scholarly journals SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate

2018 ◽  
Author(s):  
Michael J. Roy ◽  
Sandra Winkler ◽  
Scott J. Hughes ◽  
Claire Whitworth ◽  
Michael Galant ◽  
...  
Keyword(s):  
Ternary Complex ◽  
E3 Ligase ◽  
Ternary Complexes ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Dissociation Kinetics ◽  
Intracellular Degradation ◽  
The Stability ◽  
Kinetics Of

ABSTRACTBifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target:PROTAC:ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of BET proteins Brd2, Brd3 and Brd4 to the E3 ligase VHL. We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4BD2 or Brd2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilised due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

scholarly journals Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2005
Author(s):  
Irene Vorontsova ◽  
James E. Hall ◽  
Thomas F. Schilling ◽  
Noriaki Nagai ◽  
Yosuke Nakazawa
Keyword(s):  
Cell Adhesion ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Adhesion Assay ◽  
Loss Of Function ◽  
Adhesive Properties ◽  
The Difference ◽  
In Vitro Adhesion ◽  
Cell To Cell Adhesion

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

The in vitro dissociation kinetics of (R,R)-[125I]4IQNB is reflected in the in vivo washout of the radioligand from rat brain

Life Sciences ◽  
1992 ◽  
Vol 50 (9) ◽  
pp. 629-637 ◽  
Author(s):  
Raymond E. Gibson ◽  
Terry Moody ◽  
Timothy A. Schneidau ◽  
Elaine M. Jagoda ◽  
Richard C. Reba
Keyword(s):  
Rat Brain ◽  
Dissociation Kinetics ◽  
Kinetics Of

scholarly journals Characterization of the Global Stabilizing Substitution A77V and Its Role in the Evolution of CTX-M β-Lactamases

2015 ◽  
Vol 59 (11) ◽  
pp. 6741-6748 ◽  
Author(s):  
Meha P. Patel ◽  
Bartlomiej G. Fryszczyn ◽  
Timothy Palzkill
Keyword(s):  
Antibiotic Resistance ◽  
Adaptive Mutation ◽  
Common Mechanism ◽  
Antibiotic Hydrolysis ◽  
The Stability ◽  
Kinetics Of ◽  
M 14

ABSTRACTThe widespread use of oxyimino-cephalosporin antibiotics drives the evolution of the CTX-M family of β-lactamases that hydrolyze these drugs and confer antibiotic resistance. Clinically isolated CTX-M enzymes carrying the P167S or D240G active site-associated adaptive mutation have a broadened substrate profile that includes the oxyimino-cephalosporin antibiotic ceftazidime. The D240G substitution is known to reduce the stability of CTX-M-14 β-lactamase, and the P167S substitution is shown here to also destabilize the enzyme. Proteins are marginally stable entities, and second-site mutations that stabilize the enzyme can offset a loss in stability caused by mutations that enhance enzyme activity. Therefore, the evolution of antibiotic resistance enzymes can be dependent on the acquisition of stabilizing mutations. The A77V substitution is present in CTX-M extended-spectrum β-lactamases (ESBLs) from a number of clinical isolates, suggesting that it may be important in the evolution of antibiotic resistance in this family of β-lactamases. In this study, the effects of the A77V substitution in the CTX-M-14 model enzyme were characterized with regard to the kinetic parameters for antibiotic hydrolysis as well as enzyme expression levelsin vivoand protein stabilityin vitro. The A77V substitution has little effect on the kinetics of oxyimino-cephalosporin hydrolysis, but it stabilizes the CTX-M enzyme and compensates for the loss of stability resulting from the P167S and D240G mutations. The acquisition of global stabilizing mutations, such as A77V, is an important feature in β-lactamase evolution and a common mechanism in protein evolution.

scholarly journals Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity

2020 ◽  
Vol 477 (5) ◽  
pp. 905-923 ◽  
Author(s):  
Sunil Singh ◽  
J. Sivaraman
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Enzymatic Activity ◽  
Cancer Metastasis ◽  
E3 Ligase ◽  
The Other ◽  
Hect Domain ◽  
Loop Region ◽  
The Stability

The HECT family of E3 ubiquitin ligase is divided into three subfamilies: the NEDD4, the HERC, and the ‘other’. Previous studies have mostly targeted members of the NEDD4 subfamily for structural and functional analysis. The UBE3C E3 ligase is a member of the ‘other’ subfamily HECT and influences several crucial cellular processes, including innate immunity, proteasome processivity, and cancer metastasis. Here, we report the crystal structure of the HECT domain of UBE3C (amino acids (aa) 744–1083) with an additional fifty N-terminal amino acids (aa 693–743) at 2.7 Å, along with multiple in vitro ubiquitination assays to understand its enzymatic activity. The UBE3C HECT domain forms an open, L-shaped, bilobed conformation, having a large N-lobe and a small C-lobe. We show that the N-terminal region (aa 693–743) preceding the UBE3C HECT domain as well as a loop region (aa 758–762) in the N-lobe of the HECT domain affect the stability and activity of UBE3C HECT domain. Moreover, we identified Lys903 in the UBE3C HECT domain as a major site of autoubiquitination. The deletion of the last three amino acids at the C-terminal completely abrogated UBE3C activity while mutations of Gln961 and Ser1049 residues in the HECT domain substantially decreased its autoubiquitination activity. We demonstrate that these region/residues are involved in the E2–E3 transthiolation process and affect the UBE3C mediated autoubiquitination. Collectively, our study identified key residues crucial for UBE3C enzymatic activity, and it may assist in the development of suitable inhibitors to regulate its activity in multiple cancers.

