The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R–Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor–Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs. MCRs regulate energy homeostasis, pigmentation, cardiovascular function, and sexual functions.4 by reducing high levels of basal signaling activity.5Activation of MC4R by its natural agonists α-melanocyte-stimulating hormone (α-MSH) or β-MSH leads to appetite-reducing effects. In contrast, binding of the endogenous inverse agonist agouti-related peptide (AgRP) causes orexigenic effects6Besides stimulation of heterotrimeric Gsαβγ protein (Gs), MC4R can elicit other signaling pathways associated with the recruitment of arrestin, Gi, or Gq/11.To date, pharmacological approaches targeting MC4R have failed mostly due to the severe adverse effects of drug candidates caused, for instance, by a lack of MCR subtype selectivity or G-protein pathway specificity.9 is the first FDA-approved (2020) medication (brand name Imcivree) for the treatment of rare genetic conditions resulting in obesity, including pro-opiomelanocortin deficiency (POMC), proprotein subtilisin/kexin type 1 deficiency (PCSK1), and leptin receptor deficiency (LEPR).10 Setmelanotide is a cyclic high-affinity peptide with a G-protein signaling profile biased towards Gq/11 (phospholipase C (PLC) activation)11 and a 20-fold receptor subtype selectivity towards MC4R compared to natural α-MSH.12 In contrast to other MC4R agonists, setmelanotide does not cause common Gs signaling-related adverse effects, such as tachycardia or hypertension. Although rare, moderate adverse effects, such as skin hyperpigmentation, have been reported.Setmelanotide Scenesse), is a linear high-affinity analog of α-MSH13 for the treatment of MC1R-driven melanogenesis, thereby preventing skin damage from sun exposure (phototoxicity) in individuals with erythropoietic protoporphyria. Moderate adverse effects of NDP-α-MSH exposure include headache, nasopharyngitis, or back pain.14Another FDA-approved (2019), synthetic but non-selective MCR agonist, NDP-α-MSH  and the G-protein-binding cavity (GBC) is mediated by TM3 and facilitated by TM6. Our structural insights combined with signaling data from site-directed mutagenesis reveal mechanistic details of receptor activation and inhibition.Accordingly, a comprehensive understanding of MC4R-mediated signaling regulation is of fundamental importance. To explore the structural basis of agonist action and receptor-mediated signaling, we determined two cryo-electron microscopy (cryo-EM) structures of human wild-type MC4R–Gs-protein complexes bound to agonists setmelanotide and NDP-α-MSH, with resolutions of 2.6 Å and 2.9 Å, respectively. Comparison of our active structures with the recently solved MC4R crystal structure bound to an antagonistKi = 0.7 nM16 and 2.1 nM,12 respectively) compared with α-MSH (Ki = 51 nM16) which was essential for generating stable complex samples. Both ligands differ in their general peptide structure. NDP-α-MSH is a linear 13-amino acid peptide and is only modified at two positions (methionine (M4) to norleucine (Nle) and l-phenylalanine (l-F) to d-phenylalanine (d-F)) compared to α-MSH13 18 and nucleotide hydrolase apyrase, the resulting nucleotide-free agonist-bound MC4R–Gs–Nb35 complexes were extracted from membranes by solubilization and purified , several water molecules, and the agonist peptides NDP-α-MSH and setmelanotide bound to their respective LBPs  and C-termini (Ctt), the intracellular loop (IL) 1, IL3, and the extracellular loop (EL) 1, as well as the alpha-helical domain of Gαs, were not fully built into the final map .0x1R2W3 motif between the ELs and within the transmembrane bundle of the receptor  in TM3, MC4R has an aspartate (D1223.25), which forms part of a ligand and Ca2+-binding network  and C40Ntt in the N-terminus , N(D)P7.50xxY (D2987.49−P2997.50−Y3027.53 in TM7) and DR3.50Y (D1463.49−R1473.50−Y1483.51 in TM3). These active state-associated changes in the conserved motifs are related to the binding of agonists and G-protein and are linked to global helical movements in the receptor structure upon activation. These mainly include an inward twisted TM5 and a ~13 Å outward dislocated TM6 compared to the inactivated structure, a hallmark of receptor activation that opens the binding cavity for G-proteins  instead of an l-F at position x1. This substitution is important for the increased potency of NDP-α-MSH compared with α-MSH.13All three ligands share a common central amino acid motif HLBP Fig. . NDP-α-M0x1R2W3 motif forms similar interactions for both agonists, including hydrogen bonds between H0 and T1012.61 in TM2 and W3 with S188EL2 in EL2 and H2646.54 in TM6  In contrast, for MC4R no direct interactions between the ligands and W2586.48 exist  upon NDP-α-MSH, setmelanotide or α-MSH stimulation  of E100ion Fig. , S8. In ion Fig. .29 In our Gs signaling data, NDP-α-MSH displays a potency reduction of two orders of magnitude for E100N and D126S, but in contrast to the other two ligands, it only shows a slight EC50 reduction at variant D122S (9 nM instead of 1.2 nM for wild-type MC4R)  Fig. , which sThe PLC signaling data of the EDD motif mutants Fig.  revealedWe here also tested how NDP-α-MSH and setmelanotide stimulations at the wild-type MC4R differ between the two signaling pathways. Both ligands show similar potencies for the induction of cAMP accumulation and PLC activation but deviate in their efficacies . The reduced number of setmelanotide interactions is also related to a double conformation of E1002.60, in which one conformation participates in a hydrogen bond network with a water molecule and H40 , which is essential for the previously proposed “toggle switch” activation mechanism in different GPCRs.31 Comparison of the MC4R with known (in-)active class A GPCR structures revealed similar changes around the W2586.48 of the CWxP6.50 motif between the inactive or antagonized versus the active structures as observed, e.g., in rhodopsin32 and the 5-HT2A serotonin receptor26  toward the membrane is a hallmark of active-state conformations because it opens the intracellular cavity for G-protein binding Fig. . This di6.50 motif, namely F2616.51 and H2646.54. H2646.54 is in hydrogen bond distance to the ligand’s backbone oxygen of W3 near the extracellular vestibule entrance   Fig.  for NDP-50) Fig. .6.51does not directly interact with NDP-α-MSH but forms a hydrophobic interaction with the d-F1 residue in the setmelanotide–MC4R structure . This is accompanied by a reduced cell surface expression  together with I1373.40 (TM3) and M2045.50 (TM5) constitute an M5.50IF motif in MC4R, reminiscent of the class A GPCR-typical P5.50IF motif (P5.50−I3.40−F6.44) involved in the maintenance of an inactive receptor state.33 Interestingly, all substitutions of residues in the MC4R-M5.50IF motif  do not affect agonist-induced signaling  that induces a kink/bulge in the transmembrane helix34 P7.50xxY motif in TM7  and S188EL2, and R52 connects TM3−TM4 and EL2 through hydrogen bonds with S188EL2 and I1854.61  in SHU9119 at position x1 in the H0x1R2W3 motif that corresponds to d-F1 in NDP-α-MSH and setmelanotide  and the MC5R (with V1263.36)  in the α5 C-cap.Following disruption of the MC4R TM3–TM6 dual lock by ligand interaction and the TM6 outward tilt Fig. , both reα5.23 is further stabilized by a hydrogen bond to T1503.53  in agreement with the pathogenic gain-of-function mutant H158R47 and other constitutively activating mutants D146A, F149A, and F152A45 in IL2, but does not impair Gs signaling induced by NDP-α-MSH or setmelanotide 36 and MC5R (L3.36V) with neither receptor having a leucine at the reciprocal position  ultimately evidences the significance of position 3.36 for receptor activity regulation and antagonism of SHU9119 at MC4R.16Notably, extracellular interactions formed by SHU9119 are not significantly different from those formed by both agonists, but SHU9119 blocks the TM6 movement essential for MC4R signaling in the transmembrane region. SHU9119s antagonism depends on the interaction with L133119 Fig. . Finally2+-binding site, which is formed by ligand and receptor residues. These differences are potentially accompanied by local shifts in the TM3 adjustment relative to the other helices P7.50xxY motif, as well as reorientations of Y5.58 in TM5, and the conserved DR3.50Y motif in TM3 are significant for the active structures. Subsequently, the conserved DR3.50Y motif in TM3 and the adjacent IL2 constitute important parts of the interface to the Gs-protein.Compared to the antagonized SHU9119–MC4R crystal structure, we identified several active state-specific conformational changes that play a concerted role in signal transduction, which are in line with a recently reported setmelanotide–MC4R–Gs complex structure3.53 in the intracellular TM3/IL2 transition of MC4R is essential for Gs-binding as indicated by a direct hydrogen bond to Y377α5.23 in the α5 helix of Gs and supported by our mutagenesis data on substitutions against several hydrophobic residues that showed impaired signaling .T150ock Fig. . The funpes Fig. . The abepes Fig. . This alIL2 in both agonist-MC4R structures is significantly different depending on the respective ligand  and calcium. Here, the setmelanotide residue R1ne Figs. .49 and MC4R bound with endogenous ligands such as α-, β-MSH, AgRP or the cofactor protein MRAP.50 Altogether, the structural findings presented here will facilitate the development of new MCR subtype- and G-protein-selective anti-obesity drugs.To fully characterize G-protein subtype selectivity at the MC4R and to further differentiate Gq/11- and Gs-coupling, an agonist–MC4R–Gq/11 complex is required. Other open questions pertaining to the unique MC4R system await structural elucidation, such as determining the apo-state conformation, dimer-arrangements relevant to MC4R as an endocrine GPCR,Wild-type human MC4R (UniprotKB-P32245) was modified to include an