Source 1primary paper2020UNC Libraries
Toolkit/isothermal titration calorimetry
isothermal titration calorimetry
Also known as: Isothermal titration calorimetry, ITC
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Isothermal titration calorimetry (ITC) is a thermal biophysical assay that quantifies binding-associated heat changes under isothermal conditions. In the cited study, ITC was used to support thermodynamic analysis of binding between CIB1 and α-integrin cytoplasmic tails.
Usefulness & Problems
Why this is useful
ITC is useful for experimentally characterizing biomolecular interactions through direct calorimetric readout of heat released or absorbed during binding. In the cited work, it provided thermodynamic support for analysis of CIB1 interactions with α-integrin cytoplasmic tails.
Problem solved
This assay helps address the problem of determining whether CIB1 binds α-integrin cytoplasmic tails and characterizing the thermodynamic basis of those interactions. The supplied evidence links ITC measurements to conclusions about hydrophobic interaction-driven binding and dependence on residues in the CIB1 consensus binding site.
Problem links
provides experimental confirmation of direct Teniposide-STING interaction
LiteratureIt helps distinguish direct STING binding from purely indirect pathway activation.
Source:
It helps distinguish direct STING binding from purely indirect pathway activation.
Published Workflows
Objective: Identify and validate novel STING ligands, leading to selection and mechanistic characterization of Teniposide as a direct STING agonist candidate.
Why it works: The workflow combines broad in silico identification of candidate ligands with biochemical confirmation of direct binding, mutant-based specificity validation, computational binding-mode analysis, and pathway-level functional testing.
direct binding to the STING cytosolic domainactivation of STING-dependent IFN-b2 signaling independent of cGAS and IFI16high-throughput virtual screeningisothermal titration calorimetrycomputational dockingmolecular dynamics simulation
Stages
- 1.High-throughput virtual screening for potential STING ligands(in_silico_filter)
To identify candidate STING ligands before experimental testing.
Selection: Potential STING ligand identification
- 2.Biochemical confirmation of direct STING binding(secondary_characterization)
To experimentally confirm that the selected compound directly interacts with STING.
Selection: Direct binding of Teniposide to STING's cytosolic domain by ITC
- 3.Mutant-based binding validation(confirmatory_validation)
To validate that the observed binding depends on a Teniposide-sensitive STING interface.
Selection: Loss of binding with a STING double mutant unable to bind Teniposide
- 4.Computational binding-mode characterization(functional_characterization)
To characterize how Teniposide may bind STING after direct interaction was established experimentally.
Selection: Docking and molecular dynamics characterization of the Teniposide-STING binding mode
- 5.Functional signaling validation(confirmatory_validation)
To show that direct binding corresponds to pathway activation and to distinguish the mechanism from canonical upstream dsDNA-sensor activation.
Selection: Activation of the IFN-b2 signaling pathway in a STING-dependent, cGAS/IFI16-independent manner
Steps
- 1.Run high-throughput virtual screening against STING and select Teniposidescreen-selected candidate ligand
Identify potential STING ligands for downstream validation.
The source uses virtual screening as the initial candidate-narrowing step before experimental binding and signaling assays.
- 2.Confirm direct Teniposide binding to the STING cytosolic domain by ITCcandidate ligand and binding assay
Experimentally test whether the selected compound directly binds STING.
This step follows virtual screening to replace prediction with direct biochemical evidence.
- 3.Validate binding specificity using a STING double mutant unable to bind Teniposidecandidate ligand and negative-control STING construct
Test whether Teniposide binding depends on a specific STING binding interface.
The source places mutant validation after direct binding confirmation to strengthen specificity of the interaction claim.
- 4.Model the Teniposide-STING binding mode by docking and molecular dynamicsmodeled ligand
Characterize the likely binding mode after experimental binding was established.
The source uses computational analysis after biochemical confirmation to interpret how Teniposide may engage STING.
- 5.Test whether Teniposide activates IFN-b2 signaling in a STING-dependent and cGAS/IFI16-independent mannertested agonist candidate
Determine whether direct binding corresponds to functional STING pathway activation and whether the mechanism is independent of upstream dsDNA sensors.
This downstream functional test establishes biological relevance after candidate selection, direct binding confirmation, and binding-mode analysis.
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete measurement method used to characterize an engineered system.
Techniques
Functional AssayTarget processes
No target processes tagged yet.
Input: Thermal
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: multi component delivery burdenoperating role: sensorswitch architecture: multi component
The assay operates under isothermal conditions and uses calorimetric detection of binding-associated heat changes. The provided evidence does not specify instrument model, sample concentrations, buffer conditions, protein preparation, or analysis workflow.
The supplied evidence is limited to a single study context involving CIB1 and α-integrin cytoplasmic tails. No implementation parameters, performance metrics, sensitivity limits, throughput characteristics, or independent replication are provided in the evidence.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Source 2primary paper2025MED
Ranked Claims
A STING double mutant abolished Teniposide binding.
the STING double mutant abolished binding
Binding between CIB1 and α-integrin cytoplasmic tails is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Binding between CIB1 and α-integrin cytoplasmic tails is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Binding between CIB1 and α-integrin cytoplasmic tails is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Binding between CIB1 and α-integrin cytoplasmic tails is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Binding between CIB1 and α-integrin cytoplasmic tails is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Binding between CIB1 and α-integrin cytoplasmic tails is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Binding between CIB1 and α-integrin cytoplasmic tails is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Other α-integrin cytoplasmic tails compete with the αIIb cytoplasmic tail for binding to CIB1 in vitro.
