Journal: Nature Communications

Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

doi: 10.1038/s41467-025-59658-6

Figure Lengend Snippet: a Conceptual framework for an SDR assay using terminal fusion protein of interest. The equilibrium between apo ( i ) and ligand-bound protein ( ii ) can be distinguished by the sensor ( iii ) activity, where a reddish color represents a dynamic apo protein relative to a more static blue ligand-bound protein. Created in BioRender. Inglese, J. (2025) https://BioRender.com/26yvpbw . b Key steps in an SDR assay. In this study the assay was performed in a 7 μL volume (4 μL target protein/ligand plus 3 μL detection reagent) permitting 1536-well plate quantitative high-throughput screening, qHTS (see Methods for full protocol). c SDR assay proof-of-concept using a C -terminal NLuc fusion to firefly luciferase (FLuc- C -NLuc, 1 nM), or d an N - or C -terminal fusion of HiBiT to FLuc (FLuc- N- or -C -HiBiT, 10 nM). The left axes indicate RLU generated by the enzyme activity of FLuc with increasing concentrations of FLuc inhibitor PTC124, for a C -terminal NLuc fusion ( c , black circles), or an N - (black circle) or C - (gray circle) HiBiT fusion ( d ) protein. The right axes indicate ligand-dependent SDR RLU, measured from NLuc ( c ) FLuc- C -NLuc (inverted blue triangle) or reconstituted NLuc ( d ) FLuc N - (blue triangle) or C - (blue square) HiBiT, with increasing PTC124 concentration in the presence (solid symbols) or absence (open symbols) of 10 µM ATP. Error bars are SD, n = 6 experiments. e qHTS waterfall plot illustrating concentration response curves (CRCs) obtained for a library of 1,343 compounds enriched for FLuc inhibitor chemotypes. Black CRCs from FLuc enzyme assay and blue CRCs from the 10 nM FLuc- C -HiBiT SDR assay (+ ATP condition). f Representative CRC from ( e ) for indicated clades, where symbols are activity from: SDR, plus ATP (blue solid square), SDR, no ATP (blue open square), and FLuc enzyme (black circle) assays. For full SAR around clade P see Supplementary Fig. . g Comparative SDR activity of clade representative compounds using the 1 nM FLuc- C -NLuc protein with ATP (blue solid triangle), no ATP (blue open triangle), or 1 nM NLuc without ATP (open red circle), or with ATP (solid red circle). Supplementary Data , support ( e − g ). RLU, relative light units; No., number; Cpd., compound. Source data are provided in a Source Data file.

Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

Techniques: Activity Assay, High Throughput Screening Assay, Luciferase, Generated, Concentration Assay, Enzymatic Assay

Journal: Nature Communications

Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

doi: 10.1038/s41467-025-59658-6

Figure Lengend Snippet: a ABL1 kinase domain bound to PD166326 inhibitor (dark blue) and myristic acid (red) (PDB: 1OPK), Nagar B et al. b SDR output of imatinib (blue square) or ATP (blue diamond) binding to 0.5 nM (open symbol) or 10 nM (solid symbol) ABL1- N -HiBiT kinase domain or imatinib inhibition of kinase activity (black circle) using Kinase-Glo Plus reagent (KGP). Error bars represent the SD, n = 3 experiments (Imatinib SDR) or n = 4 experiments (ATP SDR and imatinib kinase activity). c Correlation plot comparing the ABL1 KGP assay pIC 50 vs pSDR 50 (10 nM enzyme without ATP) for all inhibitors identified from the 128-member kinase inhibitor library. Colored symbols reference compounds discussed in Results section. For underlying data see Supplementary Data , , and Supplementary Fig. . d , e SDR output for myristate site ligands asciminib (red solid triangle) and GNF-5 (red open triangle) compared to imatinib (blue square) and nilotinib (open square) for ABL1- C- or - N -HiBiT, respectively. f Effect of allosteric ligands on enzyme catalysis (imatinib, black circle, asciminib, solid red triangle, and GNF-5, open red triangle). Error bars for d , e , and f represent the SEM, n = 2 technical replicates. g Protein kinase A (PKA) bound to H-89 inhibitor (dark blue) (PDB: 1YDT), Engh RA et al. h PKA enzyme activity (black circle) and SDR assay (blue square) CRC for H-89. Error bars represent the SD, n = 3 experiments i Correlation plot comparing the PKA KGP assay pIC 50 to PKA SDR assay pSDR 50 (without ATP). H-89 (blue square) and staurosporine (staur., red circle) indicated. See Supplementary Data and Supplementary Fig. for underlying data and compound information. Supplementary Data – supports panels ( c , i ). Source data are provided in a Source Data file.

Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

Techniques: Binding Assay, Inhibition, Activity Assay

Journal: Nature Communications

Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

doi: 10.1038/s41467-025-59658-6

Figure Lengend Snippet: a Crystal structures of the apo (PDB 5KGL) and ipglycermide Ce-2d bound (PDB 5KGN) iPGM from Yu H et al. Metal ions are identified as purple spheres. b Saturation binding of Ce-2 to 1 nM (black circle) 0.5 nM (gray circle) or 0.1 nM iPGM (open black circle). c Relative detection sensitivity of the SDR assay (blue circle) compared to a functional couple-enzyme assay (gray/black square) for ipglycermide Ce-2 binding C. elegans iPGM. Error bars for ( b , c ) represent the SEM of 2 experiments. d Correlation plot comparing the binding potencies for ipglycermide analogs from either an FP-based competition binding (10 nM iPGM) or SDR assay (0.5 nM iPGM- C -HiBiT) format. The FP assay uses a fluorescein-labeled Ce-2d analog (Ce-2d-FL). e Correlation plot comparing the binding potencies for ipglycermide analogs between B. malayi and C. elegans iPGM orthologs using the SDR assay. Ipglycermides are identified by symbols to the right. Error bars for ( d , e ) represent the SD, n = 3 experiments. RLU, relative light units; FP, fluorescence polarization; Ce, C. elegans ; Bm, B. malayi . Source data are provided in a Source Data file.

Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

Techniques: Binding Assay, Functional Assay, Enzymatic Assay, FP Assay, Labeling, Fluorescence

Journal: Nature Communications

Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

doi: 10.1038/s41467-025-59658-6

Figure Lengend Snippet: a DNA ligase crystal structures illustrating the relative positions of the N - and C -termini, DNA and nucleotide binding sites for the E. coli ligase (MW 75 kDa) complexed with NAD + (PDB 5TT5) from Unciuleac MC et al., with NAD + and DNA (PDB 2OWO) from Nandakumar J et al., and bacteriophage T7 ligase (MW 41.1 kDa) complexed with ATP (1A0I) from Subramanya HS et al. The structure of NAD + is shown. b Agarose gel electrophoresis of Hin dIII-digested λDNA repair by ligases from E. coli and bacteriophage T7 containing N - or C -terminal HiBiT α-peptide. Ligase concentrations used: 1, NEB E. coli ligase, 0.5 U/μL; 2, E. coli N -HiBiT, 1 µM; 3, E. coli C -HiBiT, 100 nM; 4, NEB T7 ligase, 150 U/ μL; 5, T7 N -HiBiT, 100 nM; 6, T7 C -HiBiT, 1 μM. Control ligases were from NEB and used as directed. Gel is representative of 2 replicates. Concentration-response curves obtained for the SDR assay for 0.5 nM E. coli Lig- N -HiBiT (solid circle), 1 nM E. coli Lig- C -HiBiT (open circle) and 1 nM T7 Lig- N / C -HiBiT (solid/open squares, respectively). c , d a 22-mer dsDNA oligo; e , f NAD + ; g , h ATP. Error bars are the SD, n = 3 experiments. Supplementary Table supports panels ( c − h ). kb, kilobase; Lig, ligase. The uncropped gel and source data are provided in a Source Data file.

Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

Techniques: Binding Assay, Agarose Gel Electrophoresis, Control, Concentration Assay

Journal: Nature Communications

Article Title: A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

doi: 10.1038/s41467-025-59658-6

Figure Lengend Snippet: a DHFR bound to folate (red molecular surface) and NADP + (blue molecular surface) (PDB 4M6K) from Bhabha G et al. with N - and C -termini indicated. b Co-factor dependent SDR saturation binding curves for methotrexate (MTX) binding to 5 nM (solid symbol) and 0.5 nM (open symbol) DHFR- C -HiBiT in the presence (square) or absence (circle) of saturating NADPH. c SDR assay concentration response curves for MTX binding to 0.5 nM DHFR- C -HiBiT for various [NADPH]. Data normalized to 32.5 μM MTX response. d Correlation analysis of antifolate chemotherapeutic affinities as determined by a functional DHFR- C -HiBiT assay (100 nM, 75 μM NADPH) versus SDR assay using DHFR- C -HiBiT (0.5 nM, 5 μM NADPH), respectively. e SDR assay concentration response curves for MTX binding in 1:100 cellular lysate from DHFR C -terminus HiBiT gene edited HEK293 cells for indicated NADPH concentrations. Data normalized to vehicle control. f Correlation analysis of antifolate affinities determined by the SDR assay for a 1:100 lysate (~ 0.2 nM enzyme) without added NADPH or with 1 μM NADPH, respectively. The antifolates tested were methotrexate (M), γ-fluoromethotrexate (F), pralatrexate (Pr), aminopterin (A), trimetrexate (T), and pemetrexed (Pm). Error bars represent the SEM, n = 2 technical replicates, representative of 2 ( b ) or 3 ( c ) experiments, or SD, n = 3 experiments ( d , e , and f ). g Aligned minimal energy solution NMR conformer structures of apo (cyan, PDB: 2L28) and holo MTX-bound (raspberry, PDB: 1AO8) L. casei DHFR illustrating the conformational shift induced by the association of MTX (green). h Aligned solution NMR conformer ensemble structures of apo (cyan, 25 conformers, PDB: 2L28) and holo MTX-bound (raspberry, 21 conformers, PDB: 1AO8) L. casei DHFR illustrating the structural dynamics shift in DHFR upon MTX (green) binding as shown by the coalescence of conformers in the ensemble. RLU, relative light units. Source data are provided in a Source Data file.

Article Snippet: The plasmids have the following Addgene IDs: FLuc- N -HiBiT (ID 207117), FLuc- C -HiBiT (ID 207118), FLuc- C -NLuc (ID 234566), human (Hs) DHFR- C -HiBiT (ID 207119), Hs ABL1- N -HiBiT kinase domain (ID 207121), ABL1- C -HiBIT (ID 234568), Hs PKA- N -HiBiT (ID 211377), C. elegans iPGM- C -HiBiT, (ID 207120), B. malayi iPGM- C -HiBiT(ID 218483), E. coli Lig- N -HiBiT (ID 215437), E. coli Lig- C -HiBiT (ID 215438), T7 Lig- N -HiBiT (ID 215479), T7 Lig- C -HiBiT (ID 215441), and NLuc (ID 234567).

Techniques: Binding Assay, Concentration Assay, Functional Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: Inherited human ITK deficiency impairs IFN-γ immunity and underlies tuberculosis

doi: 10.1084/jem.20220484

Figure Lengend Snippet: AR ITK deficiency with TB. (A) Pedigree of the kindreds. Black symbols indicate affected individuals. Arrows indicate the probands. Genotypes for ITK are also shown. M, mutated. (B) Cranial T2-weighted MRI for P1. The high-intensity lesion in the right temporal lobe disappeared after 9 mo of anti-TB therapy (post-tx). (C) Surgical biopsy of an enlarged mesenteric lymph node of P1 showing central caseation on H&E staining and acid-fast bacilli (arrows) on Ziehl–Neelsen staining. (D) A thoracic CT scan of the lung of P3 showing tree-in-bud signs. Radiological improvement was observed after 4 mo of anti-TB therapy. (E) Variants detected by WES. (F) Sanger sequencing of the ITK variants. (G) Population genetics of ITK . The minor allele frequency (MAF) and CADD scores for all non-synonymous variants reported in the gnomAD database are shown. Three homozygous variants found in our private cohort are also shown. The CADD score of 35.0 for the c.496C>T (R166*) allele is well above the MSC of 20.7 ( ; ; horizontal dotted line). (H) Gene-level negative selection. Like other genes with mutations underlying AR IEI, ITK is not under negative selection, as shown by CoNeS . (I) Schematic representation of the ITK protein. PH, pleckstrin homology domain; SH, Src homology domain. Previously reported homozygous or compound heterozygous mutations are also shown.