N-terminal hemagglutinin signal sequence, followed by a FLAG-tag epitope (DYKDDDK). The C-terminal eGFP, followed by polyhistidine (His-) and rho-1D4 tags, is removable by HRV-3C protease cleavage (construct name: MC4R–eGFP) and was inserted into a pOET3 vector.Sf9 cells (from Spodoptera frugiperda) with pOET3_MC4R–eGFP and linearized BAC10:1629KO52 using Trans-IT Insect (Mirus Bio). Sf9 cells were cultured in SF900 II serum-free medium (Invitrogen) at 28 °C for virus generation. A 1 L preparation of Sf9 cells at 2 × 106 cells/mL were infected with 10 mL of P2 virus MC4R−eGFP virus. Cultures were grown at 27 °C, harvested by centrifugation 48 h post infection, and stored at −20 °C.For the production of MC4R–eGFP, recombinant baculovirus was generated by co-transfecting 1 (UniprotKB-P54311) and bovine Gγ2 (UniprotKB-P63212) subunits in pFastbacDual vector were previously used and described.18 Heterotrimeric Gαsβ1γ2 protein (or named Gs) was expressed in Trichoplusia ni (Tni) insect cells, maintained in ESF 921TM serum-free insect cell culture media (Expression Systems) at 28 °C. The virus was prepared using Bac-to-BacTM baculovirus expression system (Thermo Fisher Scientific). The cells were infected with both Gαs and Gβ1γ2 virus, based on small scale titrations and harvested 48 h post infection, and stored at −20 °C. Gs was purified as described previously.18 The single-domain antibody Nanobody-35 (Nb35) was previously described.18 Nb35 was expressed in E. coli strain WK6, extracted and purified by immobilized metal (Ni-NTA) affinity chromatography according to previously described methods.18Bovine Gαs-short subunit (UniprotKB-P04896-2) in pFastbac vector and rat GβSf9 membranes. Sf9 cell pellets containing MC4R–eGFP were resuspended in 20 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM CaCl2, 25 µM Tris (2-carboxyethyl) phosphine (TCEP), 25 U/mL apyrase (New England Biolabs) 2.5 mg/mL leupeptin , 0.16 mg/mL benzamidine (Sigma-Aldrich) and 1 µM of the respective agonist (in-house peptide synthesis). For 1 L of cell pellets, Gs pre-incubated with Nb35 was added and incubated overnight at 4 °C. The membrane sample containing agonist–MC4R–Gs–Nb35 complex was collected by centrifugation at 46,000× g and carefully resuspended in 20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM CaCl2, 25 µM TCEP, 2.5 mg/mL leupeptin, 0.16 mg/mL bezamidine, 1 µM agonist and 1% n-dodecyl β-d-maltoside (DDM), 0.1% cholesteryl hemisuccinate (CHS) . After 2 h, the solubilized protein was separated from insoluble remains by centrifugation at 46,000× g. The supernatant was diluted twofold with 20 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 5 mM CaCl2, 25 µM TCEP, 2.5 mg/mL leupeptin, 0.16 mg/mL benzamidine and 1 µM agonist. Anti-FLAG M1 resin (Sigma-Aldrich) was added and incubated for 2 h at 4 °C rotating. M1 resin was collected by centrifugation  and loaded into a wide-glass column and washed for 5 column volumes with wash buffer  with 0.1% DDM and 0.01% CHS, followed by a 1 h incubation in wash buffer with 0.8% lauryl maltose neopentyl glycol (LMNG), 0.08% CHS , and 0.02% DDM. Subsequently, LMNG/CHS concentration was lowered stepwise to 0.01% LMNG, 0.001% CHS for 1 h. Elution of the complex was initiated by addition of 20 mM HEPES, pH 7.5, 150 mM NaCl, 25 µM TCEP, 1 µM agonist, 0.01% LMNG, 0.001% CHS, 5 mM EDTA and 0.2 mM DYKDDDDK peptide (GenScript Biotech). C-terminal eGFP was removed by adding HRV-3C protease (in-house purified), incubated at 4 °C overnight. After concentration, the agonist–MC4R–Gs–Nb35complex was loaded onto a Superdex 200 Increase 5/150 GL (Sigma-Aldrich). Receptor-containing fractions were concentrated to 5 mg/mL and directly vitrified. One liter of MC4R–eGFP expressing cells yielded 0.25 mg complex.MC4R–Gs–Nb35 complexes with both agonists NDP-α-MSH and setmelanotide (from now on abbreviated by agonist) were formed in Vitrification of NDP-α-MSH– and setmelanotide–MC4R–Gs–Nb35 complexes was conducted immediately after sample preparation at a concentration of 1.2 mg/mL and 5 mg/mL, respectively. 3.8 µL of the sample was applied to glow-discharged holey gold grids , blotted for 4 s and plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV (Thermo Fisher Scientific) set to 10 °C and 100% humidity.−/Å2 fractionated into 33 frames. EPU 2.8 was utilized for automated data acquisition with AFIS enabled using a nominal defocus between −0.8 and −2 µm.Images were acquired using a FEI Titan Krios G3i microscope (Thermo Fisher Scientific) operated at 300 kV equipped with a FEI Falcon 3EC detector (Thermo Fisher Scientific) running in counting mode at a nominal magnification of 96,000×, giving a calibrated pixel size of 0.832 Å/px. Movies were recorded for 40.78 s accumulating a total electron dose of 40 eA total of 5618 micrographs were collected for NDP-α-MSH–MC4R–Gs–Nb35 and 7583 micrographs for setmelanotide–MC4R–Gs–Nb35. These were used for further image processing. Further details are given in Supplementary information, Table 53 Using NU-refinement, masking of the all-helical domain was not necessary to yield a high-resolution map  together with the Gsαβγ–Nb35 complex of the β2-adrenergic receptor–Gs complex (PDB ID: 3sn618) as initial models. Both MC4R complexes were built and adjusted manually using the program COOT.54 Model building for the MC4R ligands NDP-α-MSH and setmelanotide was started de novo using COOT.54 Local-refined, as well as overall cryo-EM maps were used to add water molecules. After every round of manual refinement and for the final round, Real-space refinement55 was performed with the program PHENIX56 using geometric restraints, a global minimalization protocol and B-factor refinement. Both models were additionally refined with isotropic B-factors in reciprocal space using REFMAC557 of the CCP4  software suite.58 The refinement was carried out in the resolution range of 233–2.88 Å and 233–2.6 Å for the NDP-α-MSH–MC4R and setmelanotide–MC4R complexes, respectively . MC4R: 40–108, 118–230, 239–316; NDP-α-MSH: 1–13; Gsα-protein: 13–47, 194–236, 249–280, 293–306, 322–380; Gβ1: 4–340; Gγ2: 9–63; NB35: 1–128. Structure validation was performed with PHENIX,56 MolProbity,59 SFCHECK,60 and OneDep of the Protein Data Bank.58 Potential hydrogen bonds and van der Waals contacts were analyzed using HBPLUS61 and LIGPLOT 1.45+.62 All structure superpositions of backbone α-carbon traces were performed using the CCP4 program LSQKAB.63 All molecular graphics representations in this work were created using the PyMol Molecular Graphics System Version 1.3  and UCSF Chimera.64The final model of the setmelanotide–MC4R complex includes the following amino acids . MC4R: 40–107, 117–230, 240–316; setmelanotide: 1–8; Gsα-protein: 14–47, 193–236, 248–280, 293–310, 318–351, 355–380; GβTM Luciferase; Promega)17 and cloned into pMT4 vector. Human embryonic kidney 293 (HEK293T) cells grown in DMEM/F-12 (Thermo Fisher Scientific) medium   were transiently transfected using FuGENE® HD transfection reagent (Promega). 20,000 cells per well were seeded in white corning assay 96-well plates. After 24 h, the medium was removed and replaced with 75 µL ligand serial dilutions in Opti-MEM reduced serum media (Thermo Fisher Scientific) without phenol red and incubated for 2 h. For saturation experiments, TAMRA-NDP-α-MSH (TAMRA-NDP) labeled with the fluorophore 5-carboxytetramethylrhodamine (TAMRA) was titrated from 1 µM to 1 pM. For competition experiments, each well contains 10 nM TAMRA-NDP and the competing ligand was titrated from 1 µM to 10 pM for setmelanotide. Non-specific binding was measured by adding 20 µM NDP-α-MSH, to saturate the ligand-binding pocket with the non-fluorescent ligand. After 2 h, 25 µL Furimazine (Promega) was added and incubated for 15 min. Luminescence and resulting bioluminescence resonance energy transfer (BRET) was measured in Spectramax ID3 (Molecular Devices) and CLARIOstar Plus (BMG LABTECH) plate reader with 460 nm (short-pass filter) and 610 nm (long-pass filter). The BRET ratio was calculated by the quotient of long-pass divided by short-pass.17 GraphPad PRISM 8 (GraphPad Software Inc.) was used for analysis by sigmoidal dose-response (variable slope) for dose-response measurements and one site - Fit Ki for competition experiments.Wild-type MC4R was modified to include an N-terminal hemagglutinin signal sequence, followed by Luciferase  supplemented with 5% fetal bovine serum FBS  and 1% non-essential amino acids NEA (Merck Biochrom) at 37 °C and humidified air containing 5% CO2. For cAMP accumulation and PLC activation assays, 1.5 × 104 cells per well were seeded in poly-l-lysine coated (Merck Biochrom) translucent 96-well plates  and incubated for 24 h. In an identical fashion, determination of total and cell surface expression  were performed in white opaque, poly-l-lysine-coated 96-well plates (Corning #3917).HEK293 cell line was purchased from ATCC. Cells were authenticated by single nucleotide polymorphism (SNP) analysis and regularly tested for mycoplasma contamination. Cultivation took place in ® HiBiT assays, MC4R was cloned into pBiT3.1-N (Promega) using EcoRI/BamHI restriction sites, resulting in HiBiT protein tag N-terminally spaced by eleven amino acids. All single point mutations were incorporated into the expression vectors using site-directed mutagenesis. Cloned constructs were sequenced and verified with BigDye-terminator sequencing (PerkinElmer Inc.) using an automatic sequencer . α-MSH, NDP-α-MSH and 3-Isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich.MC4R cDNA was amplified from genomic DNA and cloned into eukaryotic expression vector pcDps. The receptor was N-terminally tagged with the hemagglutinin (5′-YPYDVPDYA-3′) epitope (HA) for cAMP measurements and luciferase-based assays. For NanoGlo47 In short, low amounts of HiBiT-tagged receptor mutants were transfected (0.45 ng/well) and carrier DNA (pGEM-3Zf(+), Promega) was added to 45 ng DNA/well in total in advanced MEM  to