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Department of Chemistry

Overview Overview → Epigenetics → DNMTs → HDACs → Bromodomains → PMTs → KDMs → Conclusion Isoxazole-Derived Amino Acids are Bromodomain-Binding Acetyl- 4 5 Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3. Angew. Chem. Int. Ed. 2016, 55, 8353.

Epigenetics: Novel Therapeutics Targeting Epigenetics. J. Med. Chem. 2016, 59, 1247.

Fighting Cancer: Epigenetic Targets for Oncology

Epigenetics: Novel Therapeutics Targeting Epigenetics. J. Med. Chem. 2015, 58, 523.

Small Molecule Inhibitors of Bromodomain-Acetyl-Lysine Interactions. ACS Chem. Biol. 2015, 10, 22.

Discovery and Optimization of Small-Molecule Ligands for the CBP/P300 Bromodomains. J. Am. Chem. Soc. 2014, 136, 9308.

ACS Webinar 23rd February 2017

A Series of Potent CREBBP Bromodomain Ligands Reveals an Induced-Fit Pocket Stabilized by a Cation-π Interaction. Angew. Chem. Int. Ed. 2014, 53, 6126.

Professor Stuart Conway Department of Chemistry, Chemistry Research Laboratory University of Oxford, Mansfield Road Oxford, OX1 3TA e: [email protected] w: http://conway.chem.ox.ac.uk t: @conway_group

Phenotypic Screening and Fragment-Based Approaches to the Discovery of Small-Molecule Bromodomain Ligands. Future Med. Chem. 2014, 6, 179.

• Introduction to epigenetics and histone post-

Optimization of 3,5-Dimethylisoxazole Derivatives as Potent Bromodomain Ligands. J. Med. Chem. 2013, 56, 3217.

• Existing epigenetic drugs: DNMT inhibitors and HDAC

The Design and Synthesis of 5 and 6-Isoxazolylbenzimidazoles as Selective Inhibitors of the BET Bromodomains. Med. Chem. Commun. 2013, 4, 140.

translational modifications.

inhibitors.

• Compounds in clinical trials: bromodomain ligands, PMT inhibitors, and KDM inhibitors.

• Conclusion

Progress in the Development and Application of Small Molecule Inhibitors of Bromodomain-Acetyl-Lysine Interactions. J. Med. Chem. 2012, 55, 9393.

Bromodomains: Are Readers Right for Epigenetic Therapy? ACS Med. Chem. Lett. 2012, 3, 691.

3,5-Dimethylisoxazoles Act as Acetyl-Lysine-Mimetic Bromodomain Ligands. J. Med. Chem. 2011, 54, 6761.

Question

Epigenetics

Overview → Epigenetics → DNMTs → HDACs → Bromodomains → PMTs → KDMs → Conclusion


different genes

are expressed

different genes

are expressed

Are you familiar with the concept of epigenetics? DNA sequence is identical

different “phenotypes” “Heritable changes in phenotype that are transmitted without altering the underlying sequence of DNA bases.” Arrowsmith et al. Nature Rev. Drug Disc. 2012, 11, 384; Prinjha, R. K.; Witherington, J.; Lee, K. Trends Pharmacol. Sci. 2012, 33, 146; Bird, A. Nature 2007, 447, 396. Counterpoint: Ptashne Proc. Natl. Acad. Sci. USA 2013, 110, 7101; Berger, S. L.; Kouzarides, T.; Shiekhattar, R.; Shilatifard, A. Genes Dev. 2009, 23, 781

Dutch Hunger Winter and Breakfast at Tiffany’s

Molecular epigenetics



•

Audrey Hepburn was a survivor of the Dutch Hunger Winter in the Second World War.

•

This lasted from November 1944 to spring 1945, during this Ame people survived on ~30% of their normal daily calorie intake.

•

Surprisingly the effects of this privaAon lasted not only for the lives of the survivors, but also affected their children and grandchildren.

Carey, N. The Epigenetic Revolution; Icon Books Ltd: London, 2011.

•

If a mother was well fed at the Ame of concepAon, but malnourished at the Ame of birth, then the baby was likely to be small.

•

If the mother suffered malnutri?on for the first 3 months of pregnancy, but was then well fed, the baby was born with with normal weight.

