Positive Cooperative Binding Research Articles

Fluorescence-Based Study of Oligonucleotide Interactions With Recombinant Proteins: Insulin, Interferon α2-β, Somatotropin, and Their Receptors

Background. Oligonucleotides (OLNs) can participate in a wide range of protein-ligand interactions and perform numerous cellular functions by forming structures that enable specific interactions with DNA, RNA, and proteins, what is crucial for many biological processes. Advances in understanding these interactions could lead to the development of new technologies for treating various diseases. However, the mechanism of interaction between proteins and OLNs is complex and still requires detailed study. More research is needed to fully elucidate this process and enhance our understanding of these biomolecular interactions. Objective. The aim of this study was to synthesize, purify, and investigate the interaction of OLNs with recombinant signaling proteins interferon α2-β and insulin with their receptors and somatropin by assessing binding strength using fluorescence spectroscopy. Methods. The interactions were analyzed using the Stern–Volmer equation in both general and modified forms, as well as the Hill equation. OLNs were synthesized via the solid-phase phosphoramidite method, purified through solid-phase extraction, and subsequently verified with a spectrophotometer. Results. Fluorometric titration revealed that OLNs bind to proteins within the medium affinity range, forming non-fluorescent complexes, with the most active interactions observed with shorter OLN. Positive cooperative binding of interferon to G20 and T20, and negative cooperative binding of insulin to C20 and A20, were identified. Additionally, negative cooperative binding of somatropin to C20 was observed. Conclusions. The study demonstrated the interaction between OLNs and recombinant signaling proteins and receptors through various binding mechanisms, which could potentially affect their conformation and biolo­gical activity. These findings have implications for the therapeutic use of OLNs in the context of signaling proteins and receptors.

Dynamics and Thermodynamics Study of the Adsorption of Lysozyme to 2D SnSe Nanoflake Surfaces with Associated Unfolding

AbstractIn this work, we explore the interaction as well as corona formation and interfacial phenomena of lysozyme (LYZ) and 2D SnSe nanoflake. The optical results indicate formation of ground state complex with an apparent association constant of 1.5 × 103 (mol)−1. The dynamics of the in situ interaction exhibited surface reorganization (t2 = 73.5 min) and unfolding, which were more prominent than binding (t1 = 3 min) for tryptophan residues of LYZ. The formations of soft and hard corona of the LYZ structure were clearly observed from HRTEM. The emission quenching exhibited tertiary deformation of the LYZ structure with positive cooperative binding (n > 1) and dynamics of quenching followed the sigmoidal Boltzmann nature of the energy transfer process. The ultrafast lifetime of the bioconjugate varied from 1.9–1.35 ns with a maximum 63% energy transfer efficiency within 300 min of the corona evolution. The decrement of α helix (58.87%–21.78%) and increment of β sheet (5.27%–23.98%) were observed after interaction, with the conversion of large amount of α helix into β sheet. The ITC results showed that the injection heat results followed two‐site binding model, and the exothermic binding was dominate over the interaction. The second phase binding is driven by larger positive entropy, where the first binding site has a stronger association.

Fluorophore-Labeled Pyrrolones Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR1.

In this study, we describe the structure-based development of the first fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor type 1 (CCR1), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in inflammation and immune diseases. Starting from previously reported intracellular allosteric modulators of CCR1, tetramethylrhodamine (TAMRA)-labeled ligands were designed, synthesized, and tested for their suitability as fluorescent tracers to probe binding to the IABS of CCR1. In the course of these studies, we developed LT166 (12) as a highly versatile fluorescent CCR1 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonradioactive and high-throughput manner. Besides the detection of intracellular allosteric ligands by direct competition with 12, we were also able to monitor the binding of extracellular antagonists due to their positive cooperative binding with 12. Thereby, we provide a straightforward and nonradioactive method to easily distinguish between ligands binding to the IABS of CCR1 and extracellular negative modulators. Further, we applied 12 for the identification of novel chemotypes for intracellular CCR1 inhibition that feature high binding selectivity for CCR1 over CCR2. For one of the newly identified intracellular CCR1 ligands (i.e., 23), we were able to show CCR1 over CCR2 selectivity also on a functional level and demonstrated that this compound inhibits basal β-arrestin recruitment to CCR1, thereby acting as an inverse agonist. Thus, our fluorescent CCR1 ligand 12 represents a highly promising tool for future studies of CCR1-targeted pharmacology and drug discovery.

