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Structures and Drug Binding Potentials of HIF‐/ARNT Complexes

Fraydoon Rastinejad

SBP Medical Discovery Institute ARNT

HRE

CBP/p300

nucleusHIF‐1

Hypoxia (low oxygen stress)

Accumulation

Targetgenes

HIF targets regulate multiple steps of cancer development 

Metabolism:  (GLUT1, PDK1, LDHA)

Proliferation & Survival:  (MYC, p53, PI3K/AKT)

Angiogenesis:  (VEGF, PDFG)

Cell Infiltration:  (CXCR4, SDF1)

Invasion and Metastasis: (LOX, TWIST)

Cancer Stem Cell:   Notch, MYC, WNT pathways

PAS-BPAS-AbHLH Transactivation

HIF‐1and HIF‐2 Crystallized AsHeterodimers

+/‐ DNA

+/‐ Small‐Molecules

The Segment We Crystallized

ARNT (HIF‐1)

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bHLH

Transactivation

bHLH

Are there Any Ligand Binding Pockets ?

AreDomains Coupled ?

How do Human Mutation Alter 

Function?How do they 

Recognize DNA?

Do All Family 

Members Look the Same ?

Questions We Asked

HIF‐2ARNT

bHLH

PAS‐A

PAS‐B

bHLH

PAS‐A

PAS‐B

OX3(selective for HIF‐2)

Proflavine/Acriflavine(Inhibits both

HIF‐1 & HIF‐2

HIF‐ Inhibitors

G.L Semenza Lab (JH)

R.K.Bruick Lab (UTSW)

“Acriflavine/Proflavine inhibits HIF‐1 Dimerization, Tumor Growth and Vascularization”Lee, K, Zhang, H., Qian, DZ, Rey, S., Liu, JO, and Semenza, GL.  PNAS 2009

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PAS-BbHLHPAS-B PAS-A

bHLH

PAS-A

0X3

PAS-BbHLHPAS-B PAS-A

bHLH

PAS-A

Proflavine

PAS-BbHLHPAS-B PAS-A

bHLH

PAS-A

Space Available

PAS-BbHLHPAS-B PAS-A

bHLH

PAS-A

PAS‐A

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PAS-BbHLHPAS-B PAS-A

bHLH

PAS-A

60% of residues in PAS‐A pocket of HIF‐2 are identical in HIF‐1

PAS‐A

bHLH

bHLH

150 Å3370 Å3

110 Å3

110 Å3

60 Å3

5 Distinct Ligand‐Binding Sites

Proflavine Site is similar in HIF‐2/ARNT and HIF‐1/ARNT

Proflavine

Why does OX3 Selectively bind to HIF‐2 ? 

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Smaller pocket

HIF‐2 HIF‐1

Larger pocket

0X3

*

Three Residues Prevent 0X3 Binding to HIF‐1

*

HIF‐2ARNT+DNA

HIF‐1ARNT+DNA

PAS-BPAS-AbHLH

77% 62% 75%% identity

HIF‐1 vs. HIF‐2

5’-GCTGCGTACGTGCGGGTCGTCGACGCATGCACGCCCAGCA-5’

R102 H94 N184 K186

E98A23 R27

PAS domainExtends DNA Footprint

bHLH

Location Possible Role Primary Tissue (Subtype) Associated HistologyHIF-2αK18E bHLH α1 DNA interaction Stomach AdenocarcinomaA23V bHLH α1 DNA interaction Endometrium Endometrioid carcinomaV47M bHLH α1-α2 loop Interface 6 (bHLH/PAS-A) Central nervous system (brain) GliomaF98L PAS-A Aβ Internal stability Large intestine (colon) AdenocarcinomaR166L PAS-A Gβ Internal stability Kidney Clear cell renal cell carcinomaI223M PAS-A Iβ Interface 2 (PAS-A/PAS-A) Lung AdenocarcinomaH248N PAS-B Aβ Internal stability Large intestine (colon) AdenocarcinomaR275H PAS-B Dα-Eα loop Internal stability Cervix Squamous cell carcinomaA277P PAS-B Eα Internal stability Lung Squamous cell carcinomaE279V PAS-B Eα Internal stability Liver Hepatocellular carcinomaHIF-1αK19Q bHLH α1 DNA interaction Endometrium Endometrioid carcinomaR30Q bHLH α1 DNA interaction Skin Malignant melanomaL54I bHLH α1-α2 loop Interface 6 (bHLH/PAS-A) Kidney Clear cell renal cell carcinomaV116E PAS-A Cα Internal stability Kidney Clear cell renal cell carcinomaM120T PAS-A Cα Internal stability Large intestine (colon) AdenocarcinomaM171I PAS-A Gβ Internal stability Kidney Clear cell renal cell carcinomaM250I PAS-B Aβ-Bβ loop Internal stability Lung AdenocarcinomaL262S PAS-B Cα Internal stability Skin Malignant melanomaV341I PAS-B Iβ Internal stability Endometrium Endometrioid carcinoma

Tissue and histology data from the COSMIC database 

Missense Mutations in HIF‐1 and HIF‐2

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Endometrioid Carcinoma

Adenocarcinoma

Glioma

DNA‐Binding modifiers Adenocarcinoma

Clear Cell Renal Carcinoma

Adenocarcinoma

Squamous Cell Carcinoma

Adenocarcinoma

Squamous Cell Carcinoma

Hepatocellular Carcinoma

Domain‐Domain Junction modifiers

Glioma

Squamous Cell Carcinoma

Adenocarcinoma

Squamous Cell Carcinoma

Hepatocellular Carcinoma

PAS‐ pocket modifiers

HIF‐1/2 ARNT+++Gene expression

VEGF  GLUT1  PDGF  CAIX  CXCR4  TGFMetabolic

ReprogrammingAngiogenesis Cancer

Progression

Which Pockets Should We Target with Small‐Molecules Therapeutics?

Are there Endogenous Ligandsthat Regulate HIF Activity ?

New Questions We Are Asking

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PAS-BbHLHPAS-B PAS-A

bHLH

PAS-A

But which sites should we target?

Pocket HIF‐selective ?

Pan‐modulato

r

antagonist agonist MOA

PAS‐B Yes ? Yes ? ?

PAS‐A Yes ? ? ? ?

PAS‐B No Yes ? ? ?

PAS‐A No Yes ? ? ?

JUNCTIONAL

No ? Yes ? ?

HIF‐2ARNT

PAS‐Dom

ains

ProductiveHeterodimer

What We Learned So Far….

Domains Are Tightly Connected in HIF‐1/ARNT and HIF‐1/ARNT‐ so Ligand Binding May Influence DNA‐binding and/or Transcriptional Responses of these complexes.

HIF‐ and ARNT (HIF‐) Have Different Individual Subunit structures.

They Heterodimerize Asymmetrically to form their Quaternary Arrangement.

Mutagenesis and co‐IP studies Validate the Functional Interfaces we Observed.

Major Rearrangements in the bHLH Domains Accompany DNA Binding.

PAS Domains Extend DNA.

Human Cancer Mutations Map to Sensitive Locations in the architectures.

There are Five Different Ligand Binding Sites.

Dalei Wu

Nalini Potluri Youngchang Kim(Argonne Nat’l Labs)

ACKNOWLEDGEMENTS

Vikas ChandraJingping LuXiaoyu SuYuan Seo

Sepideh KhorasnaizadehSiobhan MalanyDavid TerryCPCCG