Development of [225Ac]Ac-DOTA-C595 as radioimmunotherapy of pancreatic cancer: in vitro evaluation, dosimetric assessment and detector calibration.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy which may benefit from radioimmunotherapy. Previously, [177Lu]Lu-DOTA-C595 has been developed as a beta-emitting radioimmunoconjugate to target cancer-specific mucin 1 epitopes (MUC1-CE) overexpressed on PDAC. However, the therapeutic effect may be enhanced by using an alpha-emitting radionuclide such as Actinium-225 (Ac-225). The short range and high linear energy transfer of alpha particles provides dense cellular damage and can overcome typical barriers related to PDAC treatment such as hypoxia. Despite the added cytotoxicity of alpha-emitters, their clinical implementation can be complicated by their complex decay chains, recoil energy and short-range impeding radiation detection. In this study, we developed and evaluated [225Ac]Ac-DOTA-C595 as an alpha-emitting radioimmunotherapy against PDAC using a series of in vitro experiments and conducted a preliminary dosimetric assessment and cross-calibration of detectors for the clinical implementation of Ac-225. RESULTS: Cell binding and internalisation of [225Ac]Ac-DOTA-C595 was rapid and greatest in cells with strong MUC1-CE expression. Over 99% of PDAC cells had positive yH2AX expression within 1 h of [225Ac]Ac-DOTA-C595 exposure, suggesting a high level of DNA damage. Clonogenic assays further illustrated the cytotoxicity of [225Ac]Ac-DOTA-C595 in a concentration-dependent manner. At low concentrations of [225Ac]Ac-DOTA-C595, cells with strong MUC1-CE expression had lower cell survival than cells with weak MUC1-CE expression, yet survival was similar between cell lines at high concentrations irrespective of MUC1-CE expression. A dosimetric assessment was performed to estimate the dose-rate of 1 kBq of [225Ac]Ac-DOTA-C595 with consideration to alpha particles. Total absorption of 1 kBq of Ac-225 was estimated to provide a dose rate of 17.5 mGy/h, confirmed via both detector measurements and calculations. CONCLUSION: [225Ac]Ac-DOTA-C595 was shown to target and induce a therapeutic effect in MUC1-CE expressing PDAC cells.

In vitro characterisation of [177Lu]Lu-DOTA-C595 as a novel radioimmunotherapy for MUC1-CE positive pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) continues to be a malignancy with an unmet clinical demand. Development of radioimmunoconjugates which target cancer-specific receptors provides an opportunity for radioimmunotherapy of both metastatic and primary PDAC. In this study, we characterised the in vitro behaviour of a novel beta-emitting radioimmunoconjugate [177Lu]Lu-DOTA-C595 as a therapeutic agent against PDAC. [177Lu]Lu-DOTA-C595 is designed to target cancer-specific mucin 1 epitopes (MUC1-CE) overexpressed on most epithelial cancers, including PDAC. RESULTS: A series of in vitro experiments were performed on PDAC cell lines (PANC-1, CAPAN-1, BxPC-3 and AsPC-1) exhibiting strong to weak MUC1-CE expression. [177Lu]Lu-DOTA-C595 bound to all cell lines relative to their expression of MUC1-CE. [177Lu]Lu-DOTA-C595 was also rapidly internalised across all cell lines, with a maximum of 75.4% of activity internalised within the PANC-1 cell line at 48 h. The expression of γH2AX foci and clonogenic survival of PANC-1 and AsPC-1 cell lines after exposure to [177Lu]Lu-DOTA-C595 were used to quantify the in vitro cytotoxicity of [177Lu]Lu-DOTA-C595. At 1 h post treatment, the expression of γH2AX foci exceeded 97% in both cell lines. The expression of γH2AX foci continued to increase in PANC-1 cells at 24 h, although expression reduced in AsPC-1. Clonogenic assays showed a high level of cell kill induced by [177Lu]Lu-DOTA-C595. CONCLUSION: [177Lu]Lu-DOTA-C595 has favourable in vitro characteristics to target and treat MUC1-CE positive PDAC. Further investigations to characterise the in vivo effects and potential value of [177Lu]Lu-DOTA-C595 in other MUC1-CE expressing malignancies such as lung, ovarian and colorectal adenocarcinoma are warranted.