•

However, the babies that were born small, stayed small all of their lives, with lower obesity rates than the general populaAon.

•

Those who were born with normal weights had higher obesity rates than the general popula?on.

Jones, P. A.; Issa, J.-P. J.; Baylin, S. Nat. Rev. Genet. 2016, 17, 630.

Histones

Histone PTMs



Histone tails

= DNA

Histone H2A

Histone H2B

Histone H3

Luger, K.; Mader, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond, T. J. Nature 1997, 389, 251.

Histone H4

Jennings, L. E.; Measures, A. R.; Wilson, B. G.; Conway, S. J. Future Med. Chem. 2014, 6, 179.

Molecular epigenetics

Clinically approved epigenetic drugs



• DNA methyltransferase inhibitors.

HDAC

D26 Y30 Zn2

D18 H18

• Histone (lysine) deacetylase (HDAC)inhibitors.

D17 H14 SAH

H14 D18 D10



DNA methylation and gene expression




• DNA methylaAon occurs on cytosine and adenine, with 5-methylcytosine being widespread in

HDAC

both eukaryotes and prokaryotes.

D26 Y30 Zn2

D18 H18


D17 H14 SAH

• Generally, DNA methylaAon is associated with long-term transcripAonal silencing of genes. • In tumours, increased DNA methylaAon occurs at the promoter regions of tumour suppressor genes, reducing their expression.

• Transfer of the methyl group from the co-factor S-adenosyl methionine (SAM) to cytosine is

H14

catalysed by one of three DNA methyltransferases in mammals: DNMT1, DNMT3A, and DNMT3B.

D18 D10

Bird, A.; Taggart, M.; Frommer, M.; Miller, O. J.; Macleod, D. A. Cell 1985, 40, 91; Herman, J. G.; Latif, F.; Weng, Y.; Lerman, M. I.; Zbar, B.; Liu, S.; Samid, D.; Duan, D. S.; Gnarra, J. R.; Linehan, W. M. Proc. Natl. Acad. Sci. USA 1994, 91, 9700; Toyota, M.; Ahuja, N.; Ohe-Toyota, M.; Herman, J. G.; Baylin, S. B.; Issa, J. P. Proc. Natl. Acad. Sci. USA 1999, 96, 8681; Esteller, M.; Silva, J. M.; Dominguez, G.; Bonilla, F.; Matias-Guiu, X.; Lerma, E.; Bussaglia, E.; Prat, J.; Harkes, I. C.; Repasky, E. A.; Gabrielson, E.; Schutte, M.; Baylin, S. B.; Herman, J. G. J. Natl. Cancer Inst. 2000, 92, 564.

DNMT mechanism of action

Clinically approved DNMT inhibitors



• AzaciAdine and decitabine are used as single agents to treat myeloid leukemias. • The drugs have at least two mechanisms of acAon. • In both cases the drugs are thought to be incorporated in to DNA (azaciAdine and decitabine) and/or RNA (azaciAdine).

• At lower concentraAons the drugs cause hypomethylaAon through DNMT inhibiAon. • At high doses these compounds are also cytotoxic, as a result of their direct incorporaAon in to DNA and/or RNA.

Yoo, C. B.; Jones, P. A. Nat. Rev. Drug Discov. 2006, 5, 37.


DNMT mechanism of action

Clinically approved DNMT inhibitors



•

AzaciAdine and decitabine funcAon by being incorporated in to DNA (azaciAdine and decitabine) and/or RNA (azaciAdine) (making them cellcycle S phase specific drugs).

•

The iniAal aXack by the enzyme cysteine thiolate can occur as expected.

•

Next methylaAon occurs on the nitrogen which is present instead of the carbon atom in cytosine.

•

No eliminaAon pathway is present, resulAng in covalent linking of the enzyme to the DNA.

•

It is possible that the DNMT undergoes degradaAon once covalently linked to the inhibitor.

Yoo, C. B.; Jones, P. A. Nat. Rev. Drug Discov. 2006, 5, 37; Taylor, S. M.; Jones, P. A. Cell 1979, 17, 771; Jones, P. A.; Taylor, S. M. Cell 1980, 20, 85.

