hypoxia & hif-1

1I M M U N O LO G Y / C A N C E R R E S E A R C H

H Y P O X I A & H I F - 1

F A L L 2 0 0 4

HYPOXIA is oxygen starving at the tissue and cellular levels. It is caused by a reduction of oxygen supply in blood and in tissues below physiological levels. Severe hypoxia can result in anoxia, a complete loss of oxygen to an area of tissue.

There are four major types of hypoxia. The fi rst type, HYPOXIC HYPOXIA, is a decrease of the fraction of inhaled oxygen possibly due to hyperventilation from respiratory depression or altitude above sea level. The second type of hy-poxia is termed ANAEMIC HYPOXIA, and is char-acterized by a decrease in the amount of hae-moglobin that binds oxygen in the blood. This

can be caused by multiple factors, including but not limited to: blood loss, reduced red blood cell production, carbon monoxide poisoning, and a genetic defect of haemoglobin. STAGNANT HYPOXIA, the third type of hypoxia that has been defi ned, results in low blood fl ow and is caused by vasoconstriction and/or heart failure. The fourth type of hypoxia is HYSTOTOXIC HYPOXIA, a poisoning of oxidative enzymes that causes vasodilation in brain arteries and veins, result-ing in more blood fl ow to the brain tissues. This response is probably mediated by nitric oxide (NO) and adenosine.

Hypoxia is a fundamental angiogenic stimu-lus. An important mediator of this primary stim-ulus is HYPOXIAINDUCIBLE TOR1α HIF1α [1]. Normally, this protein is oxidized, ubiquiti-nylated and degraded in the proteasome. In the absence of oxygen, HIF-1α levels increase and stimulate VEGF transcription. Cells of the innate immune system get energy almost entirely from glycolysis, enabling survival under a variety of harsh conditions. In a recent report HIF-1α is now placed at the center of metabolic control in these cells [2].

The regulation of most proteins required for hypoxic adaptation occurs at the gene level and involves transcriptional induction via the bind-ing of a transcription factor, hypoxia-inducible factor-1 (HIF-1), to the conserved sequence, 5-(A/T)CGTG-3, in the hypoxia response element HRE on the regulated genes [3]. To date, about 100 hypoxia-inducible genes have been found to be directly regulated by HIF-1.

CONTINUED ON PAGE 2

M I N I R E V I E W O N H Y P O X I A A N D H I F 1

Tomorrow’s Reagents Manufactured Today™

HIF-1α (120 kDa)(human)

HIF-1β (91-94 kDa)

bHLHN-Terminus C-Terminus826 aa

C-Terminus789 aa

PAS (Per-ARNT-SIM Domains)

bHLHN-Terminus PAS (Per-ARNT-SIM Domains) TAD

TAD-NODD

Dimerization

Transcriptional Modulation

P402 P564 N803

17 71 185 228 401 603

NLS

718 721

DNA Binding(HRE)

5' (A/T)CGTG 3'

531 575

K532

p300/CBP

OHOH OHCOCH3

pVHL Degradation

Hypoxic (<5% O2)

PHD1-3

O2

2-Oxoglutarate(Fe2+) ARD

FIH1

CITED2&4

K391 K477 K532

SUMO SUMO SUMO

TAD-C

C800

NO

IPAS

Normoxic (20% O2)

HYPOXIA

P R O D U C T L I N E N E W S L E T T E R

CONTENTS

HYPOXIA 1

HIF1 & PRODUC TS 2

REGULATION OF HIF1 & PRODUC TS 2/3

REGULATION BY HIF1 & PRODUC TS 4/5

CHEMIC AL INHIBITORS OF HIF1 & PRODUC TS 6

LITERATURE & SELEC TED REVIEW ARTICLES 7

HSP90 RELATED PRODUC TS 7

SMALL MOLECULE INHIBITORS 8

INCLUDES PRODUCTS FROM

Flyer_HIF_final.indd 1Flyer_HIF_final.indd 1 21.09.2004 13:35:5821.09.2004 13:35:58

I M M U N O LO G Y / C A N C E R R E S E A R C H2

For Prices visit our Online Catalog at www.alexis-e.biz, contact your Local Distributor, or call +41 61 926 89 89

Semenza and Wang [4] were the fi rst to re-port that the DNA sequence of HRE is critical for hypoxia-inducibility and that purifi ed HIF-1 binds to HRE, by using affi nity-purifi cation oli-gonucleotides with the HRE sequence [5].

HIF-1 is a heterodimer composed of a 120 kDa HIF-1α subunit and a 91-94 kDa HIF-1β subunit (fi rst described as the aryl hydrocar-bon (Ah) nuclear receptor translocator (ARNT) [6]), both of which are members of the basic helix-loop-helix (bHLH)-PAS family. PAS is an acronym for the three members fi rst recognised (Per, ARNT, and Sim). Accordingly, HIF-1α and HIF-1β each contain a bHLH domain near the N-terminus preceding the PAS domain. Whereas the basic domain is essential for DNA binding, the HLH domain and N-teminal half of the PAS domain are necessary for heterodimerization and DNA binding. Moreover, there are two tran-

scriptional activation domains (TADs) in HIF-1α, the N-terminal activation domain (TAD-N), and the C-terminal activation domain (TAD-C). In contrast, HIF-1β contains only one tran-scriptional activation domain at the C-terminus. Furthermore, HIF-1α possesses a unique oxy-gen-dependent degradation domain (ODD) that controls protein stability. A portion of the ODD overlaps with the TAD-N.

In addition to the ubiquitous HIF-1α, the HIF-1α family contains two other members, HIF-2α (also called EPAS1 [7], MOP2 [8] or HLF [9]) and HIF-3α [10], both of which have more restricted tissue expression [11]. HIF-2α and HIF-3α contain domains similar to those in HIF-1α and exhibit similar biochemical prop-erties, such as heterodimerization with HIF-1β and DNA binding to the same DNA sequence in vitro. HIF-2α is also tightly regulated by oxygen

tension and its complex with HIF-1β appears to be directly involved in hypoxic gene regulation, as is HIF-1α [12]. However, although HIF-3α is homologous to HIF-1α, it might be a negative regulator of hypoxia-inducible gene expression [13]. To activate transcription, HIF-1α must re-cruit the transcriptional adapter/histone acetyl-transferase proteins, p300 and CBP to the target gene promoters. This occurs by direct physical interactions between the transactivation domain (TAD) of HIF-1α and the fi rst cysteine-histidine (CH1) domain of the p300/CBP complex. This domain of p300/CBP also binds members of the CITED (CBP/p300 interacting transactivator with ED-rich tail) family of transcription factors. CITED2 [14] and CITED4 [15] disrupt the in-teraction between HIF-1α and p300 and inhibit transactivation by HIF-1α and gene activation by hypoxia.