Positive cooperativity in binding: Spectroscopic characterization and dissociation energy of the anisole…(CH4)2 complex

We have previously reported on the spectroscopy and binding energy of the anisole…methane complex, which exhibits a dual mode of binding that involves both CH/O and CH/π interactions. In this work, we seek to examine cooperativity in binding through a study of the isolated anisole-(methane)2 complex, using a combination of experiments that include mass-selected two-color resonant two-photon ionization spectroscopy (2CR2PI), two-color appearance potential (2CAP) measurements, and velocity mapped ion imaging (VMI) augmented with a complementary theoretical characterization. Using 2CAP and VMI, we derive the dissociation energies of the complex in ground (S0), excited (S1), and cation radical (D0) states. The experimental values from the two methods are in excellent agreement and are compared with selected theoretical values calculated using DFT and ab initio methods. The data show that the dissociation energy increases by some 10 % for the second methane relative to the first, with this trend being consistent across all three electronic states, indicating a cooperative binding effect where the initial solvation turns on binding of a second methane onto the opposite face. This lies in contrast to the aniline-(methane)2 complex, where recent studies have shown a negativity cooperativity, and these trends are examined.

Disaggregation-Activated pan-COX Imaging Agents for Human Soft tissue Sarcoma.

Cancer stem cells are pivotal players in tumors initiation, growth, and metastasis. While several markers have been identified, there remain challenges particularly in heterogeneous malignancies like adult soft tissue sarcomas, where conventional markers are inherently overexpressed. Here, we designed BODIPY scaffold fluorescence probes (BD-IMC-1, BD-IMC-2) that activate via disaggregation targeting for cyclooxygenase (COX), a potential marker for CSCs in sarcoma in clinical pathology. Based on their structures, BD-IMC-1 showcased higher susceptibility to disaggregation compared to BD-IMC-2, consistent with their selective interaction with COX. Notably, the BD-IMC-1 revealed positive cooperativity binding to COX-2 at sub-micromolar ranges. Both probes showed significant fluorescence turn-on upon LPS or PMA triggered COX-2 upregulation in live RAW264.7, HeLa, and human sarcoma cell line (Saos-LM2) up to 2-fold increase with negligible toxicity. More importantly, the BD-IMC-1 demonstrated their practical imaging for COX-2 positive cells in paraffin-fixed human sarcoma tissue. Considering the fixed tissues are most practiced pathological sample, our finding suggests a potential of disaggregation activated chemosensor for clinical applications.

Disaggregation‐Activated pan‐COX Imaging Agents for Human Soft tissue Sarcoma

AbstractCancer stem cells are pivotal players in tumors initiation, growth, and metastasis. While several markers have been identified, there remain challenges particularly in heterogeneous malignancies like adult soft tissue sarcomas, where conventional markers are inherently overexpressed. Here, we designed BODIPY scaffold fluorescence probes (BD‐IMC‐1, BD‐IMC‐2) that activate via disaggregation targeting for cyclooxygenase (COX), a potential marker for CSCs in sarcoma in clinical pathology. Based on their structures, BD‐IMC‐1 showcased higher susceptibility to disaggregation compared to BD‐IMC‐2, consistent with their selective interaction with COX. Notably, the BD‐IMC‐1 revealed positive cooperativity binding to COX‐2 at sub‐micromolar ranges. Both probes showed significant fluorescence turn‐on upon LPS or PMA triggered COX‐2 upregulation in live RAW264.7, HeLa, and human sarcoma cell line (Saos‐LM2) up to 2‐fold increase with negligible toxicity. More importantly, the BD‐IMC‐1 demonstrated their practical imaging for COX‐2 positive cells in paraffin‐fixed human sarcoma tissue. Considering the fixed tissues are most practiced pathological sample, our finding suggests a potential of disaggregation activated chemosensor for clinical applications.

Cooperative nucleic acid binding by Poly ADP-ribose polymerase 1

Poly (ADP)-ribose polymerase 1 (PARP1) is an abundant nuclear protein well-known for its role in DNA repair yet also participates in DNA replication, transcription, and co-transcriptional splicing, where DNA is undamaged. Thus, binding to undamaged regions in DNA and RNA is likely a part of PARP1’s normal repertoire. Here we describe analyses of PARP1 binding to two short single-stranded DNAs, a single-stranded RNA, and a double stranded DNA. The investigations involved comparing the wild-type (WT) full-length enzyme with mutants lacking the catalytic domain (∆CAT) or zinc fingers 1 and 2 (∆Zn1∆Zn2). All three protein types exhibited monomeric characteristics in solution and formed saturated 2:1 complexes with single-stranded T20 and U20 oligonucleotides. These complexes formed without accumulation of 1:1 intermediates, a pattern suggestive of positive binding cooperativity. The retention of binding activities by ∆CAT and ∆Zn1∆Zn2 enzymes suggests that neither the catalytic domain nor zinc fingers 1 and 2 are indispensable for cooperative binding. In contrast, when a double stranded 19mer DNA was tested, WT PARP1 formed a 4:1 complex while the ∆Zn1Zn2 mutant binding saturated at 1:1 stoichiometry. These deviations from the 2:1 pattern observed with T20 and U20 oligonucleotides show that PARP’s binding mechanism can be influenced by the secondary structure of the nucleic acid. Our studies show that PARP1:nucleic acid interactions are strongly dependent on the nucleic acid type and properties, perhaps reflecting PARP1’s ability to respond differently to different nucleic acid ligands in cells. These findings lay a platform for understanding how the functionally versatile PARP1 recognizes diverse oligonucleotides within the realms of chromatin and RNA biology.

CAMP binding to closed pacemaker ion channels is cooperative

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.

Dual molecular tweezers extending from a nanohoop.

The field of nanohoops is mature enough that synthetic protocols exists to tune their size, composition (incorporation of heteroaromatic building blocks), connectivity (para versus meta linkages), and solubility in different media (hydrophobic versus hydrophilic). Here, we report an additional dimension incorporating the concept of fullerene tweezers into a nanohoop. The resulting hybrid nanohoop is highly strained at 77 kcal mol-1, possesses a quantum yield of 0.12, emits at 584 nm, and displays a positive cooperative binding for C60 (4K2 ≫ K1).

Discovery of Potent and Selective WDR5 Proteolysis Targeting Chimeras as Potential Therapeutics for Pancreatic Cancer.

As a core chromatin-regulatory scaffolding protein, WDR5 mediates numerous protein-protein interactions (PPIs) with other partner oncoproteins. However, small-molecule inhibitors that block these PPIs exert limited cell-killing effects. Here, we report structure-activity relationship studies in pancreatic ductal adenocarcinoma (PDAC) cells that led to the discovery of several WDR5 proteolysis-targeting chimer (PROTAC) degraders, including 11 (MS132), a highly potent and selective von Hippel-Lindau (VHL)-recruiting WDR5 degrader, which displayed positive binding cooperativity between WDR5 and VHL, effectively inhibited proliferation in PDAC cells, and was bioavailable in mice and 25, a cereblon (CRBN)-recruiting WDR5 degrader, which selectively degraded WDR5 over the CRBN neo-substrate IKZF1. Furthermore, by conducting site-directed mutagenesis studies, we determined that WDR5 K296, but not K32, was involved in the PROTAC-induced WDR5 degradation. Collectively, these studies resulted in a highly effective WDR5 degrader, which could be a potential therapeutic for pancreatic cancer and several potentially useful tool compounds.

Alkali Metal Halide Ion-Pair Binding in Conformationally Dynamic Halogen Bonding Heteroditopic [2]Rotaxanes.

