Octadentate zirconium(IV)-loaded macrocycles with varied stoichiometry assembled from hydroxamic acid monomers using metal-templated synthesis

Published: February 28, 2017The reaction between Zr(IV) and the forward endo-hydroxamic acid monomer 4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoic acid (for-PBH) in a 1:4 stoichiometry in the presence of diphenylphosphoryl azide and triethylamine gave the octadentate Zr(IV)-loaded tetrameric hydroxamic acid macrocycle for-[Zr(DFOT₁)] ([M + H]⁺ calc 887.3, obs 887.2). In this metal-templated synthesis (MTS) approach, the coordination preferences of Zr(IV) directed the preorganization of four oxygen-rich bidentate for-PBH ligands about the metal ion prior to ring closure under peptide coupling conditions. The replacement of for-PBH with 5-[(5-aminopentyl) (hydroxy)amino]-5-oxopentanoic acid (for-PPH), which contained an additional methylene group in the dicarboxylic acid region of the monomer, gave the analogous Zr(IV)-loaded macrocycle for-[Zr(PPDFOT₁)] ([M + H]⁺ calc 943.4, obs 943.1). A second, well-resolved peak in the liquid chromatogram from the for-PPH MTS system also characterized as a species with [M + H]⁺ 943.3, and was identified as the octadentate complex between Zr(IV) and two dimeric tetradentate hydroxamic acid macrocycles for-[Zr(PPDFOT1D)₂]. Treatment of for-[Zr(PPDFOT₁)] or for-[Zr(PPDFOT1D)₂] with EDTA at pH 4.0 gave the respective hydroxamic acid macrocycles as free ligands: octadentate PPDFOT₁ or two equivalents of tetradentate PPDFOT1D (homobisucaberin, HBC). At pH values closer to physiological, EDTA treatment of for-[Zr(DFOT₁)], for-[Zr(PPDFOT₁)], or Zr(IV) complexes with related linear tri- or tetrameric hydroxamic acid ligands showed the macrocycles were more resistant to the release of Zr(IV), which has implications for the design of ligands optimized for the use of Zr(IV)-89 in positron emission tomography (PET) imaging of cancer.William Tieu, Tulip Lifa, Andrew Katsifis, and Rachel Cod

Endogenously produced nonclassical vitamin D hydroxy-metabolites act as "biased" agonists on VDR and inverse agonists on RORα and RORγ

The classical pathway of vitamin D activation follows the sequence D3→25(OH)D3→1,25(OH)(2)D3 with the final product acting on the receptor for vitamin D (VDR). An alternative pathway can be started by the action of CYP11A1 on the side chain of D3, primarily producing 20(OH)D3, 22(OH)D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3 and 17,20,23(OH)(3)D3. Some of these metabolites are hydroxylated by CYP27B1 at C1α, by CYP24A1 at C24 and C25, and by CYP27A1 at C25 and C26. The products of these pathways are biologically active. In the epidermis and/or serum or adrenals we detected 20(OH)D3, 22(OH)D3, 20,22(OH)(2)D3, 20,23(OH)(2)D3, 17,20,23(OH)(3)D3, 1,20(OH)(2)D3, 1,20,23(OH)(3)D3, 1,20,22(OH)(3)D3, 20,24(OH)(2)D3, 1,20,24(OH)(3)D3, 20,25(OH)(2)D3, 1,20,25(OH)(3)D3, 20,26(OH)(2)D3 and 1,20,26(OH)(3)D3. 20(OH)D3 and 20,23(OH)(2)D3 are non-calcemic, while the addition of an OH at C1α confers some calcemic activity. Molecular modeling and functional assays show that the major products of the pathway can act as “biased” agonists for the VDR with high docking scores to the ligand binding domain (LBD), but lower than that of 1,25(OH)(2)D3. Importantly, cell based functional receptor studies and molecular modeling have identified the novel secosteroids as inverse agonists of both RORα and RORγ receptors. Specifically, they have high docking scores using crystal structures of RORα and RORγ LBDs. Furthermore, 20(OH)D3 and 20,23(OH)(2)D3 have been tested in cell model that expresses a Tet-on RORα or RORγ vector and a RORE-LUC reporter (ROR-responsive element), and in a mammalian 2-hybrid model that test interactions between an LBD-interacting LXXLL-peptide and the LBD of RORα/γ. These assays demonstrated that the novel secosteroids have ROR-antagonist activities that were further confirmed by the inhibition of IL17 promoter activity in cells overexpressing RORα/γ. In conclusion, endogenously produced novel D3 hydroxy-derivatives can act both as “biased” agonists of the VDR and/or inverse agonists of RORα/γ. We suggest that the identification of large number of endogenously produced alternative hydroxy-metabolites of D3 that are biologically active, and of possible alternative receptors, may offer an explanation for the pleiotropic and diverse activities of vitamin D, previously assigned solely to 1,25(OH)(2)D3 and VDR

Improved non-invasive positron emission tomographic imaging of chemotherapy-induced tumor cell death using Zirconium-89-labeled APOMAB®