Oxygen-dependentAt normal oxygen levels NORMOXIC CONDI

TIONS, HIF-1 prolyl-hydrolases (PHD-1, -2 & -3) hydroxylate the prolyl residues at Pro 402 and 564 of the HIF-1α ODD. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF-1) hydroxylates Asp803 of the HIF-1α C-ter-minal transactivation domain. The hydroxylated peptide interacts with an E3 ubiquitin-protein ligase complex composed of the von Hippel-Lindau tumor suppressor protein VHL, elongin B & C and cullin 2. After being poly-ubiquitinized, HIF-1α is degraded by the 26S proteasome.

Under HYPOXIC CONDITIONS, HIF-1α is not hydroxylated because the major substrate, di-oxygen, is not available. The unmodifi ed protein escapes the VHL-binding, ubiquitination and deg-radation, and then dimerizes HIF-1β. The het-

erodimeric HIFs upregulate a myriad of hypoxia-inducible genes, triggering physiologic responses to hypoxia. Interestingly, PHD-1 and -2 are in-ducible by hypoxia, indicating a feedback loop to avoid excessive nuclear HIF-1 accumulation.

Oxygen-independentIn addition to mediating adaption to hypoxia,

HIF-1 also contributes to other cellular process-es that occur under normoxic conditions, such as the development of normal tissues or tumors, the determination of cell death or survival, im-mune responses and the adaption to mechanical stress. Under normoxic conditions HIF-1 can be activated by various cytokines, growth fac-tors, transition metals, iron chelation (inhibits enzymatic activity), as well as nitric oxide (NO). Degradation of HIF-1 in a VHL- and oxygen-in-

dependent manner is mediated by p53. Dephos-phorylated HIF-1α binds to p53 while phospho-rylated HIF-1α is the major form that binds to HIF-1β [16]. HIF-1α protects p53 degradation by binding to Mdm2, the principal cellular an-tagonist of p53 [17]. Also, inhibition of PI3K or mTOR prevent growth factor- and cytokine-induced HIF-1α accumulation. Generally HIF-1α induction by hypoxia is far greater than by growth factors and cytokines. The COP9 subunit CSN5 is able to stabilize HIF-1α in a pVHL-inde-pendent form aerobically by inhibiting HIF-1α prolyl-564 hydroxylation [18].

HIF-1, Nitric Oxide (NO) and ROSHypoxia stimulates nitric oxide (NO) forma-

tion through the induction of inducible nitric oxide synthase (iNOS; NOS II), the transcrip-

HIF1

REGULATION OF HIF1

MAb to HIF-1α (mgc3) ALX-804-216-R100 100 µlCLONE: mgc3. ISOT YPE: Mouse IgG1. IMMUNOGEN: Human hypoxia-inducible factor 1α (HIF-1α) (aa 530-826). SPECIFICIT Y: Recognizes human, non-human primate, bovine and pig HIF-1α. Recognizes mouse HIF-1α only in Gel Supershift assays; does not cross-react with HIF-1β (ARNT) or HIF-2α. Detects a band of ~116kDa by Western blot after hypoxic in-duction. APPLICATION: Gel Supershift assay (on mouse), ICC, IP, WB. LIT: General applicability of chicken egg yolk antibodies: the per-formance of IgY immunoglobuilins raised against thy hypoxia-in-ducible factor 1alpha: G. Camenisch, et al.; FASEB J. 13, 81 (1999)

PAb to HIF-1αALX-210-069-C050 50 µgFrom rabbit. IMMUNOGEN: Recombinant HIF-1α (aa 609-826). SPECIFICIT Y: Recognizes human and mouse HIF-1α. APPLICATION: IHC, IP, WB.

MAb to HIF-1β (2B10)[anti-Ah Receptor Nuclear Translocator (ARNT) MAb]ALX-804-108-R100 100 µlCLONE: 2B10. ISOT YPE: Mouse IgG1. IMMUNOGEN: Synthetic peptide corresponding to aa 771-789 (N771SYNNEEFPDLTMFPPFSE789) of human HIF-1β (Ah receptor nuclear translocator; ARNT). SPECIFICIT Y: Recognizes human, mouse and monkey ARNT. APPLICATION: ICC, IP, WB. LIT: Physiochemical and immunocytochemical analysis of the aryl hydrocarbon receptor translocator: Characterization of two mono-clonal antibodies to the ARNT: N.G. Hord and G. Perdew; Mol. Phar-macol. 46, 618 (1994)

PAb to HIF-1β (mouse)[anti-Ah Receptor Nuclear Translocator (ARNT) PAb]ALX-210-150-R100 100 µlFrom rabbit. IMMUNOGEN: Synthetic peptide corresponding to aa 771-789 (N771SYNNEEFPDLTMFPPFSE789) of human HIF-1β (Ah receptor nuclear translocator; ARNT). SPECIFICIT Y: Rec-ognizes mouse ARNT. APPLICATION: WB.

PAb to Ah Receptor Nuclear Translocator 3 [ARNT3][anti-BMAL1 PAb]ALX-210-245-R200 200 µlFrom rabbit. IMMUNOGEN: Synthetic peptide corresponding to aa 582-594 (D582MIDNDQGSSSPS594) of mouse ARNT3 (Ah receptor nuclear translocator 3; BMAL1). SPECIFICIT Y: Recog-nizes human and hamster ARNT3. APPLICATION: WB. BLOCKING PEPTIDE: Prod. No. ALX-152-023.

PRODUCTS


FC = Flow Cytometry; ICC = Immunocytochemistry; IP = Immunoprecipitation; IHC = Immunohistochemistry (FS = Frozen Sections, PS = Paraffin Sections); WB = Western blot


tion of which is enhanced by HIF-1. Conversly, NO affects the HIF-1-mediated induction of the hypoxia-inducible genes, such as the erythro-poietin and vascular endothelial growth factor genes [19]. However, the effect of NO on HIF-1α expression is reciprocal and may depend on the concentration of NO. NO has been shown to block the hypoxic stabilization of HIF-1α but also represses its transcriptional activity [20]. However, under different conditions NO can induce the accumulation of HIF-1α even un-der normoxic conditions by interfering with the function of PHDs [21, 22]. E.J. Yeo, et al. have

shown that YC-1, known as a NO-dependent, su-peroxide-sensitive stimulator of soluble guanylyl cyclase and non-specifi c phosphodiesterase in-hibitor, also inhibits HIF-1α activity in tumors resulting in blocked angiogenesis and an inhibi-tion of tumor growth [23, 24].

Reactive oxygen species (ROS) impair the expression of HIF-1α in hypoxic cells and the DNA-binding activity of HIF-1 [25-27]. Another hypothesis suggests that in case of oxygen defi -ciency, the last step in the respiratory chain is blocked because oxygen is essentially required for removing electrons. Electrons thus accumu-

late in the respiratory chain and produce ROS by reducing oxygen remaining in the mitochon-dria [28]. ROS generated by the mitochondrial complex III participate in HIF-1α stabilization [29]. In addition, it has been demonstrated that HIF-1α is up-regulated even under normoxic conditions by cytokines generating ROS [30, 31]. Reoxygenation of tumor cells leads to oxi-dative stress and stabilization of the HIF-1 dimer through free radical intermediates and depoly-merises the hypoxia-induced translational sup-pressors known as stress granules [32].