A series of heteroditopic halogen bonding (XB) [2]rotaxanes were prepared via a combination of passive and active metal template-directed strategies. The ability of the [2]rotaxanes to bind alkali metal halide ion-pairs was investigated by extensive 1H NMR titration studies, wherein detailed analysis of cation, anion and ion-pair affinity measurements indicate dramatic positive cooperative enhancements in halide anion association upon either Na+ or K+ pre-complexation. We demonstrate that careful consideration of multiple, parallel and competing binding equilibria is essential when interpreting observed 1H NMR spectral changes in ion-pair receptor systems, especially those which exhibit dynamic behaviour. Importantly, in comparison to XB [2]catenane analogues, these neutral XB heteroditopic [2]rotaxane host systems demonstrated that despite their relatively weaker cation and anion binding abilities, they exhibit a notably higher level of positive cooperativity for alkali metal halide ion-pair binding, highlighting the role of greater co-conformational adaptive behaviour in mechanically-bonded hosts for the purposes of charged species recognition.

Linking ATP and allosteric sites to achieve positive cooperative binding with bivalent kinase inhibitors

Linking ATP and allosteric sites to achieve positive cooperative binding with bivalent kinase inhibitors

Insight into the interactions of albumin with TiO2 nanomaterials and calcium phosphate-based biomaterials by kinetic adsorption and spectroscopic studies

Among key factors determining the fate of biomaterials in vivo are their interactions with blood serum proteins, which can lead to either successful integration or rejection/encapsulation. Although there are a number of studies investigating the interactions between proteins and bioimplants, comparable data for different types of biomaterials are lacking. To fill this gap, the adsorption kinetics and binding interactions of bovine serum albumin (BSA) with calcium phosphates (CaPs), namely hydroxyapatite (HA) and calcium deficient apatite (CaDHA), different TiO2 nanomaterials (TiNMs) presenting various morphologies such as nanoparticles (TiNPs), nanoplatelets (TiNPls), nanotubes (TiNTs) and nanowires (TiNWs), as well as their composites with CaDHA (CaDHA/TiNMs) were investigated. The kinetics of BSA adsorption on all studied materials was best described by pseudo-second order kinetics. The rate coefficient values obtained for the composites were lower than those for CaDHA and the corresponding TiNMs, while the adsorption density was higher for the composites than for CaDHA, except for the composites with TiNTs. Adsorption on TiNWs, CaDHA and all composites involved intraparticle diffusion, which was the rate-limiting step only for CaDHA/TiNTs. Fluorimetric titration experiments revealed that the number of binding sites was in larger than 1, except for TiNTs and TiNWs, indicating positive binding cooperativity. Interestingly, the values of the binding constants were lower for the TiNMs with a higher adsorption rate coefficient. Overall, BSA adsorption on the studied materials proved to be a complex process, which depended on the different surface properties of the adsorbents. Which property had a dominant role depended on the chemical identity of the adsorbent. The obtained comparable data for different types of materials point to the way of modifying their protein adsorption and binding properties.

A Pharmacometrics Model to Characterize a New Type of Target-Mediated Drug Disposition (TMDD) - Nonlinear Pharmacokinetics of Small-Molecule PF-07059013 Mediated By Its High-capacity Pharmacological Target Hemoglobin With Positive Cooperative Binding.

In general, small-molecule target-mediated drug disposition (TMDD) is caused by the interaction of a drug with its high-affinity, low-capacity pharmacological target. In the current work, we developed a pharmacometrics model to characterize a new type of TMDD, where the nonlinear pharmacokinetics (PK) is mediated by a high-capacity pharmacological target with cooperative binding instead of target saturation. The model drug we used was PF-07059013, a noncovalent hemoglobin modulator that demonstrated promising preclinical efficacy to treat sickle cell disease (SCD), and showed complex nonlinear PK in mice with the fraction of unbound drug in blood (fub) decreased with an increase in PF-07059013 concentrations/doses due to the positive cooperative binding of PF-07059013 to hemoglobin. Among the various models we evaluated, the best one is a semi-mechanistic model where only drug molecules not bound to hemoglobin were allowed for elimination, with the nonlinear pharmacokinetics being captured by incorporating cooperative binding for drug molecules bound to hemoglobin. Our final model provided valuable insight on target binding-related parameters, such as the Hill coefficient γ (estimated to be 1.6), binding constant KH (estimated to be 1450 µM), and the amount of total hemoglobin Rtot (estimated to be 2.13 µmol). As the dose selection of a compound with positive cooperative binding is tricky and challenging due to the nonproportional and steep response, our model may be valuable in facilitating the rational dose regimen selection for future preclinical animal and clinical trials for PF-07059013 and other compounds whose nonlinear pharmacokinetics are caused by similar mechanisms.