• Treatment with these drugs cause acute genome-wide demethylaAon in paAents. • Treatment resulted in demethylaAon of specific tumour suppressor gene promoters, e.g. p15.

• It is difficult to link paAent response with the varied effects of these drugs, but sustained demethylaAon of genes such as p15 correlated well with responses.

• The fact that the clinical observaAon are more consistent at lower doses, and that some paAents respond to these drugs despite resistance to cytotoxic agents, are consistent with an epigeneAc mechanism leading to the clinical responses.


DNMT inhibitors

DNMT inhibitors - pros and cons



😀 pros

• DNMT inhibitors have been approved for clinical use against MDS and AML.

• Guadecitabine (Astex, SGI-110) is a second-generaAon hypomethylaAng agent that is currently in Phase III clinical trials for acute myeloid leukaemia.

• It is a dinucleoAde of analogue of decitabine that funcAons as a prodrug for the release of decitabine, which is the acAve moiety.

•

The gradual enzymaAc cleavage of the phosphodiester bond results in the release of decitabine over an extended period of Ame, prolonging its in vivo exposure.

• Guadecitabine is resistant to cyAdine deaminase, the main enzyme responsible for decitabine degradaAon.

Issa, J.-P. J.; Roboz, G.; Rizzieri, D.; Jabbour, E.; Stock, W.; O'Connell, C.; Yee, K.; Tibes, R.; Griffiths, E. A.; Walsh, K.; Daver, N.; Chung, W.; Naim, S.; Taverna, P.; Oganesian, A.; Hao, Y.; Lowder, J. N.; Azab, M.; Kantarjian, H. Lancet Oncol. 2015, 16, 1099; Jones, P. A.; Issa, J.-P. J.; Baylin, S. Nat. Rev. Genet. 2016, 17, 630.

• The clinical effects results, at least in

part, from an epigeneAc mechanism.

• DNMT inhibitors are effecAve as

monotherapies, although combinaAon therapies are being explored.

• Guadecitabine might address some of

the issues, associated with the short halflife of azaciAdine and decitabine.

☹

cons • DNMT inhibitors are broad

reprogrammers, which tend to cause large-scale changes in gene expression.

• Primary and secondary resistance to

azaciAdine and decitabine is common.

• AcAvity in solid tumours is limited,

perhaps as a result of the short half-life of these drugs and the fact that they are S phase dependent, giving low incorporaAon into DNA in some malignancies.







HDAC

HDAC

D26 Y30 Zn2

D26 Y30

D18

Zn2

H18


D17

H18


D17

H14 SAH

H14 SAH

H14

H14

D18 D10

D18

D18 D10

Lysine acetylation

Histone deacetylases (HDACs)



HATs writing

HDACs erasing

NH3+

NH3+ H3C

S CoA O

NH

HS CoA O

H2O

CH3

HO

CH3 O

• Histone lysine acetylaAon was first reported in the 1960s, and lysine acetylaAon is now recognised as a

wide-spread protein post-translaAonal modificaAon with 3600 lysine acetylaAon sites idenAfied on 1750 proteins.

• Generally, lysine acetylaAon was associated with transcripAonal acAvaAon of genes near the acetylated nucleosome, it is now recognised that this is an over simplificaAon.

• AcetylaAon of a lysine residue removes the posiAve charge, meaning that the histone associates less Aghtly with negaAvely charged DNA, making the DNA more accessible for transcripAon factors.

• Histone/lysine acetyltransferase enzymes (HATs/KATs) transfer an acetyl group for acetyl co-enzyme A to the ε-nitrogen atom of lysine.

• Histone/lysine deacetylase enzymes (HDACs/KDACs) remove the acetyl group, returning the unadorned lysine.