A digital twin for 64Cu production with cyclotron and solid target system.

One method for finding reliable and cost-effective solutions for designing radioisotope production systems is represented by the "digital twin" philosophy of design. Looking at cyclotron solid targets, uncertainties of the particle beam, material composition and geometry play a crucial role in determining the results. The difference between what has been designed and what can be effectively manufactured, where processes such as electroplating are poorly controllable and generate large non-uniformities in deposition, must also be considered. A digital twin, where the target geometry is 3D scanned from real models, can represent a good compromise for connecting "ideal" and "real" worlds. Looking at the 64Ni(p,n)64Cu reaction, different Unstructured-Mesh MCNP6 models have been built starting from the 3D solid target system designed and put into operation by COMECER. A characterization has been performed considering the designed ideal target and a 3D scan of a real manufactured target measured with a ZEISS contact probe. Libraries and physics models have been also tested due to limited cross-section data. Proton spectra in the target volume, 3D proton-neutron-photon flux maps, average energies, power to be dissipated, shut-down dose-rate, 64Cu yield compared with various sources of experimental data and beam axial shifting impact, have been estimated. A digital twin of the 64Ni(p,n)64Cu production device has been characterized, considering the real measured target geometry, paving the way for a fully integrated model suitable also for thermal, structural or fluid-dynamic analyses.

Preliminary Development and Testing of C595 Radioimmunoconjugates for Targeting MUC1 Cancer Epitopes in Pancreatic Ductal Adenocarcinoma.

Mucin 1 is a transmembrane glycoprotein which overexpresses cancer-specific epitopes (MUC1-CE) on pancreatic ductal adenocarcinoma (PDAC) cells. As PDAC is a low survival and highly aggressive malignancy, developing radioimmunoconjugates capable of targeting MUC1-CE could lead to improvements in PDAC outcomes. The aim of this study was to develop and perform preliminary testing of diagnostic and therapeutic radioimmunoconjugates for PDAC using an anti-MUC1 antibody, C595. Firstly, p-SCN-Bn-DOTA was conjugated to the C595 antibody to form a DOTA-C595 immunoconjugate. The stability and binding affinity of the DOTA-C595 conjugate was evaluated using mass spectrometry and ELISA. DOTA-C595 was radiolabelled to Copper-64, Lutetium-177, Gallium-68 and Technetium-99m to form novel radioimmunoconjugates. Cell binding assays were performed in PANC-1 (strong MUC1-CE expression) and AsPC-1 (weak MUC1-CE expression) cell lines using 64Cu-DOTA-C595 and 177Lu-DOTA-C595. An optimal molar ratio of 4:1 DOTA groups per C595 molecule was obtained from the conjugation process. DOTA-C595 labelled to Copper-64, Lutetium-177, and Technetium-99m with high efficiency, although the Gallium-68 labelling was low. 177Lu-DOTA-C595 demonstrated high cellular binding to the PANC-1 cell lines which was significantly greater than AsPC-1 binding at concentrations exceeding 100 nM (p < 0.05). 64Cu-DOTA-C595 showed similar binding to the PANC-1 and AsPC-1 cells with no significant differences observed between cell lines (p > 0.05). The high cellular binding of 177Lu-DOTA-C595 to MUC1-CE positive cell lines suggests promise as a therapeutic radioimmunoconjugate against PDAC while further work is required to harness the potential of 64Cu-DOTA-C595 as a diagnostic radioimmunoconjugate.

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 anticancer 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 conjugate 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.

Toll-like receptors change morphine-induced antinociception, tolerance and dependence: Studies using male and female TLR and signalling gene KO mice.