CONTINUED ON PAGE 5

Growth Factors

Hypoxic (<5% O2)

Insulin, IGF-1 & -2, EGF, FGF-2

Ras

RAF

+

MEK

ERK

++ Stress

p38

JNK

+

PD98059 ---

SP600125JNKI1

---

SB203580SB220025

---

Raf1 Kinase Inhibitor ---

+

+

+

HIF-1 Degradation(26S Proteasome)

�

Oxygen SensingPHD1-3, FIH

His-Fe -HISI I

Asp

O2

2-OxoglutarateCO2

Succinate

---

N-OxalylglycineDimethyloxallylglycine

2,4-Diethylpyridine dicarboxylate

HIF-1 Transactivation�

+

+

++

Apoptosis

HIF-1�

PI(3)K (p110)�

Akt

WortmanninLY-294,002

--- PTEN

---

mTOR

GSK-3

TNF- , IL-1 , Angiotensin II, Thrombin, PDGF, HER2� �

+

p70S6K

---

++

+

HIF-1 Synthesis�

Rapamycin ---

SH-5SH-6

---

+

HIF-1�

Normoxic (20% O2)

Pro564 Pro

402

VHL

Asn803HIF-1�

OH OH

OH

Ub

Ub

Ub

Ub

HIF-1�

Bax

p53

Nucleus

p300/CBP

HIF-1�

HIF-1�

Transcription CITED2

+

---

--- OTDZTTDZD-8TIBPO

Bisindolylmaleimide V ---

U0126 ---

HIF-1�

PAb to PHD1 (BL525) BET-A300-326A 0.1 mgFrom rabbit. IMMUNOGEN: Synthetic peptide corresponding to a portion of the C-terminus of human prolyl hydroxylase domain-containing protein 2 (PHD1; EGLN2). SPECIFICIT Y: Recognizes human and mouse PHD1. APPLICATION: WB.

PAb to PHD2 (human) (BL521) BET-A300-322A 0.1 mgFrom rabbit. IMMUNOGEN: Synthetic peptide corresponding to a portion of human prolyl hydroxylase domain-containing pro-tein 2 (PHD2; EGLN1) encoded within exon 1. SPECIFICIT Y: Recognizes human PHD2. APPLICATION: WB.

PAb to PHD3 (BL526) BET-A300-327A 0.1 mgFrom rabbit. IMMUNOGEN: Synthetic peptide corresponding to a portion of human prolyl hydroxylase domain-containing pro-tein 3 (PHD3; EGLN3) encoded within exon 1. SPECIFICIT Y: Recognizes human and rat PHD3. APPLICATION: WB.

PAb to PHD4 (human) (BL529)BET-A300-330A 0.1 mg From rabbit. IMMUNOGEN: Synthetic peptide corresponding to a portion of human prolyl hydroxylase domain-containing pro-tein 4 (PHD4; EGLN4) encoded within exon 4. SPECIFICIT Y: Recognizes human PHD4. APPLICATION: WB.

PAb to Von Hippel-Lindau Disease Tumor Suppressor ProteinJBS-ABD-016 50 µgFrom rabbit. IMMUNOGEN: Recombinant human Von Hippel-Lindau (VHL) disease tumor suppressor protein. SPECIFICIT Y: Recognizes human VHL. APPLICATION: WB.

Von Hippel-Lindau Disease Tumor Suppressor Protein (human) (rec.)JBS-PR-768 5 µgProduced in Sf9 cells. PURIT Y: ≥90%, homogeneous. Contains no detectable protease, DNase and RNase activity. APPLICATION: For in vitro transcriptional activation and protein-pro-tein interaction assays.

PAb to CSN5 (human) (BL1062)BET-A300-014A 0.1 mg From rabbit. IMMUNOGEN: Synthetic peptide corresponding to a C-terminal domain of human COP9 signalosome complex subunit 5 (CSN5). SPECIFICIT Y: Recognizes human CSN5. Other species have not been tested. APPLICATION: WB.

PRODUCTS




REGULATION OF HIF1 ACTIVITYHIF-1 Prolyl-hydrolase 1 (PHD1) [1]CITED2 (P35srj) [2]IPAS [3]

ANGIOGENESISAdrenomedullin [4]LDL-receptor-related Protein 1 (LRP1) [5]VEGF [6]VEGFR-1 (Flt-1) [7]TGF-β3 [8]Leptin [9, 10]Endoglin (CD105) [11]Plasminogen-activator Inhibitor-1 (PAI-1) [12]Connective Tissue Growth Factor (CTGF) [13]Pentraxin 3 (PTX3) [14]CancerN-myc Downstream Regulated Gene 1(Cap43; NDRG1) [15]CXCR4 ↓ [16, 17]Stromal Cell-derived Factor-1 (SDF-1; CXCL12) [18]c-MET [19]ARMET [14]

DRUG RESISTANCEP-glycoprotein (MDR1) [20]

CELL PROLIFERATIONp21 (WAF-1/CIP1), p53, Bcl-2 [21]p27 [22]IGF2, IGFBP-2, IGFBP-3 [23]IGFBP-1 [24]TGF-α [25]TGF-β1, TGF-β2, TGF-β3 [8]Cyclin G2 [5]Cyclin D1 ↓ [26]hTERT ↓ [27]Nucleophosmin [28]

CELL GROWTHP58IPK [14]

APOPTOSISAdrenomedullin [4]Bcl-2 [21]Bid ↓, Bax ↓ [29]NIP3 [30]BNip3, NIX [31]RTP801 [32]NOXA [33]HGTD-P [34]REDD2 [35]

ERSTRESS GADD153 (CHOP), HERP, HEDJ, ERDJ4, GRP78 (Bip), GRP94 [14]

ALZHEIMER’S DISEASEPresenilin-1 [36]Presenilin-2 [37]

DNA REPAIRBRCA1 ↓ [27]GADD153 (CHOP) [14]

TRANSCRIPTIONAL REGULATIONDEC1 (Stra13; Sharp2) [5, 38]DEC2 [39]ETS-1 [40]NUR77 [41]ATF3 [14]

ATF4 [42]XBP1 [14]

CYTOSKELETAL STRUCTUREVimentin [43, 25]Keratin 14, Keratin 18, Keratin 19 [25]

MOTILITYLDL-receptor-related Protein 1 (LRP1) [5]TGF-α [25]Glucose-6-phosphate Isomerase (GPI, AMF, Neuroleukin) [44]

CELL ADHESIONMIC2 (CD99) [5]

