BET PROTAC ǀ MZ 1

Target protein: 
BET bromodomains
Probe Name: 
MZ 1
MOLECULAR WEIGHT [DA]: 
1002.640
In stock: 
140

Chemical structure

Highlights

MZ 1 and the negative control cis MZ 1 have been discovered by Michael Zengerle, Kwok-Ho Chan, and Alessio Ciulli1 at the Universtity of Dundee. opnMe.com is proud to be able to provide researchers with up to two batches of 5 mg of MZ 1 and one batch of cis MZ 1 for free. Larger quantities of the compounds are available from Tocris.

Inducing macromolecular interactions with small molecules to activate cellular signaling is a challenging goal. PROTACs (proteolysis-targeting chimeras) are bifunctional molecules that recruit a target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. Structural elucidation of the key ternary ligase-PROTAC-target species and its impact on target degradation selectivity remain elusive. The crystal structure of BRD4 degrader MZ 1 in complex with human VHL and the BRD4 bromodomain (BRD4BD2) offers unique insights into the first ternary complex structure of a l.m.w. degrader. The ligand folds into itself to allow formation of specific intermolecular interactions in the ternary complex. Isothermal titration calorimetry studies, supported by surface mutagenesis and proximity assays, are consistent with pronounced cooperative formation of ternary complexes with BRD4BD2. Our results elucidate how PROTAC-induced de novo contacts dictate preferential recruitment of a target protein into a stable and cooperative complex with an E3 ligase for selective degradation.1,2

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Target information

Acetylation of lysine residues is a post-translational modification with broad relevance to cellular signalling and disease biology. Inhibitors that modulate the ‘reading process’ mediated by acetyl lysines are an area of extensive research. The principal readers of ɛ-N-acetyl lysine (Kac) marks are bromodomains (BRDs), which are a diverse family of evolutionary conserved protein-interaction modules. The conserved BRD fold contains a deep, largely hydrophobic acetyl lysine binding site, which represents an attractive pocket for the development of small, pharmaceutically active molecules. Proteins that contain BRDs have been implicated in the development of a large variety of diseases.

Traditionally small molecules have been used to inhibit the action of a target protein by occupying and blocking a functional region of the protein. An alternative innovative approach is the development of proteolysis targeting chimeras (PROTACs), i.e. heterobifunctional compounds consisting of one moiety that binds a Cullin RING E3 ubiquitin ligase linked to another that binds a desired protein of interest (POI), bringing the ligase and the POI into close spatial proximity  This hijacks the intrinsic catalytic activity of the E3 ligase from the natural and directs it toward the POI as a neo-substrate, triggering its poly-ubiquitination and subsequent proteasome-dependent degradation. As a result, a PROTAC acts as a degrader of the target as opposed to just an inhibitor, enabling the effective post-translational elimination of a target gene product in living organisms. This approach presents many advantages compared to conventional target inhibition. One of the most attractive features of the approach is that a PROTAC molecule acts sub-stoichiometrically, i.e. it only needs to bind a molecule of target once to induce its degradation, and then is released and set free to bind another molecule of target and carry on, as in a catalytic cycle. For this reason, the concentrations required for PROTACs to be active in cells tend to be much lower compared to those needed to be reached and maintained with inhibitors, which can lead to fewer off-target effects and a more selective chemical intervention on the desired target.

MZ 1, 3D conformation, as observed in complex with BRD4 bromodomain and VHL (PDB 5T35)

Figure 3: MZ 1, 3D conformation, as observed in complex with BRD4 bromodomain and VHL (PDB 5T35)

In vitro Activity

The binary affinity for MZ 1 binding to BRD4BD2 and to VCB (VHL-ElonginC-EloginB) is 15 and 66 nM respectively (ITC). The affinity of the negative control “cis MZ 1” to VCB is > 15 µM (ITC). In presence of excess of BRD4BD2 MZ 1 the ternary KD for the BRD4BD2::MZ  1::VCB is 3.7 nM. MZ 1 degrades the BRD4 protein with a DC50 of 2-20 nM depending on the cell line, and with selectivity on DC50 of ~10-fold relative to BRD2 and BRD3.