Allfrey, V.; Faulkner, R.; Mirsky, A. Proc. Natl. Acad. Sci. USA 1964, 51, 786; Choudhary, C.; Kumar, C.; Gnad, F.; Nielsen, M. L.; Rehman, M.; Walther, T. C.; Olsen, J. V.; Mann, M. Science 2009, 325, 834; Brownell, J. E.; Zhou, J. X.; Ranalli, T.; Kobayashi, R.; Edmondson, D. G.; Roth, S. Y.; Allis, C. D. Cell 1996, 84, 843; Vettese-Dadey, M.; Grant, P. A.; Hebbes, T. R.; Crane- Robinson, C.; Allis, C. D.; Workman, J. L. EMBO J. 1996, 15, 2508; Kuo, M. H.; Brownell, J. E.; Sobel, R. E.; Ranalli, T. A.; Cook, R. G.; Edmondson, D. G.; Roth, S. Y.; Allis, C. D. Nature 1996, 383, 269; Taunton, J.; Hassig, C. A.; Schreiber, S. L. A Science 1996, 272, 408; Richon, V. M.; Emiliani, S.; Verdin, E.; Webb, Y.; Breslow, R.; Rifkind, R. A.; Marks, P. A. Proc. Natl. Acad. Sci. USA 1998, 95, 3003.

Arrowsmith, C. H.; Bountra, C.; Fish, P. V.; Lee, K.; Schapira, M. Nat. Rev. Drug Disc. 2012, 11, 384; Smith, B. C.; Hallows, W. C.; Denu, J. M. Chem. Biol. 2008, 15, 1002.

Histone deacetylases (HDACs)

HDAC mechanism of action




Lombardi, P. M.; Cole, K. E.; Dowling, D. P.; Christianson, D. W. Curr. Opin. Struct. Biol. 2011, 21, 735.

Clinically approved HDAC inhibitors

Clinically approved HDAC inhibitors



• There is minimal definiAve experimental evidence demonstraAng that over-expression of HDACs is oncogenic.

• Over-expression of HDAC1 in tumour cells can induce proliferaAon and dedifferenAaAon; however, there are no data showing that aberrant expression of HDACs can be a primary oncogenic effect.

• By contrast, knockdown of HDACs can induce a range of anAtumour effects such as cell cycle arrest and inhibiAon of proliferaAon, inducAon of apoptosis, differenAaAon and senescence, and disrupAon of angiogenesis.

• This provides some indicaAon that HDAC expression is required to ensure the survival and growth of a tumour cell.

• Given that HDAC knockout experiments have demonstrated an essenAal role for individual HDACs in

normal cellular and Assue development it remains unclear whether the effect of HDAC knockdown is tumour cell selecAve.

Jones, P. A.; Issa, J.-P. J.; Baylin, S. Nat. Rev. Genet. 2016, 17, 630; Falkenberg, K. J.; Johnstone, R. W. Nat. Rev. Drug Discov. 2014, 13, 673.

• HDAC inhibitors was discovered as anAcancer agents in phenotypic screens idenAfy agents that induce tumour cell differenAaAon.

• The molecular targets of these drugs were idenAfied ader the biological effects of the compounds were discovered.

• The hydroxamic acid-based Vorinostat (Zolinza; Merck & Co.), belinostat (Beleodaq; Spectrum

PharmaceuAcals) and romidepsin (Istodax; Celgene) have all been approved for the treatment of cutaneous or peripheral T cell lymphomas.

• Panobinostat (Farydak; NovarAs) was recently approved for the treatment of drug-resistant mulAple myeloma when used in combinaAon with the proteasome inhibitor bortezomib (Velcade; Millennium PharmaceuAcals).


Binding of HDAC inhibitors

HDAC inhibitor in clinical trials



PDB ID: 4LXZ

HDAC2

D269 Y308 Zn2+

Vorinostat (Zolinza, SAHA)

D181 H183 D179 H145

SAHA

H146 D186 D104

• EAnostat is a class I HDAC inhibitor that is currently in Phase III clinical trials for breast

Panobinostat (Farydak)

• HDAC inhibitors funcAon by compeAAvely inhibiAng the

cancer.

HDAC2

enzyme acAve site. They typically share similar structures, comprising a cap, a linker, and a zinc-binding group.

• While sharing a similar structure to other some of the other clinically-approved HDAC

D269 Y308

• The hydroxamic acid group, found in the prototypical HDAC

Zn2+

inhibitor SAHA, is effecAve in this role.

inhibitors, it possessed a 1,2-benzene diamine moAf that binds the acAve site Zn2+ ion.

D181 H183

• This group also displaces the acAvated water molecule, and

D179

binds to the key catalyAcally-acAve residues.