VASOMOTOR TONEEndothelin-1 [5]Adrenomedullin [4]α1β-Andrenergic Receptor (α1β-AR) [45]Inducible Nitric Oxide Synthase (iNOS; NOS II) [46]Heme Oxygenase 1 (HO-1; human: ↓, rat, bovine, monkey, hamster: ↑), Bach1 [47]Atrial Natriuretic Peptide (ANP) [71, 72]

EXTRACELLULAR MATRIX METABOLISMPlasminogen-activator Inhibitor-1 (PAI-1) [12]Prolyl-4-hydroxylase α-1 (4-PHα-1) [48]Collagen type V (α1) [5]Cathepsin D, Fibronectin 1, Matrix Metalloproteinase 2 (MMP2), Urokinase Plasminogen Activator Receptor (UPAR) [25]

COAGULATION/WOUND HEALING Transferrin (CD142; Thromoplastin) [49]Fibronectin 1 [25]Viral-like 30 Element (VL30) [50]

MUCOSAL REPAIRIntestinal Trefoil Factor (ITF) [51]

ATP/GLUCOSE/ENERGY METABOLISMGlyceraldehyde-3-P-dehydrogenase (GAPDH) [52, 53]Glucose-6-phosphate Isomerase (GPI; AMF; Neuroleukin) [44]Glucose Transporter 1 & 3 (GLUT1 & GLUT3) [54]Prolyl-4-hydroxylase α 1 (4-PHα-1) [48]Phosphofructokinase L (PFKL), Lactate Dehydrogenase A (LDHA), Aldolase A&C, Pyruvate Kinase M, Enolase 1 [55]Hexokinase 1 & 2 [56]6-Phosphofructo-2-kinase/Fructose-2,6-biphosphatase-3 (PFKFB3) [57]Phosphoglyceratekinase 1 (PGK1) [58, 55]Triose Phosphate Isomerase (TPI) [59]

DIABETESHepatic Growth Factor (HGF) [43]Leptin [9, 10]

OBESITYPPARγ2 ↓ [60]

INFLAMMATIONCOX-2 [36]Integrin β2 Subunit (CD18) [61]CYP4B1 [62]Adrenomedullin [63]

ERYTHROPOIESIS/IRON METABOLISMErythropoietin [64]Transferrin [65]Transferrin Receptor [66]

Ceruloplasmin [67]Coproporphyrinogen Oxidase [14]

NUCLEOTIDE METABOLISMAdenylate Kinase 3 (AK3) [49]Ecto-5‘-nucleotidase (5’NT, CD73) [68]

AMINO ACID METABOLISMTransglutaminase 2 [5]Asparagine Synthetase [14]

RENINANGIOTENSIN SYSTEMAminopeptidase A [5]

PH REGULATIONCarbonic Anhydrase 9 [69]

MISCELLANEOUSNorepinephrine [70]

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TABLE 1: TRANSCRIPTIONALLY REGULATED GENES BY HIF1




The HIF-1α/HIF-1β dimer recruits the coac-tivator p300/CBP. This complex then binds and activates HREs in HIF target genes to modulate transcription of those genes. A recent screen of 600,000 compounds identifi ed a single specifi c inhibitor, chetomin, of HIF-1α binding to p300 [33]. HIF-1 regulates the expression of a broad range of genes that facilitate acclimatization to low oxygen conditions. Its targets include genes that code for molecules that participate in va-somotor control, angiogenesis, erythropoiesis, iron metabolism, cell proliferation and cell cy-cle control, cell death and energy metabolism. Hypoxia is also a critical determinant of sur-vival of the activated T cells via the HIF-1α-AM cascade, defi ning a previously unknown mode of regulation of peripheral immunity [39]. For an overview of hypoxia-mediated gene ex-pression see Table 1. CONTINUED ON PAGE 6

PAb to CREB Binding Protein [CBP] ALX-210-229-C100 100 µgFrom rabbit. IMMUNOGEN: Synthetic peptide corresponding to aa 162-176 (A162TSSPATSQTGPGIC176) in the nuclear factor binding domain of human CBP (CREB binding protein). SPECIFICIT Y: Recognizes human and mouse CBP. Detects a band of ~265kDa by Western blot. APPLICATION: WB. BLOCKING PEPTIDE: Prod. No. ALX-153-023.

PAb to CREB Binding Protein [CBP] (human) (BL440)BET-A300-362A 0.1 mgFrom rabbit. IMMUNOGEN: Synthetic peptide corresponding to a portion of human CREB binding protein (CBP) encoded within exon 17. This sequence is conserved in 22 of 29 resi-dues between human and mouse. SPECIFICIT Y: Recognizes hu-man CBP. APPLICATION: IP.

PAb to CREB Binding Protein [CBP] (human) (BL441)BET-A300-363A 0.1 mgFrom rabbit. IMMUNOGEN: Synthetic peptide corresponding to a portion of human CREB binding protein (CBP) encoded within exon 33. This sequence is conserved in 28 of 31 resi-dues between human and mouse. SPECIFICIT Y: Recognizes hu-man CBP. APPLICATION: IP.

p300 (human) (rec.)JBS-PR-762 2 µgProduced in Sf9 cells. PURIT Y: >95% (SDS-PAGE). APPLICATION: For in vitro transcriptional activation, protein-protein interaction assays, in vitro acetylation assays and cell growth assays.

p300 (C-terminal Domain) (human) (rec.)JBS-PR-770 4 µgProduced in Sf9 cells. PURIT Y: >95% (SDS-PAGE). APPLICATION: For in vitro transcriptional activation, protein-protein interaction assays, in vitro acetylation assays and cell growth assays.

PAb to p300 (human) ALX-210-228-C100 100 µgFrom rabbit. IMMUNOGEN: Synthetic peptide corresponding to aa 139-151 (G139TSGPNQGPTQST151) in the nuclear factor binding domain of human p300. SPECIFICIT Y: Recognizes hu-man p300. Detects a band of ~265 kDa by Western blot. APPLICATION: WB. BLOCKING PEPTIDE: Prod. No. ALX-165-038.

PAb to p300 (human) (BL442)BET-A300-358A 0.1 mg From rabbit. IMMUNOGEN: Synthetic peptide corresponding to a portion of human E1A binding protein p300 encoded within exons 15 and 16. SPECIFICIT Y: Recognizes human p300. APPLICATION: IP.

PAb to p300 (human) (BL443)BET-A300-359A 0.1 mgFrom rabbit. IMMUNOGEN: Synthetic peptide corresponding to a portion of human E1A binding protein p300 encoded within exon 16. SPECIFICIT Y: Recognizes human p300. APPLICATION: IP.

Anacardic acid[2-Hydroxy-6-pentadecylbenzoic acid; 6-Pentadecylsalicylic acid; AA]ALX-270-381-M005 5 mgALX-270-381-M025 25 mgCell permeable salicylic acid analog that acts as a potent, non-competitive inhibitor of p300 and PCAF (p300/CBP-associated factor) HAT (histone acetyltransferase) activities (IC50~8.5µM and ~5µM, respectively).LIT: Small molecule modulators of histone acetyltransferase p300: K. Balasubramanyam, et al.; J. Biol. Chem. 278, 19134 (2003).