Amphiphilic perylene diimide-based fluorescent hemispherical aggregates as probes for metal ions

The self-assembly behaviour of a newly synthesized amphiphilic core-positioned thioester appended with carboxylic acid functionalized perylene diimide derivative is studied in different organic solvents. Fluorescent J-type hemispherical aggregates are formed in THF solution. The effect of added metal ions on these fluorescent aggregates is evaluated using spectroscopic techniques, where we found these probes bind selectively to Fe3+ and Ba2+ ions. Two equivalents of Fe3+ ions bind cooperatively to one equivalent of perylene diimide derivative in the hemispherical aggregates with a binding constant of 1.4×107 M−1 and the limit of detection (LOD) was calculated to be 8.66×10-6 M. The positive cooperative binding effect of Fe3+ ions towards hemispherical aggregates equipped with perylene diimide derivatives leads to supramolecular polymerization. Ba2+ ions showed selectivity and sensitivity towards the fluorescent aggregates in THF by quenching the fluorescence intensity completely. The linear Stern-Volmer plot with a Stern-Volmer constant value of 502.6 M−1 signifies the heavy atom effect of Ba2+ ions, leading to fluorescence quenching. The morphological transformation of the fluorescent J-type hemispherical aggregates in the presence of Fe3+ and Ba2+ was studied in detail using electron microscopy.

An NMR portrait of functional and dysfunctional allosteric cooperativity in cAMP-dependent protein kinase A.

The cAMP-dependent protein kinase A (PKA) is the archetypical eukaryotic kinase. The catalytic subunit (PKA-C) structure is highly conserved among the AGC-kinase family. PKA-C is a bilobal enzyme with a dynamic N-lobe, harbouring the Adenosine-5'-triphosphate (ATP) binding site and a more rigid helical C-lobe. The substrate-binding groove resides at the interface of the two lobes. A distinct feature of PKA-C is the positive binding cooperativity between nucleotide and substrate. Several PKA-C mutations lead to the development of adenocarcinomas, myxomas, and other rare forms of liver tumours. Nuclear magnetic resonance (NMR) spectroscopy shows that these mutations disrupt the allosteric communication between the two lobes, causing a drastic decrease in binding cooperativity. The loss of cooperativity correlates with changes in substrate fidelity and reduced kinase affinity for the endogenous protein kinase inhibitor (PKI). The similarity between PKI and the inhibitory sequence of the kinase regulatory subunits suggests that the overall mechanism of regulation of the kinase may be disrupted. We surmise that a reduced or obliterated cooperativity may constitute a common trait for both orthosteric and allosteric mutations of PKA-C that may lead to dysregulation and disease.

Micromycete Responses to Acetone: Electrochemical Express Evaluation of Acetone Metabolism.

The biosensor method has not yet been applied to study the fungus-acetone interaction. The first electrochemical (amperometric) study of Fusarium oxysporum f. sp. vasinfectum cells' responses to acetone was performed to evaluate the initial stages of the metabolism of acetone in the cells of the micromycete. Using a laboratory model of a membrane microbial sensor based on the micromycete cells, it was found that the fungus had constitutive enzyme systems that were involved in acetone transport into fungal cells. The research showed that uninduced by acetone cells had degradative activity against acetone. A positive cooperativity of acetone binding with enzymes initiating acetone degradation was revealed. Oxygen concentration affected the activation of cell enzymes initiating acetone degradation, but the cells' activity in the presence of acetone maintained even at low oxygen concentration. Kinetic parameters (the maximum rate of the cells' response to substrate and the half-saturation constant) of the processes causing the fungal cells' response to acetone were calculated. The results demonstrated the convenience of the biosensor method for assessing the potential of the micromycete as a substrate-degrading culture. In the future, the mechanism of response to acetone for microbial cells will be studied.

Membrane phospholipids induce moderate positive cooperativity in closed pacemaker channels.