Cap Linker Zn binder

H145 SAHA

Catalytic residues Zn-binding residues

H146 D186 D104

Lauffer, B. E. L.; Mintzer, R.; Fong, R.; Mukund, S.; Tam, C.; Zilberleyb, I.; Flicke, B.; Ritscher, A.; Fedorowicz, G.; Vallero, R.; Ortwine, D. F.; Gunzner, J.; Modrusan, Z.; Neumann, L.; Koth, C. M.; Lupardus, P. J.; Kaminker, J. S.; Heise, C. E.; Steiner, P. J. Biol. Chem. 2013, 288, 26926.

• A clinical trial with eAnostat showed that there might be a benefit of combining epigeneAc therapy and immunotherapy.

• A small group of paAents whose disease progressed ader low-dose treatment with a

combinaAon of azaciAdine and enAnostat, had robust and durable tumour responses when they were subsequently enrolled in a trial of immune checkpoint therapy.


Sirtuins

Sirtuins





Sirtuins mechanism of action

HDAC inhibitors - pros and cons



😀 pros

• HDAC inhibitors are broad reprogrammers,

• Panobinostat was recently approved for the

• HDAC inhibitors have been developed in a

treatment of drug-resistant mulAple myeloma when used in combinaAon with a proteasome inhibitor, broadening the applicaAon of HDAC inhibitors.

• • • • • •

variety of other acyl groups as well. SIRT1−3 can depropionylate and debutyrylate. SIRT2 funcAons as a demyristoylase and can remove the 4-oxononanoylaAon (4-ONylaAon) mark. SIRT3 has decrotonylaAon acAvity. SIRT4 acts as a lipoamidase regulaAng pyruvate dehydrogenase complex acAvity. SIRT5 can hydrolyze succinyl, malonyl, and glutaryl lysines. SIRT6 can efficiently remove long chain faXy acyl groups on TNFα.

Smith, B. C.; Hallows, W. C.; Denu, J. M. Chem. Biol. 2008, 15, 1002; Jin, J.; He, B.; Zhang, X.; Lin, H.; Wang, Y. J. Am. Chem. Soc. 2016, 138, 12304.

cons

• HDAC inhibitors have been approved for the treatment of cutaneous or peripheral T cell lymphomas.

• Most sirtuins possess NAD+-dependent protein deacylase acAvity, removing not only acetyl groups but a

☹

• HDAC inhibitors that are selecAve for a

parAcular subtype offer the potenAal of less toxic drugs, with wider therapeuAc windows.

which tend to cause large-scale changes in gene expression. largely empiric way as agents that were iniAally invesAgated to induce tumour cell differenAaAon.

• Their broad effects on chromaAn modulate the expression of many genes at the same Ame, but also likely underpin the clinical toxiciAes observed.

Epigenetic drugs in clinical trials

Epigenetic drugs in clinical trials



• Bromodomain ligands

• Bromodomain ligands

• Lysine methyltransferase inhibitors • EZH2 inhibitors • DOT1L inhibitors

• Lysine demethylase inhibitors • LDS1 inhibitors

NH3

PMT

Me Me N Me

SAM

Me Me N Me

KDM

NH3

• Lysine methyltransferase inhibitors • EZH2 inhibitors • DOT1L inhibitors

• Lysine demethylase inhibitors • LDS1 inhibitors

NH3

PMT

Me Me N Me

SAM

Me Me N Me

KDM

NH3

Writers, readers and erasers

Bromodomain structure



HDAC

inhibitors

HAT

inhibitors

Vorinostat, Romidepsin

HATs writing

HDACs erasing

αA αB αC αZ ZA/BC loop NH3+

NH3+ H3C

S CoA O

NH

HS CoA O

CH3

H2O

HO

CH3 O

bromodomain binding reading

• Bromodomains are protein modules that bind to acetylated lysine residues (KAc). • They are viewed as “readers” of the epigeneAc codes, and work by promoAng the assembly of protein complexes, which are oden involved in transcripAon.

• In humans, bromodomains exist exclusively as part of much larger proteins. Conway, S. J. ACS Med. Chem. Lett. 2012, 3, 691; Arrowsmith et al. Nature Rev. Drug Disc. 2012, 11, 384; Dhanak, D. ACS Med. Chem. Lett. 2012, 3, 521.