CTPB[N-(4-Chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentade-cyl-benzamide]ALX-420-033-M001 1 mgALX-420-033-M005 5 mgPotent activator of p300 HAT, but not of PCAF (p300/CBP-as-sociated factor) HAT (histone acetyltransferase) activities.LIT: Small molecule modulators of histone acetyltransferase p300: K. Balasubramanyam, et al.; J. Biol. Chem. 278, 19134 (2003).

PRODUCTS

REGULATION BY HIF1

VEGF-A [VEGF] (human) BioLISA Kit ALX-850-298-KI01 1 Kit QUANTIT Y: 96 wells (~80 tests). SENSITIVIT Y: 11 pg/ml. APPLICATION: For measurements of active human vascular en-dothelial growth factor A (VEGF-A; VEGF) in cell culture super-natants, human serum, plasma and other biological samples. PRODUC T DESCRIPTION: Compared to normal ELISA kits, this kit uses rsVEGFR-1 to capture the antigen instead of an anti-body, thus only biologically active VEGF-A will be measured.

sVEGFR-1 [Flt-1] (human) ELISA Kit ALX-850-264-KI01 1 Kit QUANTIT Y: 96 wells (~80 tests). SENSITIVIT Y: 100 pg/ml. APPLICATION: For measurement of total human soluble vascular endothelial growth factor receptor-1 (sVEGFR-1; Flt-1) in cell culture supernatants, human serum, heparin treated plasma and other biological samples.

PAb to VEGF-A [VEGF] (human) SIG-861-01 1 ml From rabbit. IMMUNOGEN: MAP peptide of the N-terminal sequence (aa 1-20) of human vascular endothelial growth fac-tor A (VEGF-A; VEGF). SPECIFICIT Y: Recognizes human VEGF-A. APPLICATION: IHC (PS, FS not tested), IP, ELISA, WB.

VEGFR RELATED PRODUCTS

FOR A FULL PANEL OF SYNTHETIC SMALL MOLECULES SEE PAGE 8 OR VISIT WWW.ALEXISE.BIZ

LATEST INSIGHTS

Muscle HIF-1α knockout mice show a meta-bolic shift away from glycolysis toward oxidation leading to increasing exercise times. This dem-onstrates an important role for the HIF-1 path-way in the metabolic control of muscle function. Interestingly, mice with enhanced expression of PPARδ show a similar increase in running en-durance.

HIF-2α promotes adipose differentiation by inducing PPARγ2, suggesting that HIF-2α plays a role in adipogenesis and adipocyte function in-cluding regulation of glucose uptake followed by lipid synthesis. C75 (Prod. No. ALX-270-286), a fatty acid synthase inhibitor, on the other hand, has been reported to downregulate PPARγ and to inhibit adipocyte differentiation.

LIT: Loss of Skeletal Muscle HIF-1alpha Results in Altered Exercise Endurance: S.D. Mason, et al.; PLoS Biol. 2, E288 (2004) / Regula-tion of Muscle Fiber Type and Running Endurance by PPARdelta: Y.X. Wang, et al.; PLoS Biol. 2, E294 (2004)/ EPAS1 promotes adipose differentiation in 3T3-L1 cells: S. Shimba, et al.; J. Biol. Chem. in press (2004) / Effects of a fatty acid synthase inhibitor on adipocyte differentiation of mouse 3T3-L1 cells: L.H. Liu, et al.; Acta Pharma-col. Sin. 25, 1052 (2004)

C75ALX-270-286-M001 1 mg ALX-270-286-M005 5 mg

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PRODUCT HIGHLIGHT




YC-1 (Prod. No. ALX-420-025), best described as a potent stimulator of soluble guanylyl cyclase, suppresses the cellular levels of erythropoietin and VEGF and the DNA-binding activity of HIF-1 and HIF-1α protein expression [23, 24]. Vinc-ristine (Prod. No. ALX-350-069) and 2-methoxy-estradiol, two microtubule-targeting agents, in-hibit the expression of HIF-1α in normoxic and hypoxic cancer cells [34]. The topoisomerase-I inhibitor topotecan (Prod. No. LKT-T5761) also inhibits hypoxic induction of VEGF mRNA [35]. HSP90 inhibitors 17-allylaminogeldanamycin (17-AAG) (Prod. No. ALX-380-091), geldan-amycin (Prod. No. ALX-380-054) and radicicol (Prod. No. ALX-380-092) have been shown to suppress HIF-1 activity [36-38]. Dimethyloxal-lylglycine (DMOG) (Prod. No. ALX-270-371), 2,4-diethylpyridine dicarboxylate (2,4-DPD) (Prod. No. ALX-270-372) and N-oxalylglycine (Prod. No. ALX-270-412) are inhibitors of the oxygen-sensing enzyme HIF-α prolyl hydroxyla-se (HIF-PH), which destructs HIF-1α when hy-droxylated at a specifi c proline residue (P564).

17-AAG[17-(Allylamino)-17-desmethoxygeldanamycin]ALX-380-091-C100 100 µgALX-380-091-M001 1 mgInhibitor of HSP90 binding to the N-terminal ATP-binding do-main of HSP90 family members.

2,4-Diethylpyridine dicarboxylate[2,4-DPD]ALX-270-372-M010 10 mgALX-270-372-M025 25 mgALX-270-372-M100 100 mgCell permeable, competitive inhibitor of the oxygen-sensing enzyme HIF-1α prolyl hydroxylases (PHDs).

Dimethyloxallylglycine[DMOG]ALX-270-371-M010 10 mgALX-270-371-M050 50 mgALX-270-371-M100 100 mgCell permeable, competitive inhibitor of the oxygen-sensing enzyme HIF-1α prolyl hydroxylases (PHDs).

GeldanamycinALX-380-054-C100 100 µgALX-380-054-C500 500 µgALX-380-054-M001 1 mgInhibitor of HSP90 binding to the N-terminal ATP-binding do-main of HSP90 family members.

Herbimycin AALX-350-029-C100 100 µg ALX-350-029-M001 1 mgInhibitor of HSP90 binding to the N-terminal ATP-binding do-main of HSP90 family members.

Novobiocin . sodium salt[Albamycin; Streptonivicin]ALX-380-093-G001 1 gInhibitor of HSP90 binding to the C-terminal ATP-binding do-main of HSP90.

