Background The lack of noninvasive methods for assessment of dysregulated inflammation as a major driver of fibrosis (i.e., inflammation-fibrosis axis) has been a major challenge to precision management of fibrotic lung diseases. Here, we determined the potential of very late antigen-4 (VLA-4)-targeted positron emission tomography (PET) to detect inflammation in a mouse model of bleomycin-induced fibrotic lung injury. Method Single time-point and longitudinal VLA-4-targeted PET was performed using a high-affinity peptidomimetic radiotracer, 64 Cu-LLP2A, at weeks 1, 2, and 4 after bleomycin-induced (2.5 units/kg) lung injury in C57BL/6J mice. The severity of fibrosis was determined by measuring the hydroxyproline content of the lungs and expression of markers of extracellular matrix remodeling. Flow cytometry and histology was performed to determine VLA-4 expression across different leukocyte subsets and their spatial distribution. Results Lung uptake of 64 Cu-LLP2A was significantly elevated throughout different stages of the progression of bleomycin-induced injury. High lung uptake of 64 Cu-LLP2A at week-1 post-bleomycin was a predictor of poor survival over the 4-week follow up, supporting the prognostic potential of 64 Cu-LLP2A PET during the early stage of the disease. Additionally, the progressive increase in 64 Cu-LLP2A uptake from week-1 to week-4 post-bleomycin correlated with the ultimate extent of lung fibrosis and ECM remodeling. Flow cytometry revealed that LLP2A binding was restricted to leukocytes. A combination of increased expression of VLA-4 by alveolar macrophages and accumulation of VLA-4-expressing interstitial and monocyte-derived macrophages as well as dendritic cells was noted in bleomycin-injured, compared to control, lungs. Histology confirmed the increased expression of VLA-4 in bleomycin-injured lungs, particularly in inflamed and fibrotic regions. Conclusions VLA-4-targeted PET allows for assessment of the inflammation-fibrosis axis and prediction of disease progression in a murine model. The potential of 64 Cu-LLP2A PET for assessment of the inflammation-fibrosis axis in human fibrotic lung diseases needs to be further investigated.

Topic: 26. Sickle cell disease Background: Sickle cell disease (SCD) is characterized by vaso-occlusive crises (VOC) – episodes of acute pain in areas of microvascular occlusion. Hyperadhesion of blood cells to the endothelium and to one another is critical to VOC. Very late antigen-4 (VLA-4 or integrin α4β1) mediates the adhesion of white blood cells (WBC) and reticulocytes to the endothelium, and could therefore be harnessed as an imaging biomarker of VOC. Previous work in our lab has shown that the VLA-4 peptidomimetic PET tracer 64Cu-CB-TE1A1P-LLP2A (64Cu-LLP2A) can quantitatively and in real time image VOC induced by lipopolysaccharide (LPS) in a SCD mouse model. However, VOC is frequently triggered by hypoxia in humans with SCD. Aims: To further validate our imaging strategy, we investigated whether 64Cu-LLP2A could image hypoxia-induced VOC in Townes sickle mice. We also conducted a first-in-SCD study in patients. Methods:64Cu-LLP2A was injected via the tail vein in mice (Townes sickle and non-sickle litter mates; n=10/group) at the dose of 200 µCi per animal. Baseline PET/CT images were acquired at 1h, 4h, and 24h post-injection (p.i.). After one week, all mice were challenged with 1 hour of hypoxia (FiO2=0.07) followed by 1 hour of normoxia (FiO2=0.21) in a small animal hypoxia chamber, a treatment known to cause vaso-occlusion. PET/CT images were acquired at 1h, 4h and 24 h p.i. by using a PET/CT scanner. PET image standard uptake values (SUVs) for the humerus and femur, typical sites of vaso-occlusive pain in humans, were measured using IRW software. Complete blood count analysis was conducted at baseline and after hypoxia challenge and bones were harvested and preserved. Results: The SCD mice showed uptake of 64Cu-LLP2A in the humerus and femur at both baseline and after hypoxia challenge. SUV ratios at 1 h p.i in the humerus and entire femur compared to muscle in SCD mice were increased over baseline, whereas the control mice did not show any significant change in uptake. The 1h timepoint showed the most pronounced uptake, which indicates that the effect of ischemia/reoxygenation on the expression of VLA-4 wanes after 1h. CBC data showed SCD mice had significantly higher neutrophil counts at baseline and post hypoxia. Summary/Conclusion: Our data show that 64Cu-LLP2A can image hypoxia-induced VOC in SCD mice. Increased VLA-4 uptake identifies areas of hyperadhesion and vaso-occlusion. The ongoing PET Imaging of Vaso-Occlusive Crisis (VOC) in SCD clinical study (ClinicalTrials.gov Identifier: NCT04925492, conducted under the FDA-approved IND 150142) at our institution aims to develop 64Cu-LLP2A as a quantitative PET imaging biomarker of VOC in patients with SCD. An early goal of the study is to identify an ideal post-injection time for imaging. Steady-state subjects are scanned at two points post injection which vary by subject and span a range of times, not to exceed 48 hours. Preliminary results show robust uptake in the hematopoietic marrow (Figure 1).Figure 1 – Example of 64Cu-LLP2A distribution in a human in steady-state two hours after injection of 236 MBq. A sagittal slice through the right side of the body is shown and includes a portion of the right hip and femur. Keywords: Sickle cell disease, Positron emission tomography (PET), Sickle cell adhesion