Article Snippet: The membrane was probed with the following primary antibodies: anti-ITK mAb for the N-terminal epitope (Cat: ab32039; Abcam, Clone: Y401, 1:1,000, 4 °C overnight), anti-ITK mAb for the C-terminal epitope (Cat: 77215; Cell Signaling Technology, Clone: E4X7M, 1:1,000, 4 °C overnight), anti-phospho-PLCγ1 (Y783) rabbit polyclonal antibody (Cat: 2821, 1:1,000; Cell Signaling Technology, 4 °C overnight), HRP-conjugated anti-DDK mAb (Cat: 637311, Clone: L5, 1:2,000; BioLegend, room temperature for 1 h), and HRP-conjugated anti-vinculin mAb (Cat: sc-73614, Clone: 7F9, 1:1,000; Santa Cruz, 4 °C overnight).

Techniques: Staining, Computed Tomography, Sequencing, Selection

Journal: The Journal of Experimental Medicine

Article Title: Inherited human ITK deficiency impairs IFN-γ immunity and underlies tuberculosis

doi: 10.1084/jem.20220484

Figure Lengend Snippet: Analysis of ITK expression and function. (A–C) Studies of ITK alleles in an overexpression system. (A and B) Immunoblotting of the ITK protein with two different mAbs. (C) Phosphorylation of phospholipase C-γ1. (D and E) Immunoblotting for endogenous ITK protein with two different antibodies, on total T lymphocytes from P1, P2, their father, and one healthy control (Ctrl). (F) TCR signaling assay. Total T lymphocytes from P1, P2, their father, and one healthy control were analyzed by immunoblotting for general tyrosine phosphorylation (pY) and for the phosphorylation of phospholipase C-γ1 without stimulation or after 2 or 5 min of stimulation with anti-CD3 mAb. (G) Calcium (Ca 2+ ) influx assay. Total T lymphocytes from P1, P2, their father, and one healthy control were stimulated by TCR crosslinking (with anti-CD3 and anti-IgG mAbs) or ionomycin. Intracellular Ca 2+ concentration was determined with Indo-1. Representative results from at least two independent experiments are shown. Source data are available for this figure: .

Article Snippet: The membrane was probed with the following primary antibodies: anti-ITK mAb for the N-terminal epitope (Cat: ab32039; Abcam, Clone: Y401, 1:1,000, 4 °C overnight), anti-ITK mAb for the C-terminal epitope (Cat: 77215; Cell Signaling Technology, Clone: E4X7M, 1:1,000, 4 °C overnight), anti-phospho-PLCγ1 (Y783) rabbit polyclonal antibody (Cat: 2821, 1:1,000; Cell Signaling Technology, 4 °C overnight), HRP-conjugated anti-DDK mAb (Cat: 637311, Clone: L5, 1:2,000; BioLegend, room temperature for 1 h), and HRP-conjugated anti-vinculin mAb (Cat: sc-73614, Clone: 7F9, 1:1,000; Santa Cruz, 4 °C overnight).

Techniques: Expressing, Over Expression, Western Blot, Phospho-proteomics, Control, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Inherited human ITK deficiency impairs IFN-γ immunity and underlies tuberculosis