N-Oxalyl-D-alanine[NODA; N-Oxalyl-2R-alanine]ALX-270-414-M010 10 mgALX-270-414-M050 50 mgCell permeable, potent competitive inhibitor of factor inhibit-ing HIF asparagine hydroxylase (FIH, IC50<0.4mM). Does not inhibit HIF-1α prolyl hydroxylases (PHDs).LIT: Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation: P. Jaakkola, et al.; Science 292, 468 (2001)/Structure of factor-inhibiting hypoxia-in-ducible factor (HIF) reveals mechanism of oxidative modification of HIF-1 alpha: J.M. Elkins, et al.; J. Biol. Chem. 278, 1802 (2003)

N-Oxalyl-L-alanine[NOLA; N-Oxalyl-2S-alanine]ALX-270-413-M010 10 mgALX-270-413-M050 50 mgCell permeable, competitive inhibitor of the oxygen-sensing enzyme HIF-1α prolyl hydroxylases (PHDs). Less potent in-hibitor of factor inhibiting HIF asparagine hydroxylase (FIH, IC50=2.5mM).LIT: Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation: P. Jaakkola, et al.; Science 292, 468 (2001)/Structure of factor-inhibiting hypoxia-in-ducible factor (HIF) reveals mechanism of oxidative modification of HIF-1 alpha: J.M. Elkins, et al.; J. Biol. Chem. 278, 1802 (2003)

N-Oxalylglycine[NOG]ALX-270-412-M010 10 mgALX-270-412-M050 50 mgCell permeable, competitive inhibitor of the oxygen-sensing enzyme HIF-1α prolyl hydroxylases (PHDs).

Quercetin . dihydrate[3,3‘,4‘,5,7-Pentahydroxyflavone . 2H2O]ALX-385-001-G005 5 g Potent inhibitor of factor inhibiting HIF asparagine hydroxylase (FIH, IC50=0.6mM) not dependent on FEII chelation. Inhibits the activation of the hypoxia-response element (HRE) under hypoxic conditions.LIT: The selectivity and inhibition of AlkB: R.W. Welford, et al.; J. Biol. Chem. 278, 10157 (2003)/Specific inhibition of hypoxia-in-ducible factor (HIF)-1 alpha activation and of vascular endothelial growth factor (VEGF) production by flavonoids: Y. Hasebe, et al.; Biol. Pharm. Bull. 26, 1379 (2003)

TopotecanLKT-T5761-M001 1 mg LKT-T5761-M005 5 mgInhibitor of topoisomerase-1 inhibiting hypoxic induction of VEGF mRNA.

Vincristine . sulfateALX-350-069-M001 1 mg ALX-350-069-M005 5 mgDepolymerizes microtubules and blocks binding of tubulin to microtubule proteins. Inhibits the expression of HIF-1α.

YC-1[3-(5’-Hydroxymethyl-2’-furyl)-1-benzylindazole]ALX-420-025-M001 1 mg ALX-420-025-M005 5 mgNitric oxide (NO)-independent potent stimulator of soluble guanylyl cyclase. Suppresses the cellular levels of erythropoi-etin and VEGF and the DNA-binding activity of HIF-1 and HIF-1α protein expressionLIT: Inhibitory effect of YC-1 on the hypoxic induction of erythro-poietin and vascular endothelial growth factor in Hep3B cells: Y.S. Chun, et al.; Biochem. Pharmacol. 61, 947 (2001)/YC-1: a potential anticancer drug targeting hypoxia- inducible factor 1: E.J. Yeo, et al.; J. Natl. Cancer Inst. 95, 516 (2003)

PRODUCTS

CHEMICAL INHIBITORS OF HIF1

Finally small molecule inhibitors of the HER2→PI3K→AKT→mTOR/GSK-3 pathway, Ras→Raf-1→MEK1→ERK1/2 pathway and p38→JNK

pathway (see last page), which regulate the level of HIF-1α, are of great interest in deciphering HIF-1 regulation.

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HSP90 Inhibitors

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CHEMICAL STRUCTURES

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17-AAG Geldanamycin Herbimycin A Novobiocin . Na

Topotecan Vincristine . sulfate YC-1

2,4-DPD DMOG NODA NOLA NOG Quercetin . 2H2O




[1] Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS het-erodimer regulated by cellular O2 tension: G.L. Wang, et al.; PNAS 92, 5510 (1995) [2] HIF-1alpha is essential for myeloid cell-me-diated inflammation: T. Cramer, et al.; Cell 112, 645 (2003) [3] Structural and functional analysis of hypoxia-inducible factor 1: G.L. Semenza, et al.; Kidney Int. 51, 553 (1997) [4] A nuclear factor in-duced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation: G.L. Semenza & G.L. Wang; Mol. Cell. Biol. 12, 5447 (1992) [5] Purification and characterization of hypoxia-inducible factor 1: G.L. Wang & G.L. Semenza; J. Biol. Chem. 270, 1230 (1995) [6] Cloning of a factor required for activity of the Ah (dioxin) recep-tor: E.C. Hoffman, et al.; Science 252, 954 (1991) [7] Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively ex-pressed in endothelial cells: H. Tian, et al.; Genes Dev. 11, 72 (1997) [8] Characterization of a subset of the basic-helix-loop-helix-PAS superfamily that interacts with components of the dioxin signaling pathway: J.B. Hogenesch, et al.; J. Biol. Chem. 272, 8581 (1997) [9] A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development: M. Ema, et al.; PNAS 94, 4273 (1997) [10] Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha: Y.Z. Gu, et al.; Gene Expr. 7, 205 (1998) [11] Cellular adaptation to hypoxia: O2-sensing protein hydroxylas-es, hypoxia-inducible transcription factors, and O2-regulated gene expression: R.H. Wenger; FASEB J. 16, 1151 (2002) [12] Induction of endothelial PAS domain protein-1 by hypoxia: characterization and comparison with hypoxia-inducible factor-1alpha: M.S. Wiesener, et al.; Blood 92, 2260 (1998) [13] Expression and characterization of hypoxia-inducible factor (HIF)-3alpha in human kidney: suppression of HIF-mediated gene expression by HIF-3alpha: S. Hara, et al.; BBRC 287, 808 (2001) [14] Functional role of p35srj, a novel p300/CBP binding protein, during transactivation by HIF-1: S. Bhattacharya, et al.; Genes Dev. 13, 64 (1999) [15] CITED4 inhibits hypoxia-ac-tivated transcription in cancer cells, and its cytoplasmic location in