Toll-like receptors (TLR) have been proposed as a site of action that alters opioid pharmacodynamics. However, a comprehensive assessment of acute opioid antinociception, tolerance and withdrawal behaviours in genetic null mutant strains with altered innate immune signalling has not been performed. Nor has the impact of genetic deletion of TLR2/4 on high-affinity opioid receptor binding. Here we show that diminished TLR signalling potentiates acute morphine antinociception equally in male and female mice. However, only male TIR8 null mutant mice showed reduced morphine analgesia. Analgesic tolerance was prevented in TLR2 and TLR4 null mutants, but not MyD88 animals. Withdrawal behaviours were only protected in TLR2-/- mice. In silico docking simulations revealed opioid ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. There was no binding of [3H](-)-naloxone or [3H]diprenorphine to TLR4 in the concentrations explored. These data confirm that opioids have high efficacy activity at innate immune pattern recognition binding sites but do not bind to TLR4 and identify critical pathway and sex-specific effects of the complex innate immune signalling contributions to opioid pharmacodynamics. These data further support the behavioural importance of the TLR-opioid interaction but fail to demonstrate direct evidence for high-affinity binding of the TLR4 signalling complex to ligands.

Positron Emission Tomographic Imaging of Tumor Cell Death Using Zirconium-89-Labeled APOMAB® Following Cisplatin Chemotherapy in Lung and Ovarian Cancer Xenograft Models.

PURPOSE: 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.

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]ZrIV) 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]ZrIV-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] ZrIV 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] ZrIV 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] ZrIV and DFOSq is preferred for the imminent clinical evaluation of chDAB4 as a selective tumor cell death radioligand.

89Zr-pro-MMP-9 F(ab')2 detects colitis induced intestinal and kidney fibrosis.

Intestinal fibrosis is a common complication of inflammatory bowel disease but remains difficult to detect. Matrix metalloproteases (MMPs) have key roles in fibrosis and are therefore potential targets for fibrosis detection. We determined whether immunoPET of F(ab')2 antibody fragments targeting MMPs detects colitis induced colonic fibrosis. Mice were administered 2% dextran sulfate sodium treated water for 1 cycle (inflamed) or 3 cycles (fibrotic), or were untreated (control). Colonic and kidney collagen, innate cytokine, MMPs and fecal MPO concentrations were analyzed by multiplex/ELISA. α-pro-MMP-9 F(ab')2 fragments were engineered and conjugated to 89Zr for PET imaging, ex-vivo Cherenkov analysis and bio-distribution. Colonic innate cytokine concentrations and fecal myeloperoxidase were increased in inflamed mice but not fibrotic mice, while collagen concentrations were increased in fibrotic mice. MMPs were increased in inflamed mice, but only pro-MMP-9 remained increased in fibrotic mice. 89Zr-pro-MMP-9 F(ab')2 uptake was increased in the intestine but also in the kidney of fibrotic mice, where collagen and pro-MMP-9 concentrations were increased. 89Zr-pro-MMP-9 F(ab')2 detects colitis induced intestinal fibrosis and associated kidney fibrosis.

Tumour-associated macrophages process drug and radio-conjugates of the dead tumour cell-targeting APOMAB® antibody.