Table: Binding affinities for binary and ternary complexes between MZ 1, VCB and BET bromodomains as measured by isothermal titration calorimetry (for assay conditions/details see Gadd et al, Nat Chem Biol. 2017, 13, 514–521)1

 

Probe name

MZ 1

 

 

Protein in syringe

Species in cell

Kd (nM)

α

Δp<Kd

BRD2BD1

MZ 1

62 ± 6

-

-

BRD2BD2

MZ 1

60 ± 3

-

-

BRD3BD1

MZ 1

21 ± 5

-

-

BRD3BD2

MZ 1

13 ± 3

-

-

BRD4BD1

MZ 1

39 ± 9

-

-

BRD4BD2

MZ 1

15 ± 1

-

-

BRD2BD1 KEA

MZ 1

69 ± 9

-

-

BRD4BD1 QVK

MZ 1

22 ± 8

-

-

VCBa

MZ 1a

66 ± 6

-

-

VCB

MZ 1:BRD2BD1

24 ± 8

2.9

0.4 ± 0.2

VCB

MZ 1:BRD2BD2

28 ± 3

2.3

0.36 ± 0.06

VCB

MZ 1:BRD3BD1

19 ± 4

3.5

0.5 ± 0.1

VCB

MZ 1:BRD3BD2

7 ± 2

10.7

1.0 ± 0.2

VCB

MZ 1:BRD4BD1

28 ± 6

2.3

0.4 ± 0.1

VCB

MZ 1:BRD4BD2

3.7 ± 0.7

17.6

1.24 ± 0.09

VCB

MZ 1:BRD2BD1 KEA

12 ± 7

7.9

0.8 ± 0.3

VCB

MZ 1:BRD4BD2 QVK

14.9 ± 0.1

4.2

0.62 ± 0.04

All ITC titrations were performed at 25 °C. Values reported are the mean ± S.E.M. from two independent measurements, except for VCB titration into MZ 1 (line a) for which values reported are the mean ± S.E.M. from eight independent measurements.

In vitro DMPK and CMC parameters

MZ 1 is soluble at physiological pH, metabolic stable (hepatocytes) and a PGP substrate (high Caco2 efflux ratio. Therefore, MZ 1 is not suitable for oral administration but suitable for parenteral administration (i.v., i.p. or s.c.). The compound shows moderate inhibition of Cyp3A4 and species differences in plasma protein binding.

Probe name / negative Control

MZ 1

Cis MZ 1

MW [Da]

1002.6

1002.6

Solubility @ pH 6.8 [µg/ml]

19

12 (pH 7)

CACO permeability @pH7.4 [*10-6 cm/s]

0.034

n.d.

CACO efflux ratio

481

n.d.

Microsomal stability (human/mouse/rat) [% QH]

88 / 79 / 73

>95 / >95 / 71

Hepatocyte stability 50% Serum (human/mouse/rat) [% QH]

12 / 19 / 58

n.d.

Plasma protein binding (human/mouse/rat/10%FCS) [%fu]

2.5 / 0.42 / 2.5 / 41

n.d.

CYP 3A4 (IC50)[µM]

5.5

n.d.

CYP 2C8 (IC50)[µM]

24

n.d.

CYP 2C9 (IC50)[µM]

>50

n.d.

CYP 2C19 (IC50)[µM]

>50

n.d

CYP 2D6 (IC50)[µM]

37

n.d.

 

In vivo DMPK parameters

MZ 1 is suitable for a parenteral administration (i.v., i.p. or s.c.). The compound shows high Clearance in rats and low Clearance in mice. High AUC levels can be obtained, when the compound is administered subcutaneously using a 25% HP-ß-CD formulation. Because of the high Caco2 efflux ratio, the oral exposure is very low.

MZ 1

RAT

MOUSE

Clearance [%QH]

156 a

20.7 c

Mean residence time [h]

0.61 a

0.34 c

t1/2,term [h]

1.52 a

1.04 c

Vss [l/kg]

4.05 a

0.38 c

AUC0-inf [nM.h]

1300 b

3760 d

cmax [nM]

163 b

2070 d

tmax [h]

2 b

0.5 d

t1/2,term [h]

5.13 b

2.95 d

cmax [nM]

-

<2 e

 

a rat i.v. 5 mg/kg

b rat s.c. 5 mg/kg

c mouse i.v. mg/kg

d mouse s.c. mg/kg

e mouse p.o. mg/kg

 

A

B

C

D

Figure 4: PK curves: Rat i.v. 5 mg/kg, B Rat s.c. 5 mg/kg, C mouse i.v. mg/kg, D mouse s.c. mg/kg

In vivo pharmacology

MZ 1 shows antiproliferative and Myc-suppression activity in AML MV4;11 and HL60 cells.