Brand et al., ACS Chem. Biol. 2015, 10, 22; Filippakopoulos & Knapp Nature Rev. Drug Disc. 2014, 13, 337; Hewings et al., J. Med. Chem. 2012, 55, 9393; Chung, C.-W. Prog. Med. Chem. 2012, 51, 1; Filippakopoulos & Knapp FEBS Lett. 2012, 568, 2692; Furdas et al., Med. Chem. Commun. 2012, 3, 123; Chung & Witherington J. Biomol. Screen. 2011, 16, 1170; Sanchez & Zhou Curr. Opin. Drug Disc. Develop. 2009, 12, 659.

Bromodomains

Therapeutic potential of BET inhibitors



BET bromodomains

• 61 unique bromodomains have been idenAfied in 46 separate proteins.

• PhylogeneAc profiling allows

characterisaAon of the bromodomains into subfamilies.

• The bromodomain and extra C-terminal

domain (BET) subfamily have been most studied in terms of probe development.

• BRD4, in parAcular, is involved in

condiAons such as inflammaAon and cancer.

Hewings et al., J. Med. Chem. 2012, 55, 9393; Filippakopoulos et al. Cell 2012, 149, 214.

Nicodeme et al. Nature 2010, 468, 1119. Delmore et al. Cell 2011, 146, 904. Mertz et al. PNAS 2011, 108, 16669. Zuber et al. Nature 2011, 478, 524. Dawson et al. Nature 2011, 478, 529. Matzuk et al. Cell 2012, 150, 673.

Mechanism of transcriptional regulation by BRD4

Bromodomain inhibitors - (+)-JQ1 & I-BET762



Me

N N

N

S Me

OtBu

N O

Me

Adachi, K. et al. 2006, PCT/JP2006/310709 (WO/2006/129623); Miyoshi, S. et al. 2009, PCT/JP2008/073864 (WO/2009/084693); Filippakopoulos et al. Nature 2010, 468, 1067; Zuber et al. Nature 2011, 478, 524.

(+)-JQ1

Cl

• BRD4 can recruit the mediator complex by docking to acetylated chromaAn regions, thus sAmulaAng transcripAon.

Me

• The kinase PIM1 phosphorylates histone H3 at S10, helping to recruit a 14-3-3 protein, which, in turn, • MOF acetylates histone H4, resulAng in new docking sites for BRD4, which further acts to recruit the

expression of some oncogenes in cancer.

Filippakopoulos & Knapp Nature Rev. Drug Disc. 2014, 13, 337.

N

NH

N

MeO

posiAve transcripAon elongaAon factor B (PTEFB; the complex formed by cyclin-dependent kinase 9 (CDK9) and its acAvator cyclin T) to acetylated promoter regions, leading to phosphorylaAon of the carboxyterminal heptat repeat region of RNA polymerase II (RNA Pol II).

• BRD4 is par,cularly enriched at enhancer and super-enhancer regions, which strongly s,mulates the

N N

facilitates docking of the acetyltransferase MOF.

O

Nicodeme et al. Nature 2010, 468, 1119; Chung et al. J. Med. Chem. 2011, 54, 3827; Mirguet et al. J. Med. Chem. 2013, 56, 7501.

Cl

I-BET762

Development of DMI-based bromodomain ligands

NCI-60 panel data



OH Me HO Me

IC50 vs BRD4(1) / M

382 × 10-9

pIC50 Heavy atom count clogP

• HL-60(TB) (promyelocyAc leukemia), RPMI-8226 (myeloma), SR (large cell

immunoblasAc lymphoma), and A498 and UO-31 (renal carcinomas) were parAcularly sensiAve.

• One NSCLC (HOP-92, large-cell carcinoma), once CNS cancer (SNB-75,

glioblastoma), and three breast cancers (MCF7 and MDA-MB-468, adenocarcinomas, and HS 578T, carcinoma) were also sensiAve (GI50 < 3 μM).

Hewings et al. J. Med. Chem. 2011, 54, 6761; Hewings et al. J. Med. Chem. 2013, 56, 3217.

with David Hewings.

