Can the new generation of CRBN Binding Kit usher in another spring for TPD

In the past ten years, targeted protein degradation (TPD) has jumped from a laboratory darling to a 100-billion-dollar industry. Cereblon (CRBN) is one of the most commonly used E3 enzymes, and its ligand affinity data directly determines the activity window of PROTAC molecules. However, one overlooked technical detail-the way tracers are marked-is quietly widening the distance between us and the real objective value.

 

The high-throughput CRBN Binding Kit of the imported R brand uses "thalidomide-fluorescent protein" as the tracer and is the first in the industry to establish a high-throughput screening paradigm. This solution has now been re-dismantled, verified, and upgraded by us. It is not a simple "domestic substitution", but a systematic repair of the experimental distortion problem, making IC50 closer to the objective truth value for the first time.

 

 

Distortion Source: When 105 kDa APC protein rides on the 0.25 kDa thalidomide head

 

As the first generation acceptor, XL665 is an improved allophycocyanin (APC), which couples the subunits of APC to increase stability. It is located in the infrared light region, with an excitation wavelength of 620nm and an emission wavelength of 665nm, further reducing the impact of biological solutions on experiments (biological components rarely have autofluorescence in the infrared light region). XL665 is used as an acceptor in CRBN Binding, with a molecular weight of about 105 kDa, but after coupling with thalidomide, it has the following effects on the system:

 

Steric hindrance:

✦ The diameter of APC/XL665 is ≈ 6 nm, which is equivalent to 10 times the length of thalidomide, forming a "fluorescent wall" at the entrance of the shallow pocket of CRBN;

✦ Macromolecular fluorescent labeling creates steric hindrance at the entrance to the shallow pocket of CRBN, resulting in apparent K _ d being systematically overestimated by 1.5-4 times.

 

Diffusion Rate:

✦ The overall molecular weight of protein tracer is high, and the diffusion coefficient D decreases;

✦ The diffusion coefficient of small molecule candidates (< 0.5 kDa) was much higher than that of thalidomide after labeling in kit, and the competitive environment was imbalanced.

 

Result distortion:

✦ The above influencing factors lead to the deviation between IC50 values and results, affecting the selection of candidates

 

Technological breakthrough: returning small molecules to small molecules

 

Directly labeling thalidomide with a small molecule fluorophore with a molecular weight less than 1kDa eliminates the two distortion factors from the physical level, and the competitive dynamics returns to the fair track of "small molecule to small molecule", so that every IC50 can withstand clinical torture.

 

 

Data speaks

 

(1) Standard curve

 

 

As can be seen from the graph, the signal value is reduced due to the competition between the Thalidomide standard and Thalidomide-Ac for binding to CRBN/DDB1.

 

(2) Binding activity and inhibitor validation *

 

The binding of Thalidomide-Ac to CRBN/DDB1 was verified. From the results, it can be seen that when the protein concentration remains unchanged, the detection signal increases with the increase of Thalidomide-Ac concentration. Lenalidomide and Pomalidomide and PROTAC BET Degrader-1 can compete for binding of Thalidomide-Ac to CRBN/DDB1.

 

* The data is for example reference only

 

Not only CRBN, but also PROTAC's "one-stop solution"

 

✦ TR-FRET-based screening of ternary complexes

Label song	Item number	Trade Name
	abs560016	Human BRD4/CRBN PROTAC Binding Kit
	abs560042	Human BCL-XL/CRBN PROTAC Binding Kit
	abs560044	Human BCL-XL/VHL PROTAC Binding Kit
	abs560012	Human DDB1-CRBN & GSPT1 Binding Kit
	abs560054	HumanVAV1/CRBN PROTAC Binding Kit

 

✦ PROTAC Drug target protein

 

Target abbreviation	Mainly for diseases	PROTAC	Absin	UA
AR (androgen receptor)	Prostate cancer	ARV-110		UA080416
BCL6	Lymphoma			UA080266
Bcl-X (L)	Lymphoma		abs06285
B-rafV600E	Malignant melanoma			UA080261
BRD4	Multiple cancers			UA080277
BRD9	Multiple cancers
BTK	Chronic lymphocytic leukemia	NX-2127		UA080041
EGFR	Non-small cell lung cancer		abs05321
ER (estrogen receptor)	Breast cancer	ARV-471	abs04404
IRAK4	Autoimmune disease			UA080294
KRASG12D	Solid tumor	ASP-3082		UA010733
MDM2	Multiple cancers	MD-224	abs06561
NTRK	Solid tumor		abs05487
STAT3	Lymphoma relapsed or refractory	KT-333		UA080435

 

✦ PROTAC Molecule-Positive Control

 

Item number	Trade Name	Targets
abs820796	(S,R,S)-AHPC hydrochloride	E3 ligase VHL
abs828204	ACBI1	SMARCA4:11 nM, SMARCA2:6 nM, PBRM1:32 nM
abs828800	AR Antagonist 1	Androgen Receptor:
abs825142	ARD-2128	VCaP:4 nM (IC50), LNCaP:5 μM (IC50)
abs821542	BETd-260	BRD4PROTAC
abs828404	BSJ-4-116	CDK12:6 nM
abs819411	dBET1	BRD4
abs826439	Dbet57	BRD4 (BD1):500 nM (DC50)
abs825346	dBRD9	ligand that recruits the cereblon E3 ubiquitin ligase:104 nM
abs821654	dTRIM24	TRIM24.
abs824454	LC-2	KRAS(G12C):0.25-0.76 μM (DC50)
abs819853	MD-224	MDM2
abs826178	MS4078	EML4-ALK:59 nM(DC50; NPM-ALK:11 nM(DC50);ALK:33 nM
abs828684	MT-802	BTK:1 nM(DC50)
abs821197	MZ 1	BRD4
abs821447	MZP-54	Brd4BD2
abs820470	PROTAC ERRα Ligand 1	IC50s of 0.04 and 2.8 μM for ERRα and ERRγ
abs828072	PROTAC ERRα Ligand 2	ERRα:5.67 nM
abs821731	TD-106	CRBN
abs823151	THAL-SNS-032	CDK9
abs826853	UNC6852	EED:247 nM
abs818009	VH-298	VHL:HIF-α
abs826121	YX-2-107	CDK6:4.4 nM
abs827426	ZEN-3219	BRD4BD1:0.48 μM, BRD4 BD2:0.16 μM

 

✦ PROTAC target-associated antibodies

 

Targets	Item number	Product name
Ubiquitin	abs155938	Mouse anti-Ubiquitin Monoclonal Antibody
AR	abs145396	Rabbit anti-Androgen Receptor mAb (JRMR-244)
BCL6	abs145233	Rabbit anti-BCL6 Monoclonal Antibody(JRMR-56)
Bcl-X(L)	abs159218	Rabbit anti-Bcl-XL Monoclonal Antibody(2D3)
B-rafV600E	abs173916	Rabbit anti-BRAF Recombinant mAb (S-1328-60)
BRD4	abs133244	Rabbit anti-Brd4 Polyclonal Antibody
BRD9	abs117685	Rabbit anti-BRD9 Polyclonal Antibody
More......

 

The golden age of TPD has arrived, but gold also needs an accurate scale. We believe that real data is the biggest accelerator of innovation. When the molecular weight of the tracer returns to less than 1kDa, we not only repair an experimental parameter, but also save the entire industry a lot of time, capital and trial and error lives.

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.cn

Follow us on Facebook: Absin Bio