breast cancer is associated with elevated expression of tumor cell hy-poxia-inducible factor 1alpha: S.B. Fox, et al.; Cancer Res. 64, 6075 (2004) [16] Dephosphorylated hypoxia-inducible factor 1alpha as a mediator of p53-dependent apoptosis during hypoxia: H. Suzuki, et al.; Oncogene 20, 5779 (2001) [17] Direct interactions between HIF-1 alpha and Mdm2 modulate p53 function: D. Chen, et al.; J. Biol. Chem. 278, 13595 (2003) [18] Distinct aerobic and hypoxic mechanisms of HIF-alpha regulation by CSN5: L. Bemis, et al.; Genes Dev. 18, 739 (2004) [19] Reciprocal regulation between nitric oxide and vascular endothelial growth factor in angiogenesis: H. Kimura & H. Esumi; Acta Biochim. Pol. 50, 49 (2003) [20] Inhibition of hy-poxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling: L.E. Huang, et al.; J. Biol. Chem. 274, 9038 (1999) [21] Accumulation of HIF-1al-pha under the influence of nitric oxide: K.B. Sandau, et al.; Blood 97, 1009 (2001) [22] Regulation of the hypoxia-inducible factor 1alpha by the inflammatory mediators nitric oxide and tumor necrosis fac-tor-alpha in contrast to desferroxamine and phenylarsine oxide: K.B. Sandau, et al.; J. Biol. Chem. 276, 39805 (2001) [23] Inhibitory ef-fect of YC-1 on the hypoxic induction of erythropoietin and vascular endothelial growth factor in Hep3B cells: Y.S. Chun, et al.; Biochem. Pharmacol. 61, 947 (2001) [24] YC-1: a potential anticancer drug targeting hypoxia-inducible factor 1: E.J. Yeo, et al.; J. Natl. Cancer Inst. 95, 516 (2003) [25] Effect of altered redox states on expression and DNA-binding activity of hypoxia-inducible factor 1: G.L. Wang, et al.; BBRC 212, 550 (1995) [26] Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabi-lization of its alpha subunit: L.E. Huang, et al.; J. Biol. Chem. 271, 32253 (1996) [27] Antioxidant/pro-oxidant equilibrium regulates HIF-1alpha and NF-kappa B redox sensitivity. Evidence for inhibition by glutathione oxidation in alveolar epithelial cells: J.J. Haddad, et al.; J. Biol. Chem. 275, 21130 (2000) [28] Ischemic preconditioning reduces Op6 generation and prevents respiratory impairment in the mitochondria of post-ischemic reperfused heart of rat: J.W. Park, et al.; Life Sci. 60, 2207 (1997) [29] Reactive oxygen species gener-ated at mitochondrial complex III stabilize hypoxia-inducible factor-

1alpha during hypoxia: a mechanism of O2 sensing: N.S. Chandel, et al.; J. Biol. Chem. 275, 25130 (2000) [30] A non-hypoxic, ROS-sensitive pathway mediates TNF-alpha-dependent regulation of HIF-1alpha: J.J. Haddad & S.C. Land; FEBS Lett. 505, 269 (2001) [31] Recombinant human interleukin (IL)-1 beta-mediated regulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) stabilization, nuclear translocation and activation requires an antioxidant/reactive oxygen species (ROS)-sensitive mechanism: J.J. Haddad; Eur. Cytokine Netw. 13, 250 (2002) [32] Raising the Bar: How HIF-1 Helps Determine Tumor Radiosensitivity: B.J. Moeller & M.W. Dewhirst; Cell Cycle 3 in press (2004) [33] Small molecule blockade of transcriptional coactivation of the hypoxia-inducible factor pathway: A.L. Kung, et al.; Cancer Cell 6, 33 (2004) [34] 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF: N.J. Mabjeesh, et al.; Cancer Cell 3, 363 (2003) [35] Identification of small molecule inhibitors of hypoxia-inducible factor 1 tran-scriptional activation pathway: A. Rapisarda, et al.; Cancer Res. 62, 4316 (2002) [36] Hsp90 regulates a von Hippel Lindau-independ-ent hypoxia-inducible factor-1 alpha-degradative pathway: J.S. Isaacs, et al.; J. Biol. Chem. 277, 29936 (2002) [37] Geldanamycin induces degradation of hypoxia-inducible factor 1alpha protein via the proteosome pathway in prostate cancer cells: N.J. Mabjeesh, et al.; Cancer Res. 62, 2478 (2002) [38] Reduction of hypoxia-induced transcription through the repression of hypoxia-inducible factor-1alpha/aryl hydrocarbon receptor nuclear translocator DNA bind-ing by the 90-kDa heat-shock protein inhibitor radicicol: E. Hur, et al.; Mol. Pharmacol. 62, 975 (2002) [39] Hypoxia-inducible factor regulates survival of antigen receptor-driven T cells: Y. Makino, et al.; J. Immunol. 171, 6534 (2003)

LITERATURE

SELECTED REVIEW ARTICLES

[1] REGULATION OF MAMMALIAN O2 HOMEOSTASIS BY HYPOXIAINDUCIBLE FACTOR 1: G. L. Semenza; Annu. Rev. Cell Dev. Biol. 15, 551 (1999)

[2] CELLULAR ADAPTATION TO HYPOXIA: O2SENSING PROTEIN HYDROXYLASES, HYPOXIAINDUCIBLE TRANSCRIPTION FACTORS, AND O2REGULATED GENE EXPRESSION: R. H. Wenger; FASEB J. 16, 1151 (2002)

[3] OXYGENDEPENDENT AND INDEPENDENT REGULATION OF HIF1ALPHA: Y. S. Chun, et al.; J. Korean Med. Sci. 17, 581 (2002)

[4] RECIPROCAL REGULATION BETWEEN NITRIC OXIDE AND VASCULAR ENDOTHELIAL GROWTH FACTOR IN ANGIOGENESIS: H. Kimura & H. Esumi; Acta Biochim. Pol. 50, 49 (2003)

[5] THE VON HIPPELLINDAU TUMOR SUPPRESSOR, HYPOXIAINDUCIBLE FACTOR1 HIF1 DEGRADATION, AND CANCER PATHOGENESIS: C. W. Pugh & P. J. Ratcliffe; Semin. Cancer Biol. 13, 83 (2003)

[6] THE HYPOXIAINDUCIBLE FACTORS: KEY TRANSCRIPTIONAL REGULATORS OF HYPOXIC RESPONSES: C. P. Bracken, et al.; Cell. Mol. Life Sci. 60, 1376 (2003)

[7] THE SUBTLE SIDE TO HYPOXIA INDUCIBLE FACTOR HIFALPHA REGULATION: R. L. Bilton & G. W. Booker; Eur. J. Biochem. 270, 791 (2003)

[8] HIFS AND TUMORS CAUSES AND CONSEQUENCES: G. Hopfl, et al.; Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R608 (2004)

[9] HYPOXIAINDUCIBLE FACTOR1 IN TUMOUR ANGIOGENESIS: Y. H. Shi & W. G. Fang; World J. Gastroenterol. 10, 1082 (2004)

[10] THE HIF PATHWAY AS A THERAPEUTIC TARGET: K. S. Hewit-son & C. J. Schofield; Drug Discov. Today 9, 704 (2004)

[11] HYPOXIC GENE EXPRESSION AND METASTASIS: Q. T. Le, et al.; Cancer Metastasis Rev. 23, 293 (2004)

[12] HYPOXIAINDUCIBLE FACTOR HIF1ALPHA: ITS PROTEIN STABILITY AND BIOLOGICAL FUNCTIONS: J. W. Lee, et al.; Exp. Mol. Med. 36, 1 (2004)