scholarly journals Investigations of ternary complexes of Co(II) and Ni(II) with oxydiacetate anion and 1,10-phenanthroline or 2,2′-bipyridine in solutions

2014 ◽  
Vol 12 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Dariusz Wyrzykowski ◽  
Joanna Pranczk ◽  
Dagmara Jacewicz ◽  
Aleksandra Tesmar ◽  
Bogusław Pilarski ◽  
...  
Keyword(s):  
Formation Constants ◽  
Ternary Complexes ◽  
Substitution Reactions ◽  
Constant Concentration ◽  
Experimental Conditions ◽  
Kinetic Measurements ◽  
Binary Complexes ◽  
The Stability ◽  
Kinetics Of ◽  
Complex Formation Process

AbstractPotentiometric (PT) and conductometric (CT) titration methods have been used to determine the stoichiometry and formation constants in water for a series of ternary complexes of Co(II) and Ni(II) involving the oxydiacetate anion (ODA) and 1,10-phenanthroline (phen) or 2,2′-bipyridine (bipy) ligands, namely [Co(ODA)(phen)(H2O)], [Co(ODA)(bpy)(H2O)], [Ni(ODA)(phen)(H2O)] and [Ni(ODA)(bpy)(H2O)]. The ternary complex formation process was found to take place in a stepwise manner in which the oxydiacetate ligand acts as a primary ligand and the phen or bipy ligands act as auxiliary ones. The stability of the ternary complexes formed is discussed in the relation to the corresponding binary ones. Furthermore, the kinetics of the substitution reactions of the aqua ligands in the coordination sphere of the Ni-ODA and Co-ODA complexes to phen or bipy were studied by the stopped-flow method. The kinetic measurements were performed in the 288–303 K temperature range, at a constant concentration of phen or bipy and at seven different concentrations of the binary complexes (4–7 mM). The influence of experimental conditions and the kind of the auxiliary ligands (phen/bipy) on the substitution rate was discussed.

In vivo dissociation kinetics of [3H]quinuclidinyl benzilate: relationship to muscarinic receptor concentration and in vitro kinetics

1991 ◽  
Vol 553 (1) ◽  
pp. 110-116 ◽  
Author(s):  
Raymond E. Gibson ◽  
Barry R. Zeeberg ◽  
John M. Melograna ◽  
Tessica F. Wang ◽  
Jennifer Ruch ◽  
...  
Keyword(s):  
Muscarinic Receptor ◽  
Dissociation Kinetics ◽  
Receptor Concentration ◽  
Kinetics Of ◽  
In Vitro Kinetics ◽  
Quinuclidinyl Benzilate

scholarly journals Thermodynamics of binary and ternary complexes of 3-amino-1, 2, 4-triazole and aminoacids with Ni(II) and Co(II) metal ions

2005 ◽  
Vol 70 (8-9) ◽  
pp. 1057-1066 ◽  
Author(s):  
Ayse Erçag ◽  
Tuba Sismanoglu ◽  
Suheyla Pura
Keyword(s):  
Ionic Strength ◽  
Aqueous Solutions ◽  
Glutamic Acid ◽  
Driving Force ◽  
Ternary Complex ◽  
Ternary Complexes ◽  
Binary And Ternary Complexes ◽  
Enthalpy Changes ◽  
Binary Complexes ◽  
The Stability

The stability constants of the 1:1 binary complexes of Ni(II) and Co(II) with 3-amino-1,2,4-triazole (AT), leucine (Leu) and glutamic acid (Glu), and the 1:1:1 ternary complex of them and the protonation constants of the ligands were determined potentiometrically at a constant ionic strength of I = 0.10 mol L-1 (NaClO4) in aqueous solutions at 15.0 and 25.0 ?C. The thermodynamic parameters ?Gf0, ?Hf0 and ?Sf0 are reported for the formation reactions of the complexes. The enthalpy changes of all the complexations were found to be negative but the entropy changes positive. While the driving force for the formation of the Ni(II), Co(II) ? AT complexes is the enthalpy decrease, the driving force for the ternary complexes of AT is the entropy increase.

scholarly journals A Method for Determining the Kinetics of Small-Molecule-Induced Ubiquitination

2021 ◽  
Vol 26 (4) ◽  
pp. 547-559
Author(s):  
Ellen F. Vieux ◽  
Roman V. Agafonov ◽  
Lydia Emerson ◽  
Marta Isasa ◽  
Richard W. Deibler ◽  
...  
Keyword(s):  
Ternary Complex ◽  
Ubiquitin Proteasome System ◽  
Binary Complex ◽  
Discovery Process ◽  
Drug Discovery Process ◽  
In Vitro System ◽  
Ubiquitin Proteasome ◽  
Catalytic Rate ◽  
Kinetics Of

Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin–proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.

Sign in / Sign up

Export Citation Format

Share Document