of 225Ac-DOTATATE in mice engrafted with subcutaneous H69 NETs. Therapy studies showed that 225Ac-MACROPATATE exhibits significant antitumor and survival benefit compared with saline control in mice engrafted with SSTR-positive tumors. However, the increased accumulation of 225Ac-MACROPATATE in liver and kidneys and subsequent toxicity to these organs decreased its therapeutic index compared with 225Ac-DOTATATE. Conclusion: 225Ac-MACROPATATE and 225Ac-DOTATATE exhibit favorable therapeutic efficacy in animal models. Because of elevated liver and kidney accumulation and lower administered activity for dose-limiting toxicity of 225Ac-MACROPATATE, 225Ac-DOTATATE was deemed the superior agent for targeted α-particle peptide receptor radionuclide therapy.

Rapid 13C Hyperpolarization of the TCA Cycle Intermediate α-Ketoglutarate via SABRE-SHEATH.

α-Ketoglutarate is a key biomolecule involved in a number of metabolic pathways─most notably the TCA cycle. Abnormal α-ketoglutarate metabolism has also been linked with cancer. Here, isotopic labeling was employed to synthesize [1-13C,5-12C,D4]α-ketoglutarate with the future goal of utilizing its [1-13C]-hyperpolarized state for real-time metabolic imaging of α-ketoglutarate analytes and its downstream metabolites in vivo. The signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH) hyperpolarization technique was used to create 9.7% [1-13C] polarization in 1 minute in this isotopologue. The efficient 13C hyperpolarization, which utilizes parahydrogen as the source of nuclear spin order, is also supported by favorable relaxation dynamics at 0.4 µT field (the optimal polarization transfer field): the exponential 13C polarization buildup constant Tb is 11.0 ± 0.4 s whereas the 13C polarization decay constant T1 is 18.5 ± 0.7 s. An even higher 13C polarization value of 17.3% was achieved using natural-abundance α-ketoglutarate disodium salt, with overall similar relaxation dynamics at 0.4 µT field, indicating that substrate deuteration leads only to a slight increase (∼1.2-fold) in the relaxation rates for 13C nuclei separated by three chemical bonds. Instead, the gain in polarization (natural abundance versus [1-13C]-labeled) is rationalized through the smaller heat capacity of the "spin bath" comprising available 13C spins that must be hyperpolarized by the same number of parahydrogen present in each sample, in line with previous 15N SABRE-SHEATH studies. Remarkably, the C-2 carbon was not hyperpolarized in both α-ketoglutarate isotopologues studied; this observation is in sharp contrast with previously reported SABRE-SHEATH pyruvate studies, indicating that the catalyst-binding dynamics of C-2 in α-ketoglutarate differ from that in pyruvate. We also demonstrate that 13C spectroscopic characterization of α-ketoglutarate and pyruvate analytes can be performed at natural 13C abundance with an estimated detection limit of 80 micromolar concentration × *%P13C. All in all, the fundamental studies reported here enable a wide range of research communities with a new hyperpolarized contrast agent potentially useful for metabolic imaging of brain function, cancer, and other metabolically challenging diseases.

Multivalent Gd-DOTA Decorated Oligopeptide as Sensitive MRI Molecular Probes for In Vivo Imaging of Brain Connectivity.

One of the most important goals of brain imaging is to define the anatomical connections within the brain. In addition to revealing normal circuitry, studies of neural connections and neuronal transport can show rewiring and degeneration following brain injury and diseases. In this work, a highly sensitive magnetic resonance imaging (MRI)-visible neural tracer that can be used to visualize brain connectivity in vivo is developed. It is based on an oligopeptide with gadolinium chelates appended to the peptide backbone. This peptide construct is a sensitive MRI contrast agent that was conjugated to the classical neurotracer, Cholera-toxin Subunit-B. Injection of this probe enabled it to be used to trace neural connections in vivo. This complements other MRI tracing techniques such as diffusion tensor imaging and manganese-enhanced MRI for neural tracing.