[13] HIF1: AN OXYGEN AND METAL RESPONSIVE TRANSCRIPTION FACTOR: P. Maxwell & K. Salnikow; Cancer Biol. Ther. 3, 29 (2004)

[14] HIF1 AND OXYGEN SENSING IN THE BRAIN: F. R. Sharp & M. Bernaudin; Nat. Rev. Neurosci. 5, 437 (2004)

[15] OXYGENDEPENDENT AND TISSUESPECIFIC REGULATION OF ERYTHROPOIETIN GENE EXPRESSION: J. Fandrey; Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R977 (2004)

[16] HYPOXIAINDUCIBLE FACTOR 1 HIF1 IN CANCER: M. Quintero, et al.; Eur. J. Surg. Oncol. 30, 465 (2004)

[17] HYPOXIAINDUCIBLE FACTOR 1RELATED DISEASES AND PROSPECTIVE THERAPEUTIC TOOLS: J. W. Park, et al.; J. Pharma-col. Sci. 94, 221 (2004)

[18] HIF HYDROXYLATION AND CELLULAR OXYGEN SENSING: E. Metzen & P. J. Ratcliffe; Biol. Chem. 385, 223 (2004)

[19] HYPOXIA AND THE REGULATION OF MITOGENACTIVATED PROTEIN KINASES: GENE TRANSCRIPTION AND THE ASSESSMENT OF POTENTIAL PHARMACOLOGIC THERAPEUTIC INTERVENTIONS: J. J. Haddad; Int. Immunopharmacol. 4, 1249 (2004)

[20] NEW ANTICANCER STRATEGIES TARGETING HIF1: E. J. Yeo, et al.; Biochem. Pharmacol. 68, 1061 (2004)

[21] THE ROLE OF CALCIUM IN HYPOXIAINDUCED SIGNAL TRANSDUCTION AND GENE EXPRESSION: K. A. Seta, et al.; Cell Calcium 36, 331 (2004)

[22] CALCIUM AND PH HOMEOSTASIS IN NEURONS DURING HYPOXIA AND ISCHEMIA: H. Yao & G. G. Haddad; Cell Calcium 36, 247 (2004)

HSP90 RELATED PRODUCTS

HSP90 (human) (recombinant)ALX-201-147-C025 25 µg SOURCE/HOST: Recombinant human HSP90 produced in insect cells. PURIT Y: >99%. APPLICATION: WB.

HSP90 (mouse)IMA-220-0-C100 100 µg SOURCE/HOST: Purifi ed from mouse cells. PURIT Y: >95% (SDS-PAGE).

HSP90 (E. coli) (recombinant)ALX-201-146-C025 25 µg SOURCE/HOST: Recombinant protein produced in E. coli. PURIT Y: >99%. APPLICATION: WB.

HSP90 (yeast) (recombinant)ALX-201-138-C025 25 µg SOURCE/HOST: Recombinant protein produced in yeast. PURIT Y: >99%. APPLICATION: WB.

MAb to HSP90 (3B6) ALX-804-078-R100 100 µlCLONE: 3B6. ISOT YPE: Mouse IgG2b. IMMUNOGEN: Purifi ed mouse heat shock protein 90 (HSP90). SPECIFICIT Y: Recog-nizes human, mouse and rat HSP90. Detects a band of ~90 kDa by Western blot. Does not cross-react with chicken HSP90. APPLICATION: WB. For IP use Prod. No. ALX-804-079.

MAb to HSP90 (3G3) ALX-804-079-R400 400 µl CLONE: 3G3. ISOT YPE: Mouse IgM. IMMUNOGEN: Heat shock protein 90 (HSP90) from Hepa 1 cells. SPECIFICIT Y: Recog-nizes human, mouse, rat, rainbow trout and chicken HSP90. APPLICATION: IP. For WB use Prod. No. ALX-804-078.

MAb to HSP90 (H90-10) ALX-804-808-C100 100 µg CLONE: H90-10. ISOT YPE: Mouse IgG2aκ. IMMUNOGEN: Puri-fi ed human HSP90β. SPECIFICIT Y: Recognizes human, mouse and rabbit HSP90α and HSP90β. APPLICATION: ICC, IP, WB.



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Bisindolylmaleimide V[Ro 31-6045]ALX-270-053-M001 1 mgALX-270-053-M005 5 mgSelective inhibitor of p70S6K (IC50 = 0.8µM). LIT: Ro 31-6045, the inactive analogue of the protein kinase C in-hibitor Ro 31-8220, blocks in vivo activation of p70(s6k)/p85(s6k): implications for the analysis of S6K signalling: N. Marmy-Conus, et al.; FEBS Lett. 519, 135 (2002)

JNK Inhibitor 1 (D-stereoisomer) [D-JNKI1]ALX-159-601-R050 50 µlALX-159-601-R200 200 µlCell permeable JNK (c-Jun N-terminal kinase) inhibitor. D-JNKI1 exhibits a substantially prolonged half-life for in vivo applications (days) over its L-stereoisomer (hours). FITC-con-jugated D-TAT Control Peptide (Prod. No. ALX-168-010F).LIT: Cell-permeable peptide inhibitors of JNK: novel blockers of beta-cell death: C. Bonny, et al.; Diabetes 50, 77 (2001)

JNK Inhibitor 1 (L-stereoisomer) [L-JNKI1]ALX-159-600-R100 100 µlALX-159-600-R200 200 µlCell permeable JNK (c-Jun N-terminal kinase) inhibitor.LIT: Cell-permeable peptide inhibitors of JNK: novel blockers of beta-cell death: C. Bonny, et al.; Diabetes 50, 77 (2001)

LY-294,002ALX-270-038-M001 1 mgALX-270-038-M005 5 mgALX-270-038-M025 25 mgPotent, cell permeable, highly specifi c inhibitor of phosphati-dylinositol-3-kinase (PI(3)K) (IC50 = 1.4µM) that acts on the ATP binding site of the enzyme.LIT: Specific inhibitor of phosphatidylinositol 3-kinase, 2-(4- mor-pholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002): C.J. Vla-hos, et al.; J. Biol. Chem. 269, 5241 (1994)

OTDZT[2,4-Dibenzyl-5-oxothiadiazolidine-3-thione]ALX-270-401-M001 1 mgNon-ATP competitive inhibitor of GSK-3β (IC50=10µM).LIT: First non-ATP competitive glycogen synthase kinase 3 beta (GSK-3beta) inhibitors: thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer ‘s disease: A. Martinez, et al.; J. Med. Chem. 45, 1292 (2002)

PD 98,059ALX-385-023-M005 5 mgALX-385-023-M010 10 mgALX-385-023-M100 100 mgPotent, cell permeable and selective inhibitor of MEK (MAP ki-nase kinase) (IC50 = 2