Purpose: The chimeric monoclonal antibody (mAb) chDAB4 (APOMAB®) targets the Lupus associated (La)/Sjögren Syndrome-B (SSB) antigen, which is over-expressed in tumors but only becomes available for antibody binding in dead tumor cells. Hence, chDAB4 may be used as a novel theranostic tool to distinguish between responders and nonresponders early after chemotherapy. Here, we aimed to ascertain which positron emitter, Zirconium-89 ([⁸⁹Zr]Zr(IV)) or Iodine-124 ([¹²⁴I]I), was best suited to label chDAB4 for post-chemotherapy PET imaging of tumor-bearing mice and to determine which of two different bifunctional chelators provided optimal tumor imaging by PET using [⁸⁹Zr]Zr(IV)-labeled chDAB4. Methods: C57BL/6 J mice bearing subcutaneous syngeneic tumors of EL4 lymphoma were either untreated or given chemotherapy, then administered radiolabeled chDAB4 after 24 h with its biodistribution examined using PET and organ assay. We compared chDAB4 radiolabeled with [⁸⁹Zr] Zr(IV) or [¹²⁴I] I, or [⁸⁹Zr]Zr-chDAB4 using either DFO-NCS or DFOSq as a chelator. Results: After chemotherapy, [⁸⁹Zr]Zr-chDAB4 showed higher and prolonged mean (± SD) tumor uptake of 29.5 ± 5.9 compared to 7.8 ± 1.2 for [¹²⁴I] I -chDAB4. In contrast, antibody uptake in healthy tissues was not affected. Compared to DFO-NCS, DFOSq did not result in significant differences in tumor uptake of [⁸⁹Zr]Zr-chDAB4 but did alter the tumor:liver ratio in treated mice 3 days after injection in favour of DFOSq (8.0 ± 1.1) compared to DFO-NCS (4.2 ± 0.7). Conclusion: ImmunoPET using chDAB4 radiolabeled with residualizing [⁸⁹Zr] Zr(IV) rather than [¹²⁴I] I optimized post-chemotherapy tumor uptake. Further, PET imaging characteristics were improved by DFOSq rather than DFO-NCS. Therefore, the radionuclide/chelator combination of [⁸⁹Zr] Zr(IV) and DFOSq is preferred for the imminent clinical evaluation of chDAB4 as a selective tumor cell death radioligand.Vasilios Liapis, William Tieu, Stacey E. Rudd, Paul S. Donnelly, Nicole L. Wittwer, Michael P. Brown, and Alexander H. Staudache

Fc gamma receptor is not required for in vivo processing of radio- and drug-conjugates of the dead tumor cell-targeting monoclonal antibody, APOMAB®

The Fc region of a monoclonal antibody (mAb) can play a crucial role in its biodistribution and therapeutic activity. The chimeric mAb, chDAB4 (APOMAB®), which binds to dead tumor cells after DNA-damaging anti- cancer treatment, has been studied pre-clinically in both diagnostic and therapeutic applications in cancer. Given that macrophages contribute to the tumor accumulation of chDAB4 and its potency as an antibody drug con- jugate in vivo, we next wanted to determine whether the Fc region of the chDAB4 mAb also contributed. We found that, regardless of prior labeling with chDAB4, dead EL4 lymphoma or Lewis Lung (LL2) tumor cells were phagocytosed equally by wild-type or Fcγ knock-down macrophage cell lines. A similar result was seen with bone marrow-derived macrophages from wild-type, Fcγ knock-out (KO) and NOTAM mice that express Fcγ but lack immunoreceptor tyrosine-based activation motif (ITAM) signaling. Among EL4 tumor-bearing wild-type, Fcγ KO or NOTAM mice, no differences were observed in post-chemotherapy uptake of 89Zr-labeled chDAB4. Similarly, no differences were observed between LL2 tumor-bearing wild-type and Fcγ KO mice in post-chemotherapy uptake of 89Zr-chDAB4. Also, the post-chemotherapy activity of a chDAB4-antibody drug conjugate (ADC) directed against LL2 tumors did not differ among tumor-bearing wild-type, Fcγ KO and NOTAM mice, nor did the proportions and characteristics of the LL2 tumor immune cell infiltrates differ significantly among these mice. In conclusion, Fc-FcγR interactions are not essential for the diagnostic or therapeutic applications of chDAB4 conjugates because the tumor-associated macrophages, which engulf the chDAB4-labelled dead cells, respond to endogenous ‘eat me’ signals rather than depend on functional FcγR expression for phagocytosis.Alexander H. Staudacher, Vasilios Liapis, Nicole L. Wittwer, William Tieu, Hiu Chun Lam, Jeanette Leusen, Michael P. Brow

Positron emission tomographic imaging of tumor cell death using zirconium-89-labeled APOMAB(R) following cisplatin chemotherapy in lung and ovarian cancer xenograft models