ensure comparable transfection conditions to the other performed assays. Transfection for both HiBiT-assay  was carried out simultaneously to ensure comparability.For determination of cAMP accumulation and PLC activation, HEK293 cells were transfected 24 h after seeding. Cells were transfected with 45 ng plasmid DNA and 0.45 µL Metafectene (Biontex) per well in MEM without supplements. For the NanoGlo® HiBiT assay, transfection was performed as described previously elsewhere.47 In short, 48 h after transfection, media were changed into Opti-MEM reduced serum media  without phenol red (50 µL/well) to remove background noise. Determination of cell surface expression was performed by injection of 50 µL of HiBiT extracellular substrate in the appropriate buffer supplemented with LgBiT. Total expression determination was carried out similarly, with 50 µL/well of HiBiT Lytic substrate combined with LgBiT in the appropriate buffer containing detergents to lyse the cells (Promega). After orbital shaking for 3 min at 300 cycles per minute, plates were incubated for 10 min at room temperature. Luminescence was measured using a plate reader . As background control, cells transfected with empty vector pcDNA3 were used and values were subtracted from the sample emissions.The amount of receptors expressed on the cell membrane as well as total cell expression was determined using the NanoGlo® HiBiT detection system (Promega). Assay was performed according to manufacturer’s protocol (rapid measurements) and has been described elsewhere.65 In brief, 48 h post transfection, cells were challenged with either α-MSH or NDP-α-MSH (1 µM to 0.1 nM) in stimulation buffer  containing 1 mM IBMX for 40 min for at 37 °C and 5% CO2. Incubation was stopped by freezing cells at −80 °C for 10 min prior to cAMP measurements. The determination of cAMP accumulation was performed following to the manufactures’ instructions (Perkin Elmer Life Science) and measured with a plate reader .Ligand-induced activation of MC4R was determined by using the AlphaScreen™ assay (Perkin Elmer Life Science) according to the manufacturer’s protocol and has been described elsewhere.q/11 signaling, activation of PLC-β activation was investigated using NFAT responsive element  located in the promotor region of the gene encoding a firefly luciferase and luciferase is expressed upon second messenger activation. After incubation for 48 h after transfection, cells were stimulated with NDP-α-MSH or setmelanotide (1 mM to 0.1 nM) in MEM without supplements for 6 h at 37 °C and 5% CO2. The stimulation was stopped by exchange of media with 1× passive lysis buffer  and freezing the cells at −80 °C for 10 min. Luciferase activity was then measured and provided information about the activation of the respective second messenger by transferring 10 µL lysate into a white opaque 96-well plate. Injection of 40 µL firefly luciferase substrate (Promega) and measurement of luminescence were performed with a plate reader (Mithras LB 940).As a read-out system for G∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001 and ∗∗∗∗P ≤ 0.0001. Concentration-response curves of each experiment were analyzed by fitting a non-linear regression model for sigmoidal response in GraphPad PRISM 6 (GraphPad Software Inc.) to determine EC50 values. Statistics for all functional data are given in Supplementary information, Tables Statistical analysis was performed using GraphPad Prism 6. Appropriate tests were carried out and indicated for each individual data set. Statistical significance was set at Supplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5Supplementary figure 6Supplementary figure 7Supplementary figure 8Supplementary figure 9Supplementary figure 10Supplementary figure 11Supplementary figure 12Supplementary figure 13Supplementary figure 14Supplementary figure 15Supplementary figure 16Supplementary figure 17Supplementary figure 18Supplementary figure 19Supplementary figure S20Supplementary figure S21Supplementary figure S22Supplementary figure S23Supplementary figure S24Supplementary table S1Supplementary table S2Supplementary table S3Supplementary table S4Supplementary table S5Supplementary table S6Supplementary table S7Supplementary table S8Supplementary table S9Supplementary table S10Supplementary Data S1


Background: Psoriasis is a T help 17 (Th17) cell-mediated chronic inflammatory skin disease. Recent studies have shown that dihydroartemisinin (DHA) can significantly reduce experimental autoimmune encephalomyelitis and rheumatoid arthritis by regulating Th17 cells.Objective: To verify whether DHA can improve the symptoms of psoriasis and to further explore the possible mechanism.Methods: The efficiency of DHA was preliminary detected on human keratinocytes (HaCaT) cells in psoriatic condition. Then, imiquimod-induced psoriasis-like model in BALB/c mice was established to evaluate the effects of DHA in vivo.Results: Under the stimulation of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), DHA inhibited the proliferation of HaCaT cells and significantly affected the mRNA expression levels of IFN-γ, interleukin (IL), IL-17A and IL-23. DHA treatment reduced the severity of psoriasis-like skin and resulted in less infiltration of immune cells in skin lesions. DHA restored the expression of IFN-γ, IL-17A, and IL-23 in skins, as well as a decrease of cytokines and chemokines in skin supernatant. DHA also altered the cellular composition in the spleen, which is the makeup of the T cells, dendritic cells (DCs), and macrophages. DHA recovered Th17-related profile with decreased frequency of IL-17+CD4+T cells from splenocyte of mice. Furthermore, DHA also inhibited the concentration of IL-17 from Th17 cells and the expression of Th17 cell-related transcription factors retinoid-related orphan receptor-gamma t (ROR-γt) in vitro. In addition, phosphorylation of signal transducer and activator of transcription-3 (STAT3) was significantly reduced in DHA treatment mice, suggesting that the IL-23/Th17 axis plays a pivotal role.Conclusion: DHA inhibits the progression of psoriasis by regulating IL-23/Th17 axis and is expected to be an effective drug for the treatment of psoriasis. Psoriasis is an immune-related chronic inflammatory disease characterized by excessive growth and aberrant differentiation of keratinocytes . InterleRecent studies have indicated that the IL-23/Th17 axis plays a significant role in psoriasis. Therefore, IL-23/Th17 axis has become a potential drug treatment target. At present, the anti-IL-17 antibody antagonists are approved by the Food and Drug Administration (FDA) in the United States for clinical use, including secukinumab and ixekizumab, which are superior to TNF-α antagonists in the treatment of moderate to severe plaque psoriasis, and have good safety and tolerance . SeveralDihydroartemisinin (DHA) is the main active metabolite of artemisinin compounds. Some studies have found that DHA has a potent effect on the treatment of autoimmune diseases and tumors without obvious toxic and side effects. DHA inhibits the activation of Toll-like receptor 4 signal transduction pathway and the production of type I interferon and anti-ds-DNA in splenocytes from MRL/lpr lupus mice to improve the pathological damage of lupus nephritis . Zhao YGBased on the above research, we hypothesized the inhibitory effect of DHA on psoriasis and preliminarily explored the possible mechanism of DHA regulating psoriasis based on the IL-23/Th17 axis for providing a potential therapeutic agent for psoriasis.2 atmosphere.Human keratinocytes (HaCaT cells) were purchased from FuDan IBS Cell Center . Human skin fibroblasts (HSF) were established from FuHeng Cell Center . They were maintained in DMEM medium  supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin . The cells were cultured at 37°C in 5% COThe proliferation of keratinocytes in psoriatic conditions was induced by 10 ng/ml recombinant human TNF-α and 10 ng/ml IFN-γ to further study the effects of DHA on the expression of cytokines on HaCaT cells.BALB/c female mice (8–10 w) were healthy and fertile and did not display evidence of infection or disease. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. All studies and procedures were approved by the Experimental Animal Ethical Committee of Yanbian University (SYXK (ji) 2020–0009).62.5 mg 5% imiquimod  cream was smeared to the shaved back of mice for 5 days to induce psoriasis-like skin lesions (IMQ group). Dihydroartemisinin  was applied i.g. at a dose of 25 mg/kg/d (DHA-L group) and 50 mg/kg/d (DHA-H group) 3 days in advance for 8 days. As for the control group (Con. Group), they were treated similarly with control vehicle cream and phosphate-buffered saline.The inflammation skins were graded according to the clinical Psoriasis Area and Severity Index (PASI). Erythema, scaling, and thickening were scored independently on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. The cumulative PASI score served as a measure of the severity of inflammation .4 cells per well, then incubated overnight at 37°C in 5% CO2 atmosphere, and treated with different concentrations of DHA for 24 and 48 h. Cell viability was determined using MTT assays . The absorbance was measured at a wavelength of 490 nm using a microplate reader .HaCaT cells were placed into 96-well plates at a density of 2 × 10XMG1.2), and IL-17 (eBio17B7) from eBioscience. For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences). Dead cells were detected by LIVE/DEAD Fixable Green Dead Cell Kit (Invitrogen-Molecular Probes). Multiparameter flow cytometric analysis was performed on the FACS Verse  and CytoFLEX . Data were analyzed using FlowJo software (Ver. 8.8.7).Fluorescein isothiocyanate-, R-phycoerythrin (PE)-, peridinin chlorophyll protein (PerCP)-, PE-Cy5-, PE-Cy7-, allophycocyanin (APC)-conjugated mAbs in this study include those to CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), F4/80 (BM8), CD11c (N418) from Biolegend, IFN-γ , CD4+T cells (5 × 104) were activated by 10 μg/ml anti-CD3  and 2 μg/ml anti-CD28  for 48 or 96 h with or without 0.5 μg/ml DHA. PMA  and ionomycin  were included in the culture medium for the last 5 h of incubation.Splenic T cells were purified using a CD4Tissues were harvested using a disposable, sterile 6 mm punch biopsy blade  and assessed for tissue damage and the number of infiltrating T cells, DCs, and macrophages. Four-micrometer sections were stained using haematoxylin and eosin (H&E) to identify histologic changes of dermal tissues. To identify keratinocyte proliferation, rabbit anti-mouse Ki67 mAb (D3B5) were, respectively, used on paraffin-embedded sections by immunohistochemical (IHC) staining.2). Each section was examined independently by three investigators in a blinded fashion, and the meaning was used for analysis.All cells were counted from the infection site in five serial skin sections. The numbers of infiltration cells were averaged in more than five power microscopic fields . Isolated RNA was reverse-transcribed to cDNA by using the M-MLV Reverse transcriptase system (Promega). cDNA was amplified with the ExTaq enzyme system. The sequences of primers were as follows: IFN-γ F: 5′-CGGCACAGTCATTGAAAGCCTA-3′, R: 5′-GTTGCTGATGGCCTGGATTGTC-3′; IL-4 F: 5′-ACGGAGATGGATGTGCCAAAC-3′, R: 5′-AGCACCTTGGAAGCCCTACAGA-3′; IL-6 F: 5′-CCACTTCACAAGTCGGAGGCTTA-3′, R: 5′-TGCAAGTGCATCATCGTTGTTC-3′; IL-10 F: 5′-GCCAGAGCCACATGCTCCTA-3′, R: 5′-GATAAGGCTTGGCAACCCAAGTAA-3′; IL-17A F: 5′-GAAGGCCCTCAGACTACCTCAA-3′, R: 5′-TCATGTGGTGGTCCAGCTTTC-3′; IL22 F: 5′-CCTTCCCCAGTCACCAGTTG-3′, R: 5′-CTCCACTCTCTCCAAGCTTTT-3′; IL23R F: 5′-GCTCTGAAGTGGAATTATGTGC-3′, R: 5′-CTTCTTCTGTCTCTAAACTCTTCAC-3′; IL-23 F: 5′-GACTCAGCCAACTCCTCCAGCCAG-3′, R: 5′-TTGGCACTAAGGGCTCAGTCAGA-3′; Retinoid-related orphan receptor-gammat (ROR-γt) F: 5′-A-GCTAGGTGCAGAGCTTCAG-3′, R: 5′-ATTTGTGTTCTCATGACTGAGCC-3′; β-actin F: 5′-AGGTCATCACTATTGGCAACGA-3′, R:5′-CACTTCATGATGGAATTGAATGTAGTT-3′. The relative expression levels of genes were compared with β-actin.30 mg spleen tissue was minced and homogenated, and the proteins were treated and then incubated with primary antibodies against mouse STAT3 and phosphorylation of STAT3 Ab  at 4°C overnight, followed by incubation with fluorescent secondary antibodies. Finally, the membranes were scanned using a Bio-Rad Gel imaging system  after visualization treatment using the ECL reagent. Protein expressions were analyzed with ImageJ software.50 mg depilated back skin was homogenated in PBS and 5% FBS medium, and the supernatant was obtained by centrifugation at 5000 rpm for 15 min. IL-1β, IL-6, IL-18, and Chemokine (C-X-C motif) Ligand 1 Protein (CXCL-1) in the supernatant were determined by an ELISA kit .+T cells in the culture supernatant fluid was determined by ELISA kits .IL-17 secretion from purified CD4n ≥ 4 mice/group). The significance of differences was determined using the Student’s t test and one-way ANOVA analysis of variance followed by Dunnett’s test.All data were analyzed with GraphPad Prism 9.0  and exhibited as the means ± SD (p < 0.05); the IC50 values of 24 and 48 h were, respectively, 32.51 μg/ml and 8.23 μg/ml. Moreover, DHA did not affect the proliferation of human skin fibroblasts HSF cells  through direct activation of osteoclast precursors  and affect T cell differentiation, which has a favorable application prospect in anti-inflammation , antican in vivo , 4. Morereatment , 5.−/− mice do not have Th17 cells, so leading to the immune diseases abatement (+CD4+T cells population in psoriasis groups was significantly inhibited by DHA administration (During the differentiation of Th17 cells, the signal of cytokines is transduced through STAT3, thus initiating the specific transcription program of Th17 cells. STAT3 can directly bind to IL-17A and IL-21 loci and regulate the expression of ROR-γt and IL-23R. Studies have confirmed that heterozygous STAT3 gene mutation can reduce the utilization rate of functional STAT3 dimer by 25% and consequently reduce the expression of ROR-γt by four times. The latter is a specific nuclear transcription factor necessary for Th17 cell differentiation. ROR-γtbatement . In the batement . Moreovestration . Thus, o


O-alkylated sulfonylpyrazolone, the sulfur atom lies 0.558 (1) Å out of the pyrazole ring plane. The NH2 group is involved in an intramolecular hydrogen bond to a sulfonyl oxygen atom and in a three-centre system with the two oxygen atoms of the side chain at C3, forming a ribbon structure.In this  18H17N3O4S, the pyrazole ring is planar, with the sulfur atom lying 0.558 (1) Å out of the ring plane. The NH2 group is involved in an intramolecular hydrogen bond to a sulfonyl oxygen atom; its other hydrogen atom forms an asymmetric three-centre hydrogen bond to the two oxygen atoms of the —O—CH2—C=O— grouping, via the 21 screw axis, forming a ribbon structure parallel to the b axis. Translationally adjacent, coplanar ribbons form a layer parallel to (10In the title compound, C N-sulfonyl- and N-sulfonylamino moiety, which have shown significant biological activity as novel antiviral and antimicrobial agents ethan-1-one 2 in N,N-dimethylformamide in the presence of potassium carbonate at room temperature furnished an adduct for which two possible isomers, the O-alkylated or N-alkylated N-sulfonylpyrazole structures (3 or 4) were considered. The 1H NMR spectrum of the product showed four singlet signals at δ = 2.40, 4.91, 5.45 and 6.34 ppm assigned for CH3, CH-pyrazole, CH2 and NH2 protons, in addition to signals assigned to aromatic protons. The available spectroscopic data cannot differentiate between structures 3 and 4 -1,2-dihydro-3 4 Fig. 1. Thus, t4 is shown in Fig. 2The molecular structure of x, y + z + b axis. H01 also has a short but non-linear contact to O1 (same operator), representing the weaker component of an asymmetric three-centre system -5-amino-1H-pyrazol-3-yl thiophene 2-carboxylate, refcode YILPUF , 2-bromo-1-(p-tolyl)ethan-1-one 2 (0.01 mol) and anhydrous potassium carbonate (0.01 mol) in N,N-dimethylformamide (5 mL) was stirred at room temperature for 3 h. The mixture was poured onto ice–water; the solid that formed was filtered off and recrystallized from ethanol to give pale-brown crystals in 70% yield, m.p. 445 K. IR : ν 3475, 3304 (NH2), 1690 (CO); 1H NMR (DMSO-d6): δ = 2.40 , 4.91 , 5.45 , 6.34 , 7.37 , 7.56–7.60 , 7.72–7.76 , 7.85 . Analysis calculated for C18H17N3O4S (371.41); C, 58.21; H, 4.61; N, 11.31; S, 8.63. Found: C, 58.39; H, 4.42; N, 11.65; S, 8.45%.A mixture of 5-amino-1-phenylsulfonyl-1,2-dihydro-32 group were refined freely. The methyl group was refined as an idealized rigid group allowed to rotate but not tip, with C—H = 0.98 Å and H—C—H = 109.5°. Other hydrogens were included using a riding model starting from calculated positions . The U(H) values were fixed at 1.5 or 1.2 times the equivalent Uiso value of the parent carbon atoms for methyl and non-methyl hydrogens, respectively. Six reflections were omitted because their calculated and measured Fo2 and Fc2 values differed by more than 7 s.u. The occurrence of such apparent outliers seems to be a general consequence of collecting data to high 2θ values (here 76°), whereby spherical atom scattering factors become less applicable. Special refinements using aspherical atom scattering factors can lead to greatly improved R values and thus fewer outliers, but this method is not yet widely employed. However, even for ‘normal’ refinement, it is still considered best practice to collect data to high diffraction angles wherever possible  I, global. DOI: 10.1107/S2056989021010008/yz2011Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021010008/yz2011Isup3.cmlSupporting information file. DOI: 2111897CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


P212121 chiral space group is corrected to centrosymmetric Pbca.The crystal structure of the title compound was redetermined at two temperatures [120.0 (2) and 295.0 (2) K]. The previously reported  3 in the presence of DIPEA when heated at 343 K for 8 h afforded the title hydrated salt, C7H14N3S+·Cl−·H2O, in 84% yield. This course of the tosylation reaction differs from the result of tosylation obtained for this substrate at room temperature, when only 2-amino-8-thia-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium tosylate was isolated in 56% yield. The structure of the reaction product was established by physicochemical methods, spectroscopy, and X-ray diffraction. The single-crystal data demonstrated that the previously reported crystal structure of this compound  had been refined in a wrong space group. In the extended structure, the chloride anions, water molecules and amine groups of the cations form two-periodic hydrogen-bonded networks with the fes topology.The reaction of β-(thiomorpholin-1-yl)propioamidoxime with tosyl chloride in CHCl In our opinion, the source of hydrate formation was air moisture, since the formation of single crystals took place over a long time under conditions of natural evaporation of the solvent for crystallization with air access. This result of the amidoxime (1) tosylation differs from the result of the same reaction performed at room temperature, when the main kinetic product of the reaction was 2-amino-8-thia-1,5-diazaspiro[4.5]dec-1-en-5-ium tosylate (2) -1,5-diazaspiro[4.5]dec-1-en-5-ium arylsulfonates as the main products -1,2,4-oxadiazoles  K for 3, thus a single crystal of the reaction product was also determined at 295.0 (2) K and the resulting crystal structures were compared with the previously reported one.Spiropyrazolinium chloride monohydrate 3 is shown in Fig. 27H14N3S+ cation adopt chair and envelope conformations, respectively. It may be noted that in respect to a chair conformation of the six-membered ring, atom N2 can be situated in the equatorial and axial positions of the N1 atom; however, in this and previously reported salts, only the axial disposition of the N2 atom is observed. This is in accord with our B3LYP/6-31++G calculations of standard Gibbs free energies of reactions leading to the formation of various products. We established that the axial stereoisomer is more stable than the equatorial one : it is equal to 0.401 (1) Å, and the two molecular conformers (corresponding to different directions of this carbon atom shifted in respect to the N—N=C—C mean plane) are equally present in this centrosymmetric crystal. However, the previously reported crystal structure  plane are observed  and 295.0 (2) K Tables 1 and 2 ▸ PLATON package . Melting points were determined on a TPL apparatus . The progress of the reaction was monitored using Sorbfil TLC plates  coated with CTX-1A silica gel, grain size 5–17 µm, UV-254 indicator. The spots were developed in I2 vapours and in the UV light of a chromatoscope (λ = 254 nm) TSX 254/365 (PETROLASER). The eluent for the analysis was a mixture of EtOH:benzene = 1:1 + a few drops of a 25% aqueous solution of NH3. Microanalysis according to the Pregl method was carried out on an elemental analyser with the absorption of CO2 and O2 isolated during combustion with a two-degree repetition of combustion.IR spectra were obtained on a Thermo Scientific Nicolet 5700 FTIR instrument in KBr pellets. 