APOMAB (chDAB4) is a dead tumour cell-targeting antibody which has been used preclinically as a diagnostic agent and therapeutically as a radioimmunotherapy and antibody drug conjugate (ADC). However, little is known of the intra-tumour processing of chDAB4 when bound to dead tumour cells. In this study we examine the role of macrophages in the in vitro and in vivo processing of radiolabelled chDAB4 and a chDAB4 ADC. We found that chDAB4 binds to macrophages in vitro, resulting in the killing of macrophages when using the ADC, chDAB4-SG3249. Free drug released by the macrophage processing of chDAB4-SG3249 could result in killing of 'bystander' Lewis lung (LL2) carcinoma cells. Furthermore, macrophages phagocytosed chDAB4-bound dead LL2 cells and were killed when they phagocytosed chDAB4-SG3249-bound dead LL2 cells in vitro. In vivo, we found markedly different tumour retention of chDAB4 in the LL2 tumour model depending on whether it was radiolabelled with a residualising radionuclide (89Zr), which is retained intracellularly, or a non-residualising radionuclide (124I), which can diffuse out of the cell. This prolonged retention of 89Zr vs124I indicated intra-tumoral processing of chDAB4 in vivo. The tumour uptake of 89Zr-chDAB4 was reduced after macrophage depletion, which also reduced the efficacy of the chDAB4 ADC in vivo. This study shows that macrophages can process chDAB4 and chDAB4 ADC in vitro and shows the importance of tumour-associated macrophages in the tumour retention of chDAB4 and the efficacy of chDAB4 ADC in vivo.

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.

Rapid and automated production of [68Ga]gallium chloride and [68Ga]Ga-DOTA-TATE on a medical cyclotron.

INTRODUCTION: The demand for Gallium-68 (68Ga) for labelling PET radiopharmaceuticals has increased over the past few years. 68Ga is obtained through the decayed parent radionuclide 68Ge using commercial 68Ge/68Ga generators. The principal limitation of commercial 68Ge/68Ga generators is that only a limited and finite quantity of 68Ga (<1.85 GBq at start of synthesis) may be accessed. The focus of this study was to investigate the use of a low energy medical cyclotron for the production of greater quantities of 68Ga and to develop an automated and rapid procedure for processing the product. METHODS: Enriched ZnCl2 was electrodeposited on a platinum backing using a NH4Cl (pH 2-4) buffer. The Zn target was irradiated with GE PETtrace 880 at 35 µA and 14.5 and 12.0 MeV beam energy. The irradiated Zn target was purified using octanol resin on an automated system. RESULTS: Following the described procedure, 68Ga was obtained in 6.30 ±â€¯0.42 GBq after 8.5 min bombardment and with low radionuclidic impurities (66Ga (<0.005%) and 67Ga (<0.09%)). Purification on a single octanol resin gave 82% recovery with resulting [68Ga]GaCl3 obtained in 3.5 mL of 0.2 M HCl. [68Ga]GaCl3 production from irradiation to final product was <45 min. To highlight the utility of the automated procedure, [68Ga]Ga-DOTA-TATE labelling was incorporated to give 1.56 GBq at EOS of the labelled peptide with RCY of >70%. CONCLUSIONS: A straightforward procedure for producing 68Ga on a low energy medical cyclotron was described. Current efforts are focus on high activity production and radiolabelling using solid target produced 68Ga.

Sulfonamide-Based Inhibitors of Biotin Protein Ligase as New Antibiotic Leads.

Here, we report the design, synthesis, and evaluation of a series of inhibitors of Staphylococcus aureus BPL (SaBPL), where the central acyl phosphate of the natural intermediate biotinyl-5'-AMP (1) is replaced by a sulfonamide isostere. Acylsulfamide (6) and amino sulfonylurea (7) showed potent in vitro inhibitory activity (Ki = 0.007 ± 0.003 and 0.065 ± 0.03 µM, respectively) and antibacterial activity against S. aureus ATCC49775 with minimum inhibitory concentrations of 0.25 and 4 µg/mL, respectively. Additionally, the bimolecular interactions between the BPL and inhibitors 6 and 7 were defined by X-ray crystallography and molecular dynamics simulations. The high acidity of the sulfonamide linkers of 6 and 7 likely contributes to the enhanced in vitro inhibitory activities by promoting interaction with SaBPL Lys187. Analogues with alkylsulfamide (8), ß-ketosulfonamide (9), and ß-hydroxysulfonamide (10) isosteres were devoid of significant activity. Binding free energy estimation using computational methods suggests deprotonated 6 and 7 to be the best binders, which is consistent with enzyme assay results. Compound 6 was unstable in whole blood, leading to poor pharmacokinetics. Importantly, 7 has a vastly improved pharmacokinetic profile compared to that of 6 presumably due to the enhanced metabolic stability of the sulfonamide linker moiety.