Hyperpolarization-activated cyclic nucleotide-gated ion channels (HCN channels) play pivotal role in pacemaking activity in the heart and brain. Previous studies have shown the effects of phospholipids like PIP2 on voltage dependent activation of HCN channels. Similar lipid dependent effects on channel activity have also been reported for other ion channels at an ensemble level. However, there is a lack of information regarding how ligand binding to membrane proteins itself is affected by membrane phospholipids at a molecular level. Here, we probe cAMP binding to full length HCN1 channel reconstituted into nanodiscs containing soy polar lipids to mimic a membrane environment. HCN1 nanodiscs were immobilized on zero mode waveguides (ZMWs) and binding of fluorescently labelled cAMP (fcAMP) was monitored over time using total-internal-reflection-fluorescence microscopy (TIRF). Surprisingly, we observed a moderate positive cooperativity in fcAMP binding to the closed HCN1 channel. The observed bound probability distributions deviated from the expected binomial distribution from an identical and independent binding model. These results are significantly different from the cAMP binding to the closed HCN1 channel in detergent micelles.2 Our results shed new light on the contribution of membrane phospholipids to cooperativity in ligand binding to HCN channels.

Assembly of Metal-Organic Frameworks on Transition Metal Phosphides as Self-Supported Electrodes for High-Performance Hybrid Supercapacitors.

In this work, the composite electrode composed of metal-organic frameworks and transition metal phosphides is first assembled on the nickel foam substrate. The as-prepared NiCo-MOF-74@Ni12P5/NF exhibits excellent performances with ultrahigh specific capacitance (12.8 F/cm2 at 1 mA/cm2), stable charge-discharge rate (82.8%), and excellent cycling stability (reserve 73.90% after 5000 charge and discharge cycles at 30 mA/cm2), which are better than those of NiCo-MOF-74@NF without phosphating treatment of nickel foam. The corresponding hybrid supercapacitor (SC) device (NiCo-MOF-74@Ni12P5/NF//AC) delivers high storage capability (44.33 W·h/kg at 800 W/kg) and distinguished operating durability (83.04% after 5000 cycles). In addition, an all-solid-state hybrid SC successfully lit the LED for more than 2 min, which means that there is viable potential for practical applications in energy storage. The improved electrical properties are mainly due to the 3D heterostructure, the positive cooperative binding of nickel and cobalt elements, and the excellent electrical conductivity of the phosphide. As a result, this study proves the possibility of practical applications of NiCo-MOF-74@Ni12P5/NF electrodes for energy storage in hybrid SCs.

Modeling the Effect of Cooperativity in Ternary Complex Formation and Targeted Protein Degradation Mediated by Heterobifunctional Degraders.

Chemically induced proximity between certain endogenous enzymes and a protein of interest (POI) inside cells may cause post-translational modifications to the POI with biological consequences and potential therapeutic effects. Heterobifunctional (HBF) molecules that bind with one functional part to a target POI and with the other to an E3 ligase induce the formation of a target-HBF-E3 ternary complex, which can lead to ubiquitination and proteasomal degradation of the POI. Targeted protein degradation (TPD) by HBFs offers a promising approach to modulate disease-associated proteins, especially those that are intractable using other therapeutic approaches, such as enzymatic inhibition. The three-way interactions among the HBF, the target POI, and the ligase-including the protein-protein interaction between the POI and the ligase-contribute to the stability of the ternary complex, manifested as positive or negative binding cooperativity in its formation. How such cooperativity affects HBF-mediated degradation is an open question. In this work, we develop a pharmacodynamic model that describes the kinetics of the key reactions in the TPD process, and we use this model to investigate the role of cooperativity in the ternary complex formation and in the target POI degradation. Our model establishes the quantitative connection between the ternary complex stability and the degradation efficiency through the former's effect on the rate of catalytic turnover. We also develop a statistical inference model for determining cooperativity in intracellular ternary complex formation from cellular assay data and demonstrate it by quantifying the change in cooperativity due to site-directed mutagenesis at the POI-ligase interface of the SMARCA2-ACBI1-VHL ternary complex. Our pharmacodynamic model provides a quantitative framework to dissect the complex HBF-mediated TPD process and may inform the rational design of effective HBF degraders.