doi: 10.1084/jem.20220484

Figure Lengend Snippet: Impaired production of IFN-γ and TNF by ITK-deficient T lymphocytes. (A and B) PBMCs from P1 and P2 (obtained at the ages of 19 and 18 yr, respectively) and their father were stimulated for (A) 18 h or (B) 3 or 5 d with IL-2. Secreted cytokine levels were determined in LEGENDplex assays. Technical duplicates were prepared for (A) P1 or (B) P1, P2, and their father (except P2 for 3 d). (C and D) Expanded T cell blasts (T-blasts) from P1 and P2 were stimulated for 2 h with various reagents and then for an additional 5 h in the presence of a secretion inhibitor. Secreted cytokines were determined with the LEGENDplex assay, and intracellular cytokine levels were determined by flow cytometry. Technical duplicates were prepared for the unstimulated and anti-CD3 mAb-conjugated bead-stimulated samples. (E) Lentiviral rescue. P2’s T-blasts underwent lentiviral transduction with the EV or the WT ITK CDS. Cells were selected on puromycin. Cells were either left unstimulated or stimulated with anti-CD3 mAb-conjugated beads for 5 h. Cytokine production was assessed by flow cytometry. (F) Pharmacological ITK inhibition. Magnetically enriched CD4 + T-blasts from four healthy donors were incubated for 1 h with DMSO or an ITK inhibitor (BMS509744) and were then stimulated for 6 h. Secreted cytokines were determined in LEGENDplex assays. In A–D and F, bars represent the mean and SEM. In A–D, statistical significance was determined for differences between the two ITK-deficient patients on the one hand, and local and familial controls on the other. *, P < 0.05; **, P < 0.01; ***, P < 0.001, and two-tailed Wilcoxon’s rank sum tests with FDR adjustment.

Article Snippet: The membrane was probed with the following primary antibodies: anti-ITK mAb for the N-terminal epitope (Cat: ab32039; Abcam, Clone: Y401, 1:1,000, 4 °C overnight), anti-ITK mAb for the C-terminal epitope (Cat: 77215; Cell Signaling Technology, Clone: E4X7M, 1:1,000, 4 °C overnight), anti-phospho-PLCγ1 (Y783) rabbit polyclonal antibody (Cat: 2821, 1:1,000; Cell Signaling Technology, 4 °C overnight), HRP-conjugated anti-DDK mAb (Cat: 637311, Clone: L5, 1:2,000; BioLegend, room temperature for 1 h), and HRP-conjugated anti-vinculin mAb (Cat: sc-73614, Clone: 7F9, 1:1,000; Santa Cruz, 4 °C overnight).

Techniques: Flow Cytometry, Transduction, Inhibition, Incubation, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Inherited human ITK deficiency impairs IFN-γ immunity and underlies tuberculosis

doi: 10.1084/jem.20220484

Figure Lengend Snippet: Impaired production of IL-9 by ITK-deficient T lymphocytes. (A) T-blasts from P1 and P2 were stimulated for 2 h with various reagents. Secreted cytokines were determined with the LEGENDplex assay. Technical duplicates were prepared for the unstimulated and anti-CD3 mAb-conjugated bead-stimulated samples. (B) Expression of IRF4. T-blasts from P2, her mother, and local controls were either left unstimulated or were stimulated with an anti-CD2/CD3/CD28 mAb cocktail for 24 h and analyzed by flow cytometry. For pharmacological ITK inhibition, T-blasts from controls were cultured in the presence of DMSO or an ITK inhibitor (BMS509744). Bars represent the mean and SEM. Statistical significance was determined for differences between the two ITK-deficient patients on the one hand, and local and familial controls on the other. *, P < 0.05, two-tailed Wilcoxon’s rank sum tests with FDR adjustment. Representative results from two independent experiments are shown.

Article Snippet: The membrane was probed with the following primary antibodies: anti-ITK mAb for the N-terminal epitope (Cat: ab32039; Abcam, Clone: Y401, 1:1,000, 4 °C overnight), anti-ITK mAb for the C-terminal epitope (Cat: 77215; Cell Signaling Technology, Clone: E4X7M, 1:1,000, 4 °C overnight), anti-phospho-PLCγ1 (Y783) rabbit polyclonal antibody (Cat: 2821, 1:1,000; Cell Signaling Technology, 4 °C overnight), HRP-conjugated anti-DDK mAb (Cat: 637311, Clone: L5, 1:2,000; BioLegend, room temperature for 1 h), and HRP-conjugated anti-vinculin mAb (Cat: sc-73614, Clone: 7F9, 1:1,000; Santa Cruz, 4 °C overnight).

Techniques: Expressing, Flow Cytometry, Inhibition, Cell Culture, Two Tailed Test