Exploring hydroxamic acid inhibitors of HDAC1 and HDAC2 using small molecule tools and molecular or homology modelling.

Hydroxamic acid compounds 1-10 containing a N-hydroxycinnamamide scaffold and a 4-(benzylamino)methyl cap group that was either unsubstituted (1) or substituted with one (2-4) or two (5-10) methoxy groups in variable positions were prepared as inhibitors of Zn(II)-containing histone deacetylases (HDACs). The 3,4- (9) and 3,5- (10) bis-methoxy-substituted compounds were the least potent against HeLa nuclear extract, HDAC1 and HDAC2. Molecular modelling showed methoxy groups in the 3-, 4- and 5-position, but not the 2-position, had unfavourable steric interactions with the G32-H33-P34 triad on a loop at the surface of the HDAC2 active site cavity. An HDAC1 homology model showed potential ionic (E243..K288) and cation-pi (K247..F292) interactions between helix 10 and helix 11 that were absent in HDAC2 ((G243..K288) and (K247..V292)). This surface-located interhelical constraint could inform the design of bitopic HDAC1 and HDAC2 selective ligands using an allosteric approach, and/or protein-protein interaction (PPI) inhibitors.

Analogues of desferrioxamine B (DFOB) with new properties and new functions generated using precursor-directed biosynthesis.

Desferrioxamine B (DFOB) is a siderophore native to Streptomyces pilosus biosynthesised by the DesABCD enzyme cluster as a high affinity Fe(III) chelator. Although DFOB has a long clinical history for the treatment of chronic iron overload, limitations encourage the development of new analogues. This review describes a recent body of work that has used precursor-directed biosynthesis (PDB) to access new DFOB analogues. PDB exploits the native biosynthetic machinery of a producing organism in culture medium augmented with non-native substrates that compete against native substrates during metabolite assembly. The method allows access to analogues of natural products using benign methods, compared to multistep organic synthesis. The disadvantages of PDB are the production of metabolites in low yield and the need to purify complex mixtures. Streptomyces pilosus medium was supplemented with different types of non-native diamine substrates to compete against native 1,5-diaminopentane to generate DFOB analogues containing alkene bonds, fluorine atoms, ether or thioether functional groups, or a disulfide bond. All analogues retained function as Fe(III) chelators and have properties that could broaden the utility of DFOB. These PDB studies have also added knowledge to the understanding of DFOB biosynthesis.

Immuno-PET of Innate Immune Markers CD11b and IL-1ß Detects Inflammation in Murine Colitis.

Inflammatory bowel disease (IBD) is a chronic relapsing and remitting inflammatory disease of the gastrointestinal tract. The diagnosis and monitoring of IBD are reliant on endoscopy, which is invasive and does not provide information on specific mediators. Symptom flare in IBD is associated with increased activation of innate immune pathways. Immuno-PET approaches have previously demonstrated the ability to detect colitis; however, a direct comparison of antibodies targeted to innate immune mediators and cells has not been done. We aimed to compare immuno-PET of antibodies to IL-1ß and CD11b against standard 18F-FDG and MRI approaches to detect colonic inflammation. Methods: Colonic concentrations of IL-1ß and myeloperoxidase were determined by ELISA, and colonic infiltration by CD11b-positive CD3-negative innate immune cells was determined by flow cytometry and compared between healthy and dextran sodium sulphate-treated colitic mice. PET of 89Zr-lα-IL-1ß, 89Zr-α-CD11b, and 18F-FDG was compared by volume-of-interest analysis and with MRI by region-of-interest analysis. Imaging results were confirmed by ex vivo biodistribution analysis. Results: Colonic inflammation was associated with impaired colonic epithelial barrier permeability, increased colonic IL-1ß and myeloperoxidase concentrations, and increased CD11b-positive CD3-negative innate immune cell infiltration into the colon. 89Zr-α-IL-1ß and 89Zr-α-CD11b immuno-PET detected colonic inflammation, as did 18F-FDG, and all PET tracers were more sensitive than MRI. Although 18F-FDG volumes of interest correlated with colitis severity and a strong trend was observed with 89Zr-α-IL-1ß, no correlation was observed for 89Zr-α-CD11b or MRI. 89Zr-α-IL-1ß was distributed mainly to the gastrointestinal tract, whereas 89Zr-α-CD11b was distributed to more tissue types. Conclusion: Immuno-PET using antibodies directed to innate immune markers detected colonic inflammation, with 89Zr-α-IL-1ß providing a more tissue-specific signal than 89Zr-α-CD11b. Development of these technologies for human subjects will potentially provide a less invasive approach than endoscopy for diagnosing and monitoring IBD.