The lack of techniques for noninvasive imaging of inflammation has challenged precision medicine management of acute respiratory distress syndrome (ARDS). Here, we determined the potential of positron emission tomography (PET) of chemokine-like receptor-1 (CMKLR1) to monitor lung inflammation in a murine model of lipopolysaccharide-induced injury. Lung uptake of a CMKLR1-targeting radiotracer, [ 64 Cu]NODAGA-CG34, was significantly increased in lipopolysaccharide-induced injury, correlated with the expression of multiple inflammatory markers, and reduced by dexamethasone treatment. Monocyte-derived macrophages, followed by interstitial macrophages and monocytes were the major CMKLR1-expressing leukocytes contributing to the increased tracer uptake throughout the first week of lipopolysaccharide-induced injury. The clinical relevance of CMKLR1 as a biomarker of lung inflammation in ARDS was confirmed using single-nuclei RNA-sequencing datasets which showed significant increases in CMKLR1 expression among transcriptionally distinct subsets of lung monocytes and macrophages in COVID-19 patients vs. controls. CMKLR1-targeted PET is a promising strategy to monitor the dynamics of lung inflammation and response to anti-inflammatory treatment in ARDS.

In sickle cell disease (SCD), very late antigen-4 (VLA-4 or integrin α4β1) mediates the adhesion of reticulocytes to inflamed, proinflammatory endothelium, a key process in promoting vaso-occlusive episodes (VOEs). We hypothesized that a radionuclide tracer targeting VLA-4 could be harnessed as a positron emission tomography (PET) imaging biomarker of VOEs. We tested the VLA-4 peptidomimetic PET tracer 64Cu-CB-TE1A1P-PEG4-LLP2A (64Cu-LLP2A) for imaging hyper-adhesion-associated VOEs in the SCD Townes mouse model. With lipopolysaccharide (LPS)-induced VOEs, 64Cu-LLP2A uptake was increased in the bone marrow of the humeri and femurs, common sites of VOEs in SCD mice compared with non-SCD mice. Treatment with a proven inhibitor of VOEs (the anti-mouse anti-P-selectin monoclonal antibody [mAb] RB40.34) during LPS stimulation led to a reduction in the uptake of 64Cu-LLP2A in the humeri and femurs to baseline levels, implying blockade of VOE hyper-adhesion. Flow cytometry with Cy3-LLP2A demonstrated an increased percentage of VLA-4-positive reticulocytes in SCD vs non-SCD mice in the bone and peripheral blood after treatment with LPS, which was abrogated by anti-P-selectin mAb treatment. These data, for the first time, show in vivo imaging of VLA-4-mediated hyper-adhesion, primarily of SCD reticulocytes, during VOEs. PET imaging with 64Cu-LLP2A may serve as a valuable, noninvasive method for identifying sites of vaso-occlusion and may provide an objective biomarker of disease severity and anti-P-selectin treatment efficacy in patients with SCD.

Trigonostemons G and H, two novel dimeric dinorditerpenoids, were isolated from the stem barks of Trigonostemon chinensis. Their planar structures and relative configurations were established by extensive analysis of spectroscopic data. Trigonostemons G and H possess a homodimeric biaryl skeleton obtained from two rearranged chiral nonracemic abietane-type dinorditerpenes through an axially chiral biaryl 11,11'-linkage. Torsional scan and computation of the transition states were carried out to estimate the rotational energy barrier, and the axial chirality (aS) was determined by time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations. The positive n-π* ECD transitions of the isolated carbonyl chromophore above 300 nm could be used to determine the central chirality of trigonostemon G independently by ECD calculations of the diastereomers.

Inflammation plays a central role in the pathogenesis of acute lung injury (ALI) during both the acute pneumonitis stage and progression into the chronic fibroproliferative phase, leading to pulmonary fibrosis. Currently, there is an unmet clinical and research need for noninvasive ways to monitor lung inflammation through targeting of immunoregulatory pathways contributing to ALI pathogenesis. In this study, we evaluated the role of targeted imaging of very late antigen-4 (VLA-4), as a key integrin mediating the adhesion and recruitment of immune cells to inflamed tissues, in quantifying lung inflammation in a mouse model of lipopolysaccharide-induced ALI. Methods: ALI was induced by a single intratracheal administration of lipopolysaccharide (10, 20, or 40 μg per mouse) in C57BL/6J mice. Control mice were intratracheally instilled with sterile phosphate-buffered saline. VLA-4–targeted PET/CT was performed 24 h after intravenous injection of a 64Cu-labeled high-affinity peptidomimetic ligand referred to as 64Cu-LLP2A, which is conjugated with the chelator (1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) and a polyethylene glycol 4 linker, at day 2 after the induction of ALI. Ex vivo biodistribution of 64Cu-LLP2A was determined by γ-counting of harvested organs. The severity of lung inflammation was assessed histologically and by measuring the expression of inflammatory markers in the lung tissue lysates using reverse transcription quantitative polymerase chain reaction. Results: Intratracheal lipopolysaccharide instillation led to an acute inflammatory response in the lungs, characterized by increased expression of multiple inflammatory markers and infiltration of myeloid cells, along with a significant and specific increase in 64Cu-LLP2A uptake, predominantly in a peribronchial distribution. There was a strong correlation between the lipopolysaccharide dose and 64Cu-LLP2A uptake, as quantified by in vivo PET (R = 0.69, P < 0.01). Expression levels of both subunits of VLA-4, that is, integrins α4 and β1, significantly correlated with the expression of multiple inflammatory markers, including tumor necrosis factor-α, interleukin-1β, and nitric oxide synthase-2, highlighting the potential of VLA-4 as a surrogate marker of acute lung inflammation. Notably, in vivo 64Cu-LLP2A uptake significantly correlated with the expression of multiple inflammatory markers and VLA-4. Conclusion: Our study demonstrates the feasibility of molecular imaging of VLA-4, as a mechanistically relevant target in ALI, and the accuracy of VLA-4–targeted PET in quantification of ongoing lung inflammation in a murine model.

The cyanobacterial prenyltransferase AmbP3 catalyzes the reverse prenylation of the tetracyclic indole alkaloid hapalindole U at its C-2 position. Interestingly, AmbP3 also accepts hapalindole A, a halogenated C-10 epimer of hapalindole U, and catalyzes normal prenylation at its C-2 position. The comparison of the two ternary crystal structures, AmbP3-DMSPP/hapalindole U and AmbP3-DMSPP/hapalindole A, at 1.65-2.00 Å resolution revealed two distinct orientations for the substrate binding that define reverse or normal prenylation. The tolerance of the enzyme for these altered orientations is attributed to the hydrophobicity of the substrate binding pocket and the plasticity of the amino acids surrounding the allyl group of the prenyl donor. This is the first study to provide the intimate structural basis for the normal and reverse prenylations catalyzed by a single enzyme, and it offers novel insight into the engineered biosynthesis of prenylated natural products.

In vitro reconstitution of a newly discovered isonitrile synthase (AmbI1 and AmbI2) and the detection of an elusive intermediate (S)-3-(1H-indol-3-yl)-2-isocyanopropanoic acid 1 in indolyl vinyl isocyanide biogenesis are reported. The characterization of iron/2-oxoglutarate (Fe/2OG) dependent desaturases IsnB and AmbI3 sheds light on the possible mechanism underlying stereoselective alkene installation to complete the biosynthesis of (E)- and (Z)-3-(2-isocyanovinyl)-1H-indole 2 and 5. Establishment of a tractable isonitrile synthase system (AmbI1 and AmbI2) paves the way to elucidate the enigmatic enzyme mechanism for isocyanide formation.

Enzymatic installation of chlorine/bromine into unactivated carbon centers provides a versatile, selective, and environmentally friendly alternative to chemical halogenation. Iron(II) and 2-(oxo)-glutarate (FeII/2OG)-dependent halogenases are powerful biocatalysts that are capable of cleaving aliphatic C-H bonds to introduce useful functional groups, including halogens. Using the structure of the Fe/2OG halogenase, WelO5, in complex with its small molecule substrate, we identified a similar N-acyl amino acid hydroxylase, SadA, and reprogrammed it to halogenate its substrate, thereby generating a new chiral haloalkyl center. The work highlights the potential of FeII/2OG enzymes as platforms for development of novel stereospecific catalysts for late-stage C-H functionalization.

Hapalindole U (4) is a validated biosynthetic precursor to ambiguine alkaloids (Angew. Chem. Int. Ed. 2016, 55, 5780), of which biogenetic origin remains unknown. The recent discovery of AmbU4 (or FamC1) protein encoded in the ambiguine biosynthetic pathway (J. Am. Chem. Soc. 2015, 137, 15366), an isomerocyclase that can rearrange and cyclize geranylated indolenine (2) to a previously unknown 12-epi-hapalindole U (3), raised the question whether 3 is a direct precursor to 4 or an artifact arising from the limited in vitro experiments. Here we report a systematic approach that led to the discovery of an unprecedented calcium-dependent AmbU1-AmbU4 enzymatic complex for the selective formation of 4. This discovery refuted the intermediacy of 3 and bridged the missing links in the early-stage biosynthesis of ambiguines. This work further established the isomerocyclases involved in the biogenesis of hapalindole-type alkaloids as a new family of calcium-dependent enzymes, where the metal ions are shown critical for their enzymatic activities and selectivities.

The in vitro biochemical characterization revealed that iron/2-oxoglutarate (Fe/2OG)-dependent aliphatic halogenase WelO5* in Hapalosiphon welwitschii IC-52-3 has an enhanced substrate specificity towards 12-epi-hapalindole C (1) in comparison to WelO5 in H. welwitschii UTEX B1830. This allowed us to define the origin of the varied chlorinated versus dechlorinated alkaloid structural diversity between the two welwitindolinone producers. Furthermore, this study, along with the recent characterization of the AmbO5 protein, collectively confirmed the presence of a signature sequence motif in the C-terminus of this newly discovered halogenase enzyme family that confers substrate promiscuity and specificity. These observations may guide the rational engineering and evolution of these proteins for biocatalyst application.

Heterologous expressions and purifications of all WelU proteins from the welwitindolinone pathways in Hapalosiphon welwitschii UTEX B1830 and IC-52-3 led to the discovery that WelU1 and WelU3 selectively assemble 12-epi-fischerindole U (2) and 12-epi-hapalindole C (1), respectively, from 3-geranyl 3-isocyanovinyl indolenine (4) via an enzymatic cascade featuring the Cope rearrangement, stereoselective aza-Prins cyclization and regioselective carbocation deposition. In combination with the in vitro characterization of WelU1/WelU3-homolog AmbU4 for the biogenesis of 12-epi-hapalindole U, this study provide a unified view on the origin of the early stage structural diversifications in hapalindole-type alkaloid biosynthesis, post common intermediate 4.

WelO5 is an iron- and 2-oxoglutarate-dependent halogenase involved in welwitindolinone biosynthesis. A collection of X-ray crystal structures reveals the active-site geometry and ligand dynamics required for small-molecule halogenation by WelO5. A 2.4-Å-resolution X-ray crystal structure of the carrier-protein-independent halogenase WelO5 in complex with its welwitindolinone precursor substrate, 12-epi-fischerindole U, reveals that the C13 chlorination target is proximal to the anticipated site of the oxo group in a presumptive cis-halo-oxo-iron(IV) (haloferryl) intermediate. Prior study of related halogenases forecasts substrate hydroxylation in this active-site configuration, but X-ray crystallographic verification of C13 halogenation in single crystals mandates that ligand dynamics must reposition the oxygen ligand to enable the observed outcome. S189A WelO5 produces a mixture of halogenation and hydroxylation products, showing that an outer-sphere hydrogen-bonding group orchestrates ligand movements to achieve a configuration that promotes halogen transfer.