BET Reduction by MZ 1 in HeLa Cells, Antiproliferative Activity, and BRD4/cMyc Reduction in AML Cellsa.3

MZ 1

 

pDC50/Dmax (%) in HeLa cells

pIC50

Emax (%)

BRD4/cMyc depletion (%)

(α)

 

BRD4

short

BRD4

long

BRD3

BRD2

MV4;11

HL60

MV4;11

HL60

MV4;11

HL60

 

MZ 1

8.1/ 98

8.6/ 100

7.0/ 100

7.4/ 98

7.57 ±0.03

6.66 ±0.05

96.1 ±0.3 

92.1 ±0.4

 96/ 84

82/ 68

7.4

aDC50: concentration in molar causing 50% reduction of protein level relative to vehicle control treatment in 24 h. Dmax: maximum reduction of protein level relative to vehicle control treatment. pIC50 was measured after 48 h treatment. Errors on pIC50 values reflect the quality of the curve fitting. Protein depletion % are for 50 nM treatments (4 h) in MV4;11/HL60. For more details please refer to reference 3.

Negative control

MOLECULAR WEIGHT OF NEGATIVE CONTROL [DA]: 
1002.640

“Cis MZ 1” is the (S) hydroxy diastereoisomer of MZ 1. While exhibiting MZ 1 comparable Bromodomain binding affinity it no longer is able to bind and recruit VCB and therefore is not degrading BET proteins in cells. The affinity of the “cis MZ 1” to VCB is > 15 µM (ITC).

Figure 5: “cis MZ 1”  which serves as a negative control

Figure 5: “cis MZ 1” which serves as a negative control

Selectivity

No data available

MZ 1

Selectivity data available

Cerep®

No

Panlabs®

No

Invitrogen®

No

DiscoverX®

No

Dundee

No

 

Co-crystal structure of the BI probe compound and the target protein

The X-ray of the PROTAC MZ 1 in complex with the second bromodomain of BRD4 and pVHL:ElonginC:ElonginB is available in the PDB: 5T35

reference molecules

dBET (CRBN based PROTAC) is BET bromodomain degrader which is less specific than MZ 1.4,5

Summary

MZ 1 is a first in class l.m.w. degrader that tethers JQ1 to a VHL E3 ubiquitin ligase ligand, aimed at triggering the intracellular destruction of BET proteins. MZ 1 potently and rapidly induces reversible, long-lasting, and preferential removal of BRD4 over BRD2 and BRD3 in cells.

MZ 1 and the negative control cis MZ 1 have been discovered by Michael Zengerle, Kwok-Ho Chan, and Alessio Ciulli1 at the Universtity of Dundee. opnMe.com is proud to be able to provide researchers with up to two batches of 5 mg of MZ 1 and one batch of cisMZ 1 for free. Larger quantities of the compounds are available from Tocris.

Supplementary data

References

  1. Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4

    Michael Zengerle, Kwok-Ho Chan, and Alessio Ciulli

    ACS Chem Biol. 2015, 10, 1770–1777.

  2. Structural basis of PROTAC cooperative recognition for selective protein degradation

    Morgan S Gadd, Andrea Testa, Xavier Lucas, Kwok-Ho Chan, Wenzhang Chen, Douglas J Lamont, Michael Zengerle and Alessio Ciulli

    Nat. Chem. Biol. 2017, 13, 514–521.

  3. Impact of Target Warhead and Linkage Vector on Inducing Protein Degradation: Comparison of Bromodomain and Extra-Terminal (BET) Degraders Derived from Triazolodiazepine (JQ1) and Tetrahydroquinoline (I-BET726) BET Inhibitor Scaffolds

    Kwok-Ho Chan, Michael Zengerle, Andrea Testa, and Alessio Ciulli

    J. Med. Chem. 2018, 61, 504-513.

  4. DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation

    Georg E. Winter, Dennis L. Buckley, Joshiawa Paulk, Justin M. Roberts, Amanda Souza, Sirano Dhe-Paganon, James E. Bradner

    Science 2015, 348, 1376-1381.

  5. Selective degradation of BET proteins with dBET1, a proteolysis-targeting chimera, potently reduces pro-inflammatory responses in lipopolysaccharide-activated microglia

    Kelly M. DeMars, Changjun Yang, Carolina I. Castro-Rivera and Eduardo Candelario-Jalil

    Biochem Biophys Res Commun. 2018, 497, 410-415.