O N

OXFBD 02

LE (LLE) RMM H-bond donors H-bond acceptors

6.42 22 3.63 0.41 (2.8) 295 2 4

TPSA

66.49

clogD7.4

3.62

#Ar rings SFI (clogD7.4 + #Ar)

3 6.62

NCI-60 panel data

BET bromodomain ligands


Overview → Epigenetics → DNMTs → HDACs → Bromodomains → PMTs → KDMs → Conclusion Me N N

OH

Me

NH

O

N N Me

N N

Me

OXFBD 02 IC50 vs BRD4(1) / M pIC50

clogP

panel (median GI: OXFBD 02 = 52.9%, PFI-1 = 59.2%; median GI50: OXFBD02 = 6.5 μM, PFI-1 = 5.5 μM)

• There is a very good correlaAon (Pearson’s product-moment

correlaAon coefficient, r = 0.82) in pGI50 values between OXFBD 02 and PFI-1.

with David Hewings.

Ph

OMe H N S O O

pIC50 Heavy atom count clogP LE (LLE)

Mount Sinai: MS417 R = Me BRD4(1) IC50 = 30 nM (Fluorescence Anisotropy) N O N

Me RO

0.41 (2.8)

N N H

Me

Me O

6.66 24 2.11 0.39 (4.6)

Me O

O N

Resverlogix: RVX-208 BRD4(1) IC50 = 1800 nM (FRET) BRD4(2) IC50 = 40 nM (FRET) J. Am. Coll. Cardiol. 2010, 55, 2580 PLoS ONE 2013, 8, e83190

O O S N H

Me

N O N Me GSK: I-BET151 BRD4(1/2) IC50 = 36 nM (Fluorescence Anisotropy) BRD4(1/2) KD = 100 nM (SPR) Nature 2011, 478, 529 ChemMedChem 2014, 9, 580

OMe H N S O O

O N

N N H

Me O

CN

SGC / Oxford Chemistry BRD4(1) IC50 = 220 nM (AlphaScreen) Med. Chem. Commun. 2013, 4, 140

Pfizer / SGC: PFI-1 BRD4(1) IC50 = 220 nM (AlphaScreen) J. Med. Chem. 2012, 55, 9831 Cancer Res. 2013, 73, 3336

Cl HO2C

S

Me

N Me

S O

220 × 10-9

N Me

HN

Oxford Chemistry / SGC: OXFBD02: R = H RD4(1) IC50 = 382 nM (AlphaScreen) OXFBD03: R = Ac RD4(1) IC50 = 371 nM (AlphaScreen) J. Med. Chem. 2011, 54, 6761 J. Med. Chem. 2013, 56, 3217

PFI-1 IC50 vs BRD4(1) / M

OMe O Cl

Constellation SGC / Broad: R = tBu: (+)-JQ1 BRD4(1) IC50 = 77 nM (AlphaScreen) BRD4(1) IC50 = 26 nM (AlphaScreen) ACS Med. Chem. Lett. 2013, Nature 2010, 468, 1067 4, 835

OH

22 3.63

LE (LLE)

Me

Me

OH

Me

10-9

6.42

Heavy atom count

• PFI-1 and OXFBD 02 have similar average acAvity value across the

382 ×

N

MeO

NH

Cl

GSK: I-BET762 (GSK525762A) BRD4(1/2) IC50 = 36 nM (FRET) Nature 2010, 468, 1119 J. Med. Chem. 2011, 54, 3827 J. Med. Chem. 2013, 56, 7501

O N

Me O

Me

H3CO

NH2

O

N

S

N

Cl

HO

N O Me

S Me

Me

R O

O

N

NH H N

NH2

N N

HO O

S Chinese Academy of Sciences BRD4(1) IC50 = 230 nM (Fluorescence Anisotropy) J. Med. Chem. 2013, 56, 3833

Brand et al., ACS Chem. Biol. 2015, 10, 22.

HN

N O

Me

O N S NH O

Me

Me

HO Et HN

O Me

N H

O

O

Et S N O Et

Me

GSK: I-BET726 BRD4(1/2) IC50 = 22 nM (FRET) PLoS ONE 2013, 8, e72967

Mount Sinai: MS436 BRD4(1) KD =

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