solid-phase competitive binding assays showing that other α-integrin CTs compete with the αIIb CT for binding to CIB1 in vitro
Other α-integrin cytoplasmic tails compete with the αIIb cytoplasmic tail for binding to CIB1 in vitro.
solid-phase competitive binding assays showing that other α-integrin CTs compete with the αIIb CT for binding to CIB1 in vitro
Other α-integrin cytoplasmic tails compete with the αIIb cytoplasmic tail for binding to CIB1 in vitro.
solid-phase competitive binding assays showing that other α-integrin CTs compete with the αIIb CT for binding to CIB1 in vitro
Other α-integrin cytoplasmic tails compete with the αIIb cytoplasmic tail for binding to CIB1 in vitro.
solid-phase competitive binding assays showing that other α-integrin CTs compete with the αIIb CT for binding to CIB1 in vitro
Other α-integrin cytoplasmic tails compete with the αIIb cytoplasmic tail for binding to CIB1 in vitro.
solid-phase competitive binding assays showing that other α-integrin CTs compete with the αIIb CT for binding to CIB1 in vitro
Other α-integrin cytoplasmic tails compete with the αIIb cytoplasmic tail for binding to CIB1 in vitro.
solid-phase competitive binding assays showing that other α-integrin CTs compete with the αIIb CT for binding to CIB1 in vitro
Other α-integrin cytoplasmic tails compete with the αIIb cytoplasmic tail for binding to CIB1 in vitro.
solid-phase competitive binding assays showing that other α-integrin CTs compete with the αIIb CT for binding to CIB1 in vitro
Docking models predicted that multiple α-integrin cytoplasmic tails can bind the same hydrophobic binding pocket on CIB1.
We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics.
Docking models predicted that multiple α-integrin cytoplasmic tails can bind the same hydrophobic binding pocket on CIB1.
We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics.
Docking models predicted that multiple α-integrin cytoplasmic tails can bind the same hydrophobic binding pocket on CIB1.
We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics.
Docking models predicted that multiple α-integrin cytoplasmic tails can bind the same hydrophobic binding pocket on CIB1.
We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics.
Docking models predicted that multiple α-integrin cytoplasmic tails can bind the same hydrophobic binding pocket on CIB1.
We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics.
Docking models predicted that multiple α-integrin cytoplasmic tails can bind the same hydrophobic binding pocket on CIB1.
We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics.
Docking models predicted that multiple α-integrin cytoplasmic tails can bind the same hydrophobic binding pocket on CIB1.
We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics.
Key residues in the CIB1 binding site on αIIb are well conserved across α-integrin cytoplasmic tails, enabling delineation of a consensus binding site I/L-x-x-x-L/M-W/Y-K-x-G-F-F.
A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site on αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F).
Key residues in the CIB1 binding site on αIIb are well conserved across α-integrin cytoplasmic tails, enabling delineation of a consensus binding site I/L-x-x-x-L/M-W/Y-K-x-G-F-F.
A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site on αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F).
Key residues in the CIB1 binding site on αIIb are well conserved across α-integrin cytoplasmic tails, enabling delineation of a consensus binding site I/L-x-x-x-L/M-W/Y-K-x-G-F-F.
A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site on αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F).
Key residues in the CIB1 binding site on αIIb are well conserved across α-integrin cytoplasmic tails, enabling delineation of a consensus binding site I/L-x-x-x-L/M-W/Y-K-x-G-F-F.
A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site on αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F).
Key residues in the CIB1 binding site on αIIb are well conserved across α-integrin cytoplasmic tails, enabling delineation of a consensus binding site I/L-x-x-x-L/M-W/Y-K-x-G-F-F.
A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site on αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F).
Key residues in the CIB1 binding site on αIIb are well conserved across α-integrin cytoplasmic tails, enabling delineation of a consensus binding site I/L-x-x-x-L/M-W/Y-K-x-G-F-F.
A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site on αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F).
Key residues in the CIB1 binding site on αIIb are well conserved across α-integrin cytoplasmic tails, enabling delineation of a consensus binding site I/L-x-x-x-L/M-W/Y-K-x-G-F-F.
A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site on αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F).
Approval Evidence
2 sources1 linked approval claimfirst-pass slugs isothermal-titration-calorimetry, isothermal-titration-calorimetry-for-teniposide-sting-binding
Direct binding of Teniposide to STING's cytosolic domain was confirmed via isothermal titration calorimetry (ITC).
Source:
Isothermal titration calorimetry measurements indicated
Source:
binding mechanismsupports
Binding between CIB1 and α-integrin cytoplasmic tails is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site.
Source:
Comparisons
Source-backed strengths
ITC provides a direct thermal measurement of binding-associated heat changes without requiring an optical reporter in the supplied description. In the cited study, ITC measurements supported mechanistic interpretation of CIB1 promiscuity toward multiple α-integrin cytoplasmic tails.
Compared with cellular thermal shift assay
isothermal titration calorimetry and cellular thermal shift assay address a similar problem space.
Shared frame: same top-level item type; same primary input modality: thermal
Strengths here: appears more independently replicated; looks easier to implement in practice.
Compared with CRISPR/Cas-hybrid assays
isothermal titration calorimetry and CRISPR/Cas-hybrid assays address a similar problem space.
Shared frame: same top-level item type; same primary input modality: thermal
Strengths here: appears more independently replicated; looks easier to implement in practice.
Compared with next-generation sequencing
isothermal titration calorimetry and next-generation sequencing address a similar problem space.
Shared frame: same top-level item type; same primary input modality: thermal
Strengths here: appears more independently replicated; looks easier to implement in practice.
Ranked Citations
- 1.
- 2.