Published online 06 July 2021Purpose Early detection of tumor treatment responses represents an unmet clinical need with no approved noninvasive methods. DAB4, or its chimeric derivative, chDAB4 (APOMAB®) is an antibody that targets the Lupus associated antigen (La/SSB). La/SSB is over-expressed in malignancy and selectively targeted by chDAB4 in cancer cells dying from DNA-damaging treatment. Therefore, chDAB4 is a unique diagnostic tool that detects dead cancer cells and thus could distinguish between treatment responsive and nonresponsive patients. Procedures In clinically relevant tumor models, mice bearing subcutaneous xenografts of human ovarian or lung cancer cell lines or intraperitoneal ovarian cancer xenografts were untreated or given chemotherapy followed 24h later by chDAB4 radiolabeled with [⁸⁹Zr]ZrIV. Tumor responses were monitored using bioluminescence imaging and caliper measurements. [⁸⁹Zr]Zr-chDAB4 uptake in tumor and normal tissues was measured using an Albira SI Positron-Emission Tomography (PET) imager and its biodistribution was measured using a Hidex gamma-counter. Results Tumor uptake of [⁸⁹Zr]Zr-chDAB4 was detected in untreated mice, and uptake significantly increased in both human lung and ovarian tumors after chemotherapy, but not in normal tissues. Conclusion Given that tumors, rather than normal tissues, were targeted after chemotherapy, these results support the clinical development of chDAB4 as a radiodiagnostic imaging agent and as a potential predictive marker of treatment response.Vasilios Liapis, William Tieu, Nicole L. Wittwer, Tessa Gargett, Andreas Evdokiou, Prab Takhar, Stacey E. Rudd, Paul S. Donnelly, Michael P. Brown, Alexander H. Staudache

Tetraspanin (TSP-17) Protects Dopaminergic Neurons against 6-OHDA-Induced Neurodegeneration in <i>C. elegans</i>

Parkinson's disease (PD), the second most prevalent neurodegenerative disease after Alzheimer's disease, is linked to the gradual loss of dopaminergic neurons in the substantia nigra. Disease loci causing hereditary forms of PD are known, but most cases are attributable to a combination of genetic and environmental risk factors. Increased incidence of PD is associated with rural living and pesticide exposure, and dopaminergic neurodegeneration can be triggered by neurotoxins such as 6-hydroxydopamine (6-OHDA). In C. elegans, this drug is taken up by the presynaptic dopamine reuptake transporter (DAT-1) and causes selective death of the eight dopaminergic neurons of the adult hermaphrodite. Using a forward genetic approach to find genes that protect against 6-OHDA-mediated neurodegeneration, we identified tsp-17, which encodes a member of the tetraspanin family of membrane proteins. We show that TSP-17 is expressed in dopaminergic neurons and provide genetic, pharmacological and biochemical evidence that it inhibits DAT-1, thus leading to increased 6-OHDA uptake in tsp-17 loss-of-function mutants. TSP-17 also protects against toxicity conferred by excessive intracellular dopamine. We provide genetic and biochemical evidence that TSP-17 acts partly via the DOP-2 dopamine receptor to negatively regulate DAT-1. tsp-17 mutants also have subtle behavioral phenotypes, some of which are conferred by aberrant dopamine signaling. Incubating mutant worms in liquid medium leads to swimming-induced paralysis. In the L1 larval stage, this phenotype is linked to lethality and cannot be rescued by a dop-3 null mutant. In contrast, mild paralysis occurring in the L4 larval stage is suppressed by dop-3, suggesting defects in dopaminergic signaling. In summary, we show that TSP-17 protects against neurodegeneration and has a role in modulating behaviors linked to dopamine signaling

Advanced resistance studies identify two discrete mechanisms in staphylococcus aureus to overcome antibacterial compounds that target biotin protein ligase

Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound (<10-9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.Andrew J. Hayes, Jiulia Satiaputra, Louise M. Sternicki, Ashleigh S. Paparella, Zikai Feng, Kwang J. Lee ... et al

D-β-Hydroxybutyrate Is Protective in Mouse Models of Huntington's Disease

Abnormalities in mitochondrial function and epigenetic regulation are thought to be instrumental in Huntington's disease (HD), a fatal genetic disorder caused by an expanded polyglutamine track in the protein huntingtin. Given the lack of effective therapies for HD, we sought to assess the neuroprotective properties of the mitochondrial energizing ketone body, D-β-hydroxybutyrate (DβHB), in the 3-nitropropionic acid (3-NP) toxic and the R6/2 genetic model of HD. In mice treated with 3-NP, a complex II inhibitor, infusion of DβHB attenuates motor deficits, striatal lesions, and microgliosis in this model of toxin induced-striatal neurodegeneration. In transgenic R6/2 mice, infusion of DβHB extends life span, attenuates motor deficits, and prevents striatal histone deacetylation. In PC12 cells with inducible expression of mutant huntingtin protein, we further demonstrate that DβHB prevents histone deacetylation via a mechanism independent of its mitochondrial effects and independent of histone deacetylase inhibition. These pre-clinical findings suggest that by simultaneously targeting the mitochondrial and the epigenetic abnormalities associated with mutant huntingtin, DβHB may be a valuable therapeutic agent for HD