1) was performed in dried CHCl3 with tosyl chloride in the presence of DIPEA, purchased from Sigma–Aldrich and used without purification. Solvents for synthesis, recrystallization and TLC analysis  were purified according to the standard procedures described for each solvent.The tosylation of β-(thiomorpholin-1-yl)propioamidoxime :1) in 40 ml of CHCl3, 0.92 ml (0.0053 mol) of DIPEA were added. The reaction mixture was cooled to 272 K, and a solution of 1.01 g (0.00530 mol) of tosyl chloride in 4 ml of CHCl3 was added dropwise under stirring. The reaction mixture was stirred for 1 h at room temperature and was then heated and stirred at the reflux temperature of CHCl3 for 8 h until the completion of the reaction, the progress of the reaction being monitored by TLC. The formed white precipitate of the chloride hydrate 3 was filtered off and recrystallized from 2-PrOH solution. The yield of 3 was 1.01 g (84%), m.p. 575 K, Rf 0.08. Found, %: C 37.67, H 7.49. C7H16ClN3OS. Calculated, %: C 37.24, H 7.14. IR, cm−1: 1659 (ν C=N); 1612 ; 670 (ν S—C); 3135, 3230, 3380, 3384 . 1H NMR, δ, ppm : 2.88 , 3.14 , 3.14 §, 3.37 , 3.62 , 3.74 , 3.88 , 7.48 . The signals for the methylene protons of the N(+)CH2CH2 group in 3 coincide with the signals of the S(CHax)2 group at δ 3.14 ppm.To a solution of 1.00 g (0.0053 mol) of β-(thiomorpholin-1-yl)propioamidoxime (Uiso(H) = 1.5Ueq(O) and 1.2Ueq(X) for the other atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022000111/hb8000sup1.cifCrystal structure: contains datablock(s) 3_LT, 3_RT. DOI: 10.1107/S2056989022000111/hb80003_LTsup2.hklStructure factors: contains datablock(s) 3_LT. DOI: 10.1107/S2056989022000111/hb80003_RTsup3.hklStructure factors: contains datablock(s) 3_RT. DOI: 2106799, 2106798CCDC references:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


While amyloid-β4–15 the next most abundant N-terminus found in Alzheimer’s disease brain, was only ∼50% isomerized at Asp-7 in Alzheimer’s disease. Further investigations into different biochemically defined amyloid-β-pools indicated a distinct pattern of accumulation of extensively isomerized amyloid-β in the insoluble fibrillar plaque and membrane-associated pools, while the extent of isomerization was lower in peripheral membrane/vesicular and soluble pools. This pattern correlated with the accumulation of aggregation-prone amyloid-β42 in Alzheimer’s disease brains. Isomerization significantly alters the structure of the amyloid-β peptide, which not only has implications for its degradation, but also for oligomer assembly, and the binding of therapeutic antibodies that directly target the N-terminus, where these modifications are located.Plaques that characterize Alzheimer’s disease accumulate over 20 years as a result of decreased clearance of amyloid-β peptides. Such long-lived peptides are subjected to multiple post-translational modifications, in particular isomerization. Using liquid chromatography ion mobility separations mass spectrometry, we characterized the most common isomerized amyloid-β peptides present in the temporal cortex of sporadic Alzheimer’s disease brains. Quantitative assessment of amyloid-β N-terminus revealed that > 80% of aspartates (Asp-1 and Asp-7) in the N-terminus was isomerized, making isomerization the most dominant post-translational modification of amyloid-β in Alzheimer’s disease brain. Total amyloid-β et al. report more than 80% of amyloid β N-terminus accumulating in sporadic Alzheimer’s disease is isomerized at Asp-1 and Asp-7 using ion mobility mass spectrometry. The discovery has implications for drug discovery, particularly immunotherapeutic strategies that often target the N-terminus of the peptide.Mukherjee   Analytical chromatographic separation of N-terminal isomers and epimers of Aβ and their simultaneous characterization using MS/MS fragmentation techniques have been investigated.,The antibodies currently in clinical trials target multiple forms (soluble oligomeric and insoluble fibrils) of Aβ due to their potential roles in the pathogenesis and disease progression.1–15 and its associated modified isomers. We also quantified the total levels of the most abundant isomers of Aβ4–15. As an extension of the previously reported biochemical method,42 and Aβ40post-mortem brains is partitioned in the insoluble/fibrillar and detergent soluble phase, while only <7% is extractable as vesicular and soluble.,1–15 and Aβ4–15 in the insoluble/fibrillar and membrane pool, with a comparatively lower extent of isomerization in the vesicular and soluble pools. This data allowed us to estimate the accurate biochemical identity and distribution of the spontaneously isomerized Aβ in post-mortem brain. This PTM is intricately associated with slow turnover rates and degradation of Aβ which accumulates over decades in sporadic Alzheimer’s disease.In this article, we have identified, characterized and quantified the most common isomers of Aβ isoforms extracted from the temporal cortex of Alzheimer’s disease brains by using liquid chromatography (LC) coupled to drift tube IMS-QTOF MS. In particular, we determined Alzheimer’s disease-specific changes of the Aβ N-terminal pool in comparison to age-matched control brains; we report the total levels of Aβ2CO3, Tris buffers were purchased from Merck-Sigma or ThermoFischer Scientific. EDTA-free protease inhibitors from Roche. Bond-Breaker™ tris(2-carboxyethyl)phosphine (TCEP) Solution, neutral pH was from ThermoFischer Scientific. MS-grade metalloprotease LysN from Grifola frondosa, and dithiothreitol (DTT) were purchased from ThermoFischer Scientific. Biomasher were purchased from Omni International. The MS vials, Advanced Bio Peptide Mapping C18 Column  and ESI low concentration tune mix used for instrument calibration were obtained from Agilent Technologies . Oasis HLB µElution 96 well-plates were purchased from Waters. Affinity purification was performed using PureProteome NHS FlexiBind Magnetic Beads from Millipore. Stable isotope standards (SIS) of Aβ peptides DAEF(R + 10)HDSGYEVHHQ, F(R + 10)HDSGYEVHHQ, (K + 8)GAIIGLMVGGVV, (K + 8)GAIIGLMVGGVVIA and K(+8)LVFFAEDVGSN were purchased from New England Peptides  and their concentration determined by amino acid analysis. Stock solutions of SIS Aβ peptides were prepared in 2% ACN, 0.05% TFA to a final concentration of 200 fmol/µL and stored at −20˚C. All the isomerized Aβ peptide standards were commercially synthesized and purchased from JPT Peptide Technologies (Germany). All the SIS isomeric Aβ peptides were resuspended in 30% ACN, 0.1% FA at 0.2 nmol/µL which were subsequently diluted to ∼2 pmol/µL in 15% ACN, 0.1% FA and stored at −20˚C.All LC-MS grade solvents, acetonitrile (ACN), formic acid (FA), trifluoroacetic acid (TFA), acetic acid, isopropanol and urea, thiourea, N, N-Bis(2-hydroxyethyl)glycine (Bicine), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), iodoacetamide (IAA), tri-ethyl ammonium bicarbonate buffer, NaCl, Nan = 9), where the number of plaques and tangles were histopathologically analyzed and well below the cut-off values for Alzheimer’s disease. No other major neuropathological disease was present. Alzheimer’s disease brains (n = 11) met the standard criteria for Alzheimer’s disease neuropathological diagnosis . In detail, the cohort consisted of age-matched healthy control brains . Sections were deparaffinised, endogenous peroxidase blocked with 5% aqueous hydrogen peroxide (5 min), treated (5 min) with 98–100% FA (Scharlau AC10852500), rinsed and immersed in Tris buffer (0.5 M pH 7.6). Sections were incubated in a 1/100 dilution of Dako anti-amyloid antibody (MO872—clone 6 F/3D) for 60 min at room temperature. Positively labelled Aβ was detected with a peroxidase labelled streptavidin/biotin system (Dako K0675) with a diaminobenzidine chromogen. Sections were counterstained with Harris’s haematoxylin, dehydrated and cover-slipped for imaging. Low and high magnification images were obtained with a Leica ICC50 HD camera on a Leica DM 750 binocular microscope.