Crystal structure of highly glycosylated human leukocyte elastase in complex with an S2' site binding inhibitor.

Glycosylated human leukocyte elastase (HLE) was crystallized and structurally analysed in complex with a 1,3-thiazolidine-2,4-dione derivative that had been identified as an HLE inhibitor in preliminary studies. In contrast to previously described HLE structures with small-molecule inhibitors, in this structure the inhibitor does not bind to the S1 and S2 substrate-recognition sites; rather, this is the first HLE structure with a synthetic inhibitor in which the S2' site is blocked that normally binds the second side chain at the C-terminal side of the scissile peptide bond in a substrate protein. The inhibitor also induces the formation of crystalline HLE dimers that block access to the active sites and that are also predicted to be stable in solution. Neither such HLE dimers nor the corresponding crystal packing have been observed in previous HLE crystal structures. This novel crystalline environment contributes to the observation that comparatively large parts of the N-glycan chains of HLE are defined by electron density. The final HLE structure contains the largest structurally defined carbohydrate trees among currently available HLE structures.

The chemical biology and coordination chemistry of putrebactin, avaroferrin, bisucaberin, and alcaligin.

Dihydroxamic acid macrocyclic siderophores comprise four members: putrebactin (putH2), avaroferrin (avaH2), bisucaberin (bisH2), and alcaligin (alcH2). This mini-review collates studies of the chemical biology and coordination chemistry of these macrocycles, with an emphasis on putH2. These Fe(III)-binding macrocycles are produced by selected bacteria to acquire insoluble Fe(III) from the local environment. The macrocycles are optimally pre-configured for Fe(III) binding, as established from the X-ray crystal structure of dinuclear [Fe2(alc)3] at neutral pH. The dimeric macrocycles are biosynthetic products of two endo-hydroxamic acid ligands flanked by one amine group and one carboxylic acid group, which are assembled from 1,4-diaminobutane and/or 1,5-diaminopentane as initial substrates. The biosynthesis of alcH2 includes an additional diamine C-hydroxylation step. Knowledge of putH2 biosynthesis supported the use of precursor-directed biosynthesis to generate unsaturated putH2 analogues by culturing Shewanella putrefaciens in medium supplemented with unsaturated diamine substrates. The X-ray crystal structures of putH2, avaH2 and alcH2 show differences in the relative orientations of the amide and hydroxamic acid functional groups that could prescribe differences in solvation and other biological properties. Functional differences have been borne out in biological studies. Although evolved for Fe(III) acquisition, solution coordination complexes have been characterised between putH2 and oxido-V(IV/V), Mo(VI), or Cr(V). Retrosynthetic analysis of 1:1 complexes of [Fe(put)]+, [Fe(ava)]+, and [Fe(bis)]+ that dominate at pH < 5 led to a forward metal-templated synthesis approach to generate the Fe(III)-loaded macrocycles, with apo-macrocycles furnished upon incubation with EDTA. This mini-review aims to capture the rich chemistry and chemical biology of these seemingly simple compounds.