The elucidation of enigmatic enzymatic chlorination timing in ambiguine indole alkaloid biogenesis led to the discovery and characterization of AmbO5 protein as a promiscuous non-heme iron aliphatic halogenase. AmbO5 was shown capable of selectively modifying seven structurally distinct ambiguine, fischerindole and hapalindole alkaloids with chlorine via late-stage aliphatic C-H group functionalization. Cross-comparison of AmbO5 with a previously characterized aliphatic halogenase homolog WelO5 that has a restricted substrate scope led to the identification of a C-terminal sequence motif important for substrate tolerance and specificity. Mutagenesis of 18 residues of WelO5 within the identified sequence motif led to a functional mutant with an expanded substrate scope identical to AmbO5, but an altered substrate specificity from the wild-type enzymes. These observations collectively provide evidence on the evolvable nature of AmbO5/WelO5 enzyme duo in the context of hapalindole-type alkaloid biogenesis and implicate their promise for the future development of designer biocatalysis for the selective late-stage modification of unactivated aliphatic carbon centers in small molecules with halogens.

The anion promiscuity of a newly discovered standalone aliphatic halogenase WelO5 was probed and enabled the selective synthesis of 13R-bromo-12-epi-fischerindoleU via late-stage enzymatic functionalization of an unactivated sp(3) C-H bond. Pre-saturating the WelO5 active site with a non-native bromide anion was found to be critical to the highly selective in vitro transfer of bromine, instead of chlorine, to the target carbon center and also allowed the relative binding affinity of bromide and chloride towards the WelO5 enzyme to be assessed. This study further revealed the critical importance of halogen substitution on modulating the antibiotic activity of fischerindole alkaloids and highlights the promise of WelO5-type aliphatic halogenases as enzymatic tools to fine-tune the bioactivity of complex natural products.

Eine promiskuitive aliphatische Halogenase, …… AmbO5, wurde in Biogenesestudien der Ambiguin-Alkaloide entdeckt. X. Liu et al. zeigen in ihrer Zuschrift auf S. 5874 ff., dass AmbO5 sieben strukturell verschiedene Ambiguin-, Fischerindol- und Hapalindol-Alkaloide durch späte C-H-Funktionalisierung chlorieren kann. Die Charakterisierung eines C-terminalen Sequenzmotivs in AmbO5, das für Substrattoleranz und Spezifität wichtig ist, belegt die evolvierbare Natur dieser neu entdeckten Halogenasefamilie für die Halogenierung niedermolekularer Verbindungen.

The hapalindole-type alkaloids naturally show striking late stage diversification of what was believed to be a conserved intermediate, cis-indolyl vinyl isonitrile (1a). Here we demonstrate enzymatically, as well as through applying a synthetic biology approach, that the pathway generating 1a (itself, a potent natural broad-spectrum antibiotic) is also dramatically flexible. We harness this to enable early stage diversification of the natural product and generation of a wide range of halo-analogues of 1a. This approach allows the preparatively useful generation of a series of antibiotics with increased lipophilicity over that of the parent antibiotic.

The hapalindole-type alkaloids naturally show striking late stage diversification of what was believed to be a conserved intermediate, cis-indolyl vinyl isonitrile (1a). Here we demonstrate enzymatically, as well as through applying a synthetic biology approach, that the pathway generating 1a (itself, a potent natural broad-spectrum antibiotic) is also dramatically flexible. We harness this to enable early stage diversification of the natural product and generation of a wide range of halo-analogues of 1a. This approach allows the preparatively useful generation of a series of antibiotics with increased lipophilicity over that of the parent antibiotic.

A new lanostan-type triterpenoid with hydroperoxy group, toosendanin A (1), together with two known triterpenoids, meliastatin 3 (2) and ursolic acid (3), were isolated and identified from the stems of Melia toosendan. The structures of these compounds were elucidated by 1D- and 2D-NMR spectra and other spectroscopic studies. These compounds were assayed for the antibacterial activities against some hospital pathogenic bacteria. Toosendanin A (1) exhibited strong antibacterial activity against K. pneumoniae.