-mortem brain tissue was processed as previously described with some modifications.g for 1 min at room temperature. To the bio-mashed tissue, Tris-buffered saline  containing EDTA-free protease inhibitors (Roche) was added at a ratio of 1:4 . This solution was transferred to ultracentrifuge tubes (Beckman Coulter) and centrifuged (Optima MAX-XP from Beckman Coulter) at 100 000 g for 30 min at 4°C. The supernatant was collected, referred to as TBS fraction henceforth, and stored on ice until freezing.Hemisected freshly frozen post2CO3 pH 11  and incubated for 20 min on ice before another ultracentrifugation step at 100 000 g was carried out for 30 min at 4°C. The supernatant containing peripheral membrane and vesicular material was recovered into an Eppendorf tube, referred to as Na2CO3 fraction.The resulting pellet was then resuspended in 100 mM Na2CO3 fractionation was resuspended with urea−detergent buffer  and spun at 100 000 g for 30 min at 4°C. The supernatant was aspirated out, referred to as urea−detergent fraction. These three biochemical fractions were then snap frozen in liq. N2 and stored at −80°C until further processing.The pellet resulting from Nag for 15 min at 4°C and supernatant was collected. The FA fractions  were aliquoted into 10 µL portions and snap frozen in liquid N2, freeze dried in a lyophilizer and stored at −80°C. A summary of the biochemical fractionation procedure can be found in The residual pellet was finally incubated in 200 µL 70% glass-distilled FA (GDFA) for 2 h at room temperature in a fume hood. The FA fractions were spun at 13 200 −detergent fractions were re−suspended/diluted to 100 µL in 100 mM tri-ethyl ammonium bicarbonate buffer (TEAB), pH 8.5. Next, the samples were reduced by incubating with dithiothreitol (DTT) to a final concentration of 20 mM at 37°C for 30 min, followed by alkylation using 25 mM iodoacetamide (IAA) in the dark for another 30 min. The samples were then diluted to 200 µL with 100 mM TEAB buffer, pH 8.5 and digested overnight by incubation at 37°C after adding LysN metalloprotease at enzyme: protein ratio of 1:100. The same in-solution digestion process was performed with 50 µL for the Na2CO3 and 100 µL for the TBS fractions. The Na2CO3 fraction was diluted to 100 µL and the TBS fraction to 170 µL with 8 M urea, 100 mM TEAB buffer (pH 8.5), respectively. Sample reduction and alkylation were carried out as described above. Finally, the two fractions were diluted to 250 µL for proteolytic digestion with LysN. All the proteomic sample processing was performed at pH 8.5. The digestion reaction was quenched by adding 10% FA to a final concentration of 0.1%. The FA and urea−detergent samples were then spiked with 10 µL of SIS Aβ peptides mixture (200 fmol/µL of Aβ NEP peptides), while only 5 µL was spiked into the Na2CO3 and TBS samples. The acidified samples were finally loaded onto an Oasis HLB µElution 96 well-plate (Waters). The wells were washed with 250 µL of 0.1% FA, followed by 250 µL of 5% methanol, 0.1% FA. The peptides were finally eluted with two sequential washes of 25 µL of 75% ACN, 0.1% FA. The eluent was lyophilized and stored at −20°C until further processing. The FA and urea−detergent samples were re-constituted in 25 µL of 2% ACN, 0.05% TFA, vortexed for 30 min on ice and sonicated for 2 min. The re-constituted samples were centrifuged at 10 000 g for 5 min and the supernatant was aliquoted in MS vials (Agilent Technologies) for analysis.A total of 10 µL of both lyophilized FA and urea1–40 and Aβ1–42 peptide sequences  was used to generate affinity purified rabbit polyclonal antibodies against Lys-N cleaved Aβequences , i.e. AβEnrichment experiments were performed in a round-bottom 96-well polypropylene plates using the magnetic bead protocol. The NEP Aβ anti-peptide antibodies were coupled to PureProteome NHS FlexiBind Magnetic Beads (Millipore) according to the manufacture’s protocol. At first, the capture efficiencies of the anti-peptide antibodies were determined in a complex background. Lyophilized LysN-digested pooled brain homogenate  was resuspended in 200 µL with PBS, 0.03% CHAPS pH 7.5 buffer along with the 500 fmol of respective SIS Aβ peptides and 1 µg of specific antibody . These aFor the multiplexed experiment, 1 µg of each antibody was added to the sample mixture and 1 M Tris–HCl pH 7.5 to a final concentration of 0.2 mM. To this mixture, 500 fmol SIS Aβ peptides were added. The mixture was incubated overnight at 4°C with shaking at 800 rpm. After overnight incubation the magnetic beads were magnetized, and the supernatant was discarded. Next, the magnetic beads were manually washed three times with 0.1 M ammonium acetate, 0.5 M NaCl, 0.03% CHAPS (pH 7.5) followed by another three washes with 0.1 M ammonium acetate, 15% ACN, pH 7.5. Finally, the captured peptides were eluted from the magnetic beads with 25 µL of 5% acetic acid, 15% ACN with shaking at 600 rpm and 2 min incubation.2CO3/TBS brain fractions for Aβ enrichment.This SISCAPA process was used only on the LysN-digested lyophilized Na18 Column  through ultra-low dispersion kit (5067–5963 Agilent Technologies), maintained at 60°C in thermostatted column compartment (TCC) and eluted at 0.4 mL/min flow rate with the following linear gradient: t (min), % B: 0, 2.5; 5,6; 64, 22; 85, 29; 90, 34; 95, 81; 97, 81; 97, 2.5; stop time, 99 min. The ESI source parameters operating in positive ion mode were as follows; gas temp., 300°C; drying gas, 6 L/min; nebulizer, 35 psi; sheath gas temp., 275°C, sheath gas flow, 12 L/min; Vcap, 4500 V. The peptides were analyzed in the positive 4-bit multiplexing IM-QTOF mode in the m/z range of 290–1700 with a maximum drift time of 50 ms using nitrogen as drift gas, trap fill time of 3.2 ms; trap release time of 0.3 ms, and acquisition rate of 1 IM frame/s. The drift tube was operated with an absolute entrance voltage of 1700 V and an exit voltage of 250 V (drift field 18.529 V/cm) and the trapping funnel RF was set at 150 V. An Agilent ESI-Low Calibration mixture was injected both before the analysis to tune the instrument in the m/z range of 100–1700 and at the start of the worklist to perform single-field Collisional Cross Section (DTCCSN2) recalibration. The drift gas upgrade kit maintained both the drift tube and trap funnel pressure at constant 3.94 ± 0.01 and 3.80 ± 0.02 Torr, respectively, while the drift tube ambient temperature was stable at 23.5 ± 0.3°C across all the acquisition runs.An Agilent 1290 Infinity series UHPLC system coupled to Agilent 6560 Drift Tube Ion Mobility QToF high-resolution MS  was used for UHPLC-ESI-IM-MS separations. 0.1% FA in water (mobile phase A) and 0.1% FA in 100% ACN (mobile phase B) were used as a solvent system. Samples were loaded onto an Agilent Advanced Bio Peptide Mapping C18 Column  maintained at 55°C in TCC and eluted at 0.4 mL/min flow rate with the following gradient, 2.5% B, 0 min; 6% B, 5 min; 9% B, 20 min; 22% B, 25 min; 29% B, 35 min; 34% B, 37 min; 81% B, 38 min; 81% B, 40 min; 2.5% B, 41 min with a post-run equilibration for 2 min. The list of transitions along with their retention times (RT) are presented in 2CO3 SISCAPA samples were injected on to the columns.An Agilent 1200 Infinity series UHPLC system connected to 6495 QQQ  was used for the LC-ESI-QQQ-MRM assay. Mobile phase A consisted of 0.1% FA in water and mobile phase B of 0.1% FA in 100% ACN. Samples were loaded onto an Advanced Bio Peptide Mapping CDTCCSN2 calibration and feature finding was carried out using IM-MS browser and Mass Profiler from MassHunter Suite . Following post-processing, the raw data were imported into Skyline (v4.2) with formula annotations of the targeted peptides added to the method. Data for each peptide was extracted in the software in a MS1 filtering mode1–15, Aβ4–15, Aβ16–27, Aβ28–40 and Aβ28–42 peptides in this study were performed on the first three peaks of the isotope cluster. The peak areas of the endogenous peptides and their heavy analogues  were extracted to derive the light-to-heavy ratios. The absolute quantification was determined by comparing the peak areas of the SIS peptides.The IMS-MS data files collected using 4-bit multiplexing mode were first de-multiplexed using vendor-supplied software without any smoothing applied.R and resolution r are defined as R = td/w and r = 1.18 * (td1 − td2)/(w1 + w2), where td is the drift time of the ion and w is the full peak width at half-maximum (FWHM). To determine statistically significant differences between potential biomarkers , the unpaired independent sample t-test was used, while Pearson’s correlation was used to assess correlation between different cell fractions. For the total amount of isomers and their normalized ratio, adjusted P values were calculated with one-way analysis of variance (ANOVA), corrected for multiple comparison false discovery rate (P < 0.05) with Benjamini–Hochberg correction. The means of most common isomers of Aβ1–15 and Aβ4–15 were summarized as pie charts for Alzheimer’s disease and control brains, respectively, obtained from different biochemical fractions.For the drift tube IMS, the resolving power post-mortem temporal cortex tissue using anti-Aβ immunohistochemistry (IHC) and semi-quantitatively scored by an independent assessor (CAM) after anti-Aβ  staining. The scoring system comprised of; the following four categories: −, absent or not discernible, +, slight; ++, moderate; +++, severe. The semi-quantitative IHC scores were compared with the quantitative results obtained using IMS-MS in this study.The degree of amyloid pathology was assessed in the Patient and post-mortem brain tissue demographics, experimental details of nano-LC-ESI-MS/MS for ETD-PRM, MRM transition list for Aβ SISCAPA on QQQ, age-of-death correlation with absolute quantity of Aβ peptides and the respective isomers are provided in the supporting information. Additional data related to this article may be requested from the authors.1–15, Aβ2–15, Aβ3Glu-15, Aβ4–15 and the two canonical C-terminal peptides, Aβ28–40 and Aβ28–42  would provide the required analytical resolution, even if at modest R ∼ 50, to resolve the N-terminal Aβ isomers/racemers derived from Alzheimer’s disease brains and improve the detection/quantification limits of these complex biological samples. We applied DT-IMS in combination with chromatography and synthetic heavy labelled Aβ standards to characterize the identity of isomerized Aβ1–15, Aβ2–15, Aβ3Glu-15 and Aβ4–15 from Alzheimer’s disease brain.Qualitative bottom-up proteomic identification of Aβ peptides in Alzheimer’s disease brain tissue demonstrated a range of N-terminal truncations including Aβ Aβ28–42 .13 Previ1–15 has two Asp residues (Asp-1 and Asp-7) that can undergo individual isomerization/epimerization events, each peptide having combination of their l/d and/or iso-l/iso-d forms resulting in total 16 Aβ1–15 isomers. Hence, we systematically characterized the liquid chromatography retention time (LC-RT) and DTCCSN2 properties of possible synthetic SIS Aβ1–15 peptides (4+m/z 457.4515 ion and the predicted SIS isomer [M + 4H]4+m/z 459.9535 ion in IMS-MS (1–15 isomer. Based on chromatographic RT and averaged DTCCSN2 (1–15 found in the FA fraction of Alzheimer’s disease brain (with increasing RT) as 1-l, 7-l-Asp (1); 1-iso-l, 7-l-Asp (4); 1-l, 7-iso-l-Asp (5); 1-iso-l, 7-iso-l-Asp (10); 1-iso-d, 7-iso-l-Asp (11); 1-iso-l, 7-iso-d-Asp (12) and 1-iso-d, 7-iso-d-Asp (13). Minor singly enantiomerized Aβ1–15 epimers present in the Alzheimer’s disease FA fraction (with increasing RT) were 1-d, 7-l-Asp (2) and 1-iso-l, 7-d-Asp (3); 1-iso-d, 7-d-Asp (6), 1-d, 7-iso-l-Asp (7) and 1-iso-d, 7-l-Asp (8)  peptide. However, internal Asp-7 isomerization not only influences the RT of the isomerized species compared to the natural 1-l, 7-l-Asp (1) Aβ1–15 peptide, but importantly leads to a significant increase in DTCCSN2  . The DTCtructure  comparedl, 7-l-Asp (1) and 1-d, 7-d-Asp (15) epimers had similar RT (DTCCSN2 606 Å2 (2 (r ∼ 0.4 (R ∼ 60) between 1-l, 7-l-Asp (1) (td = 20.2 ms) and 1-d, 7-d-Asp (15) (td = 19.7 ms) epimers , the presence of both of these species would have led to a DT peak broadening for the endogenous [M + 4H]4+m/z 457.4515 ion. However, DT-IMS results indicated doubly racemized endogenous Aβ1–15 , 7-iso-l-Asp (18) and 7-iso-d-Asp (19) are the primary isomers of Aβ4–15 present in Alzheimer’s disease brain  and 7-iso-d-Asp (23) along with unmodified 7-l-Asp (21) , we predicted the major isomers of AβpGlu3–15 as 7-l-Asp (25), 7-iso-l-Asp (26) and 7-iso-d-Asp (27) with increasing RT   . Based oasing RT . This wa1–15 isomers, internal Asp-7 isomerization of Aβ2–15 led to a larger DTCCSN2 for the [M + 4H]4+m/z 428.6934 compared to unmodified 7-l-Asp (21) peptide. In contrast, the [M + 4H]4+m/z 378.6791 iso-Asp-7 Aβ4–15 peptide isomer was more compact  compared to its unmodified version  (28–40 (DTCCSN2 ∼ 372 Å2) and Aβ28–42 (DTCCSN2 ∼ 406 Å2) were detected in Alzheimer’s disease brain  . Similarse brain .1–15 and Aβ4–15 peptides in each of the four biochemical fractions of Alzheimer’s disease and control brain tissues was estimated using SIS peptides (1–15 was significantly increased in Alzheimer’s disease brains (12-fold) compared to control brains, while 14-fold increase was documented for the total Aβ4–15 , while 15-fold (P = 0.0008) and 4-fold (P = 0.0021) increase were observed in urea-detergent fraction and Na2CO3 fraction (P = 0.0365) elevation of Aβ1–15 in Alzheimer’s disease brains  in FA fraction, ∼25-fold increase (P = 0.0001) in urea-detergent fraction and ∼12-fold increase (P = 0.0004) in Na2CO3 fraction (P = 0.0263) in Aβ4–15 levels in the pooled Alzheimer’s disease TBS fraction  N-terminus in Alzheimer’s disease, reaching almost equal concentration as the BACE1 cleaved Aβ N-terminus (Asp-1), making it the next most abundant N-termini present in Alzheimer’s disease brains.Absolute quantitation of the Aβpeptides . As expel Aβ4–15 . Compariractions . Quantitfraction , Fig. 3Ae brains . Similarfraction , Fig. 3Bfraction . The Aβ4was ∼0.1 . This in1–15 and Aβ4–15 was isomerized in each biochemical fraction in Alzheimer’s disease in comparison to the control tissue. As expected, we observed significant elevation in the total amount of the isomers of Aβ1–15 and isomers of Aβ4–15 in each of the Aβ rich fractions from Alzheimer’s disease tissue  peptide, but statistically significant elevation for the singly isomerized 1-iso-l, 7-l-Asp (4), 1-l, 7-iso-l-Asp (5) and doubly isomerized 1,7-iso-l-Asp Aβ1–15 (10) peptides  peptide with the concomitant statistically significant increase in 1-iso-l-Asp, 7-iso-l-Asp Aβ1–15 (10) isomer in all the biochemical fractions in Alzheimer’s disease   compared to only ∼30% in controls  . These dcontrols .4–15 was elevated in Alzheimer’s disease brain (iso-l-Asp Aβ4–15 (18 in P = 0.0016) and Na2CO3  fractions. Most importantly, there was an increase (∼21%) in the amount of 7-iso-l-Asp Aβ4–15 . In the urea-detergent fraction, we documented only a 3-fold increase (P = 0.0207) of Aβ28–42 (28–42 SISCAPA quantitation in the Na2CO3 fraction indicated a ∼3.5-fold increase (P = 0.0001)  , Fig. 4Cmg brain  comparedractions . Strong ractions , while A7 levels . This wa42/Aβ40 in the biological fluids (CSF/blood) has been shown to inversely correlate with brain amyloid PET imaging,,28–42 and Aβ28–40  was consistent in Alzheimer’s disease patients  evaluation of the isomer ratios of Aβ1–15 and Aβ4–15, with significant elevation in doubly isomerized Aβ1–15 and isomerized Aβ4–15 levels in the insoluble/fibrillar and membrane pools, while the sparsely populated vesicular and soluble Aβ pools have lower proportion of these PTMs; (iv) brain-derived Aβ primarily has Ala-42 as the C-terminus which is significantly increased in Alzheimer’s disease, while Aβ with Val-40 C-terminus is increased in Alzheimer’s disease but does not reach statistical significance compared to control brains.The slowly progressive nature of Alzheimer’s disease with almost 20 years of Aβ accumulation from threshold to the onset of dementia predisposes the depositing Aβ peptide to undergo multiple biochemical changes at the molecular level. We used a quantitative proteomics approach coupled with ion mobility mass spectrometry to unravel the diversity of isomerized Aβ N-termini found in the Alzheimer’s disease brain. Our major findings include (i) characterization of isomerization of the Asp residues (Asp-1 and Asp-7) in four common sequentially truncated N-termini of Aβ found in Alzheimer’s disease brain tissues; (ii) quantitative estimation of the level of Aβ,iso-Asp formation in model peptides is considerably slower  compared to asparagine deamidation/isomerization . In vitro, the N-terminus of Aβ has been documented to undergo such spontaneous isomerization at Asp1 and Asp7 residues.t1/2 ∼ 231 days, Asp-1; t1/2 ∼ 462 days, Asp-7 for Aβ1–40)1–42 deposition),1–15 (one t1/2) indicate that the observed mild Aβ deposit (diffuse plaques) is nearly 8 months old. In contrast, data from the ∼85% isomerized Aβ1–15 (three t1/2) in Alzheimer’s disease  from SH-SY5Y neuroblastoma cell-culture models.,,1–15 derived from Alzheimer’s disease brain is significantly isomerized  compared to soluble Aβ1–15  in age-matched control brains  N-terminus of Aβ is conformationally flexible allowing spontaneous reactions to occur and (ii) contrary to previous reports,,R ∼ 50 for our DT-IMS-MS method, it is not possible to rule out any other amino acid (such as Ser) isomerization on this peptide. Future investigations with techniques like SLIM-IMS providing higher resolution (R > 300),While we documented abundant N-terminal Asp-1 and Asp-7 isomerization/racemization in all the four different biochemical pools in both Alzheimer’s disease and control brains , surprisobserved . The pre,,42 being the dominant neuronal peptide form accumulating in Alzheimer’s disease brain with Aβ40 levels increasing with perivascular amyloidosis.4–42.42 while the vessels had less aggregation prone Aβ40.42 and Aβ40 in biochemically defined pools from the temporal cortex of sporadic Alzheimer’s disease revealed less than 0.01% of total Aβ42 and 0.3% of total Aβ40 are in the soluble cytosolic TBS fraction with the rest being distributed in either the vesicular , membranous  and/or insoluble fully polymerized fibrillar phase . In contrast, total Aβ42 in control brain tissue (0.5 ± 1.8 pmol/mg brain) is much lower concentrated compared to Alzheimer’s disease brain (3.2 ± 1.8 pmol/mg brain). Again, most of the Aβ42 is still partitioned in the membranous (57.2%) and insoluble/fibrillar fraction (41.2%). Drugs that can target the C-terminus,42 for clearance have much better chance to exploit the equilibrium of amyloid deposition in Alzheimer’s disease brain.The data presented here and by others42 in the brain—a key distinguishing signature from freshly generated Aβ. Future studies will be required to understand the role of these isomers in the disease, but this is clearly an important question to answer due to the >80% abundance of isomerized Aβ in Alzheimer’s disease brain.Development of therapeutic drugs and interventions for ameliorating or decreasing the progress of Alzheimer’s disease requires techniques that can accurately and quantitatively monitor the changes in the amyloid biomarkers in the CSF and/or the blood along with PET imaging. Our results show that isomerized Aβ is intricately associated with the accumulation of Aβ1–15, while vesicular and soluble fractions have lower percentage of isomerization. The extent of isomerization on Aβ extracted from Alzheimer’s disease brains is 3 years older than the Aβ found in age-matched control brains. Quantitatively, BACE-1-cleaved Asp-1 N-terminus is present in almost equimolar amounts with Phe-4 truncated N-terminus, an interesting Aβ metabolic by-product of unclear origin. Our data provide the link between older isomerized Aβ and the consequences it might have in the disease aetiology, such as oligomers that diffuse out of these plaques into soluble pool will be neurotoxic due to their inherent resistance to lysosomal degradation. Strategies in designing better immunotherapeutic must take into consideration of the extensive PTMs of the N-terminus of Aβ and specifically target older isomerized Aβ species for better target engagement and clearance.In summary, in this study we have shown that different biochemical pools of Aβ has different amounts of N-terminus isomerization. Insoluble plaques and membrane fractions in Alzheimer’s disease brains have ∼85% isomerized AβBrain Communications online.fcab028_Supplementary_DataClick here for additional data file.


While immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics.111In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy.To introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with A set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets.The online version contains supplementary material available at 10.1186/s12951-022-01272-5. Over the past decade, immunotherapy, in particular blockade of immune checkpoint molecules such as Programmed Death 1 (PD-1) and its ligand PD-L1 using antibodies, has revolutionized the field of cancer therapeutics due to unparalleled responses in a range of cancers . DespiteSeveral preclinical –21 and cHere, we demonstrate that combining our CRISPR/HDR hybridoma engineering technology with chemical synthesis provides a plug-and-play platform to create molecularly defined antibody-based multimodal, multiscale PD-L1 imaging agents. To this end, we functionalized engineered mouse IgG1 (mIgG1) and Fab aPD-L1 site-specifically with a peptide containing both the radionuclide chelator diethylenetriaminepentaacetic acid anhydride (DTPA) and near-infrared (NIR) dye sulfo-Cy5 , while blood concentration and kidney uptake were significantly lower . Site-specifically conjugated isotype-switched mIgG1 with either DTPA alone (mIgG1-IH20) or with the additional fluorophore (mIgG1-IH18) did not show a significant change in blood clearance or liver uptake, compared to randomly labeled WT rIgG2a antibody. Spleen uptake of mIgG1-IH18 was increased compared to mIgG1-IH20 . Duodenum uptake was slightly decreased for IH18- and IH20-labeled antibodies  compared to the MIH5 WT antibody (10.94 ± 0.52% ID/g). Taken together, isotype switching and site-specific conjugation of PD-L1 targeting antibody with IH18 and IH20 was feasible without major changes in the tumor targeting.First, using our molecular toolbox Fig. A and B wIH18, we next aimed to use our platform to produce multimodal imaging conjugates with different pharmacokinetic properties. In addition to mIgG1-IH18, we produced c-terminal sortag-histag carrying aPD-L1 Fab fragments 2+) 659.28, found 659.30. C52H84N16O20S23+ ([M + 3H]3+): 439.85, found 439.94. After purification and lyophilization, IH20 was obtained as a white powder (yield 43%).The resulting IH18 by modification of IH20 with sulfo-Cy5, IH20  was dissolved in DMF (60 mg/mL) and mixed with 1.1 eq.  sulfo-cyanine-5-maleimide  in PBS . This mixture was agitated overnight at RT. The resulting IH18 peptide was purified using RP-HPLC (5–40% ACN/H2O in 35 min), and analyzed using LC–MS. HPLC rt: 27.27. LC–MS (ESI +) m/z calcd for C90H128N20O29S42+ ([M + 2H]2+) 1041.40, found 1041.80. C90H128N20O29S43+ ([M + 3H]3+): 694.60 found: 694.84. After lyophilization, IH18 was obtained as a blue powder .To yield IH18 or IH20 for mIgG1 PD-L1 and 50 eq of IH18 or IH20 for Fab PD-L1. After termination of the reaction with EDTA , the reaction mixture was incubated with 200 µl Ni–NTA beads for 20 min at RT, in order to remove unreacted antibody and Sortase. Beads were separated from the reaction mixture using empty spin columns , and washed twice with wash buffer  and twice with PBS. Reaction mixture and wash fractions were combined and purified using size-exclusion chromatography (SEC), with 1 mM EDTA in PBS as buffer (10 mL/min). Fractions containing the product were combined and concentrated using Amicon Ultra-15 Centrifugal Filter Units, MWCO 10 kDa . Buffer exchange was performed using sterile PBS. Antibody concentration was determined on NanoDrop, using the UV–vis program and measuring at 280 nm for IH20 and at 280 and 646 nm for IH18. Protein purity was assessed under reducing conditions using SDS-PAGE gel electrophoresis (12% acrylamide), comparing starting material, product and product clicked with 5 kDa mPEG-DBCO to verify functionalization of all heavy chains. Per construct, several batches were produced and these batches were combined before performing in vitro and in vivo experiments to guarantee batch uniformity. Quantification of protein purity was achieved using densitometry. Densitometry was performed in ImageJ, and defined as (product bands − background)/(non-product bands − background) × 100%. Purity mIgG1 PD-L1-IH18 = 85%, Fab PD-L1-IH18 = 96%.After optimization, batch sortagging was carried out using 0.5–1.0 mg of antibody (fragment) and 25 eq of 20kDa = 89%.Fab PD-L1-IH18 was conjugated to 20 kDa PEG by adding 10 Eq. (94.0 nmol) of mPEG-DBCO  in PBS (3.33 mM) directly to Fab PD-L1-IH18 in PBS . This mixture was incubated for 90 min at 37 °C, buffer exchange was performed with 0.02 M acetate buffer  using Amicon Ultra-4 Centrifugal Filter Units, MWCO 10 kDa  and subsequently purified using a 1 mL HiTrap SP FF  cation exchange column on an NGC Quest Plus Bio-Rad system operating at 0.5 mL/min. After equilibrating with buffer A for 8 column volumes, a linear gradient of 0.5 M NaCl in buffer A was applied (0–0.5 M NaCl in 30 column volumes). PEGylated Fab PD-L1-IH18 were typically eluted after about 8 column volumes (0.15 M NaCl). Fractions containing the purified product were concentrated using Amicon Ultra-4 Centrifugal 10 kDa MWCO filter units  and sterile PBS as a buffer, and analyzed by 12% SDS-PAGE. The concentration was determined using Nanodrop. This method was verified as reliable by use of a BCA assay . Quantification of protein purity was achieved by performing densitometry, as described above. Purity Fab PD-L1-IH18-PEG3 in PBS, pH 5.5 were added to each antibody and incubated for 1 h at RT. Non-conjugated p-SCN-DTPA was removed from the reaction mixture by dialysis against 5 L 0.25 M NH4Ac  using Slide-a-Lyzer Cassettes (ThermoFisher Scientific). After dialysis, concentration in all samples was determined via spectrophotometer.To prevent contaminants from disturbing radiolabeling, 1 mg of mIgG1 PD-L1-srt-his and rIgG2a WT antibodies were dialyzed against 5 L sterile PBS  using Slide-a-Lyzer Cassettes (ThermoFisher Scientific). Subsequently, a 15 eq. of S-2-(4-Isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid  and 1/10 reaction volume 1 M NaHCO111In  in 0.5 M MES buffer (pH 5.4) for 20 min at room temperature under metal-free conditions as described previously [111In was complexed by adding 50 mM EDTA to a final concentration of 5 mM. Labeling efficiency was determined using thin-layer chromatography on silica gel chromatography strips (Agilent Technologies), using 0.1 M citrate buffer  as mobile phase. Samples with a labeling efficiency below 95% were purified using a PD-10 column  eluted with PBS. Radiochemical purity of 111In-labeled antibody-IH18 conjugates exceeded 95% in all experiments.Antibody-IH18 conjugates were incubated with eviously . Non-che20kDa or a mIgG1 isotype control . Concentrations of these constructs ranged from 0.1 pM to 1000 nM for antibodies and 0.4 pM to 6000 nM for Fabs. After 20 min of incubation at 4 °C, 30 μL of commercially available MIH5 PD-L1-PE  was added . After another 30 min of incubation at 4 °C, cells were washed and resuspended in PBA and measured on FACS. The IC50 was defined as the antibody-conjugate concentration that was required to inhibit binding of the commercially available fluorescently-labeled antibody by 50%.30,000 Renca cells were seeded per well in a 96-well V-bottom plate, and stained with 30 μl of mIgG1 PD-L1-IH18, Fab PD-L1-IH18, Fab PD-L1-IH18-PEG2. Non-specific binding and uptake were determined by co-incubation with 16 nM unlabeled WT PD-L1 (rIgG2a). After incubation, cells were washed and incubated with acid wash buffer  for 10 min at 37 °C to remove the membrane-bound fraction of the antibody conjugates. Cells were washed with PBS, and the acid wash and PBS were combined into one fraction containing the membrane-bound activity. Finally, cells containing the internalized activity were lysed with 0.1 M NaOH and harvested. Samples were measured in a gamma counter  to determine the internalized and membrane-bound activity.Renca cells were cultured to confluency in 6 wells plates and incubated for 1, 3 or 24 h with 105 pM radiolabeled mIgG1, 323 pM Fab or 86 pM PEGylated Fab in RPMI-1640 containing 1% BSA at 37 °C in a humidified atmosphere with 5% CO6 4T1 cells (ATCC) by injection into the mammary fat pad. Mice were block-randomized across the experimental groups based on tumor size. Experiments started when average tumor size reached 0.3 cm3.All experiments were performed in accordance with the revised Dutch Act on Animal Experimentation (2014) approved by the central authority for scientific procedures on animals and animal welfare body of the Radboud University, Nijmegen. Mice were housed in individually ventilated cages with a filter top  under pathogen-free conditions with cage enrichment present , and were fed and watered ad libitum. Biotechnicians were blinded to all groups and compounds administered. Studies were performed with 6–8 weeks old BALB/c mice . In all experiments mice received 1 × 10111In-labelled tracer . At 1, 2 and 24 h after injection with Fab PD-L1-IH18 or Fab PD-L1-IH18-PEG20kDa, or 4, 24 and 72 h for mIgG1, mice were sacrificed using CO2/O2-asphyxiation. To determine the biodistribution of each construct, tumor and normal tissues  were harvested, weighed and measured in a gamma counter, as well as 1% standards of the injected dose. Values are reported as percentage injected dose per gram (%ID/g).For each construct, 3 groups of 5 mice received an intravenous injection of 0.4 pmol 111In-labelled tracer . For Fab, scans were acquired at 4 h and 24 h post-injection (p.i.), for Fab-PEG at 4 h, 24 h and 48 h, and the full length antibodies were imaged at 24 and 72 h p.i.. Images were acquired for 45 min under general anesthesia  with the U-SPECT-II/CT  using a 1.0 mm diameter pinhole mouse high sensitivity collimator, followed by CT scan  for anatomical reference. Scans were reconstructed with MILabs reconstruction software using a 16-subset expectation maximization algorithm, with a isotropic voxel size of 0.2 mm and 1 iteration. SPECT/CT scans were analyzed and maximum intensity projections (MIP) were created using Inveon Research Workplace software (Siemens).For each construct, mice (n = 5/group) received 0.4 pmol Snap frozen, unstained tumor section (10 µm) were scanned on a fluorescence flat-bed scanner at pixel resolution of 4 µm and with an excitation/filter setting of 690/700 nm, quality setting ‘high’  and subsequent sections were exposed to a Fujifilm BAS cassette 2025 (Fuji Photo Film) for 3 days. Phosphor luminescent plates were scanned using a phosphor imager  at a pixel size of 25 × 25 µm. Images were acquired with Aida Image software.PD-L1 expression in tumors was determined by chromogenic detection using fresh frozen tissue sections fixed in PBS 45% acetone 10% formalin. In short, sections were pre-incubated with 10% normal rabbit serum, endogenous biotin and avidin were blocked, and endogenous peroxidase activity was blocked with 3% H2O2. Subsequently, anti–mPD-L1  was applied and tissues were incubated overnight at 4 °C. Next, sections were incubated with biotinylated rabbit anti-goat IgG  followed by incubation with avidin–biotin-enzyme complexes . Finally, 3’,3’-diaminobenzidine (DAB) was used to develop the tumor sections prior to mounting slides.Furthermore, fluorescence detection was performed to co-localize mIgG1 PD-L1-IH18 with PD-L1 expression. Fresh frozen tissue sections were fixed using buffered formalin-acetone fixative and pre-incubated with 10% normal donkey serum or 10% normal goat serum. Subsequently, slides were incubated overnight at 4 °C with anti-mPD-L1  or isotype control goat IgG . Next, donkey-anti-goat-Alexa488  was applied for 30 min and DAPI  for 2 min at RT. Sections were mounted with fluoromount  and fluorescence imaging was performed with a Leica DMI6000 epi-fluorescence microscope fitted with a 63 × 1.4 NA oil immersion objective.Statistical analyses were performed using GraphPad Prism version 5.03  for Windows. Data are presented as mean ± standard deviation. Differences in uptake of the radiolabeled tracers were tested for significance using a one-way ANOVA with a Bonferroni test. A p-value below 0.05 was considered significant.Additional file 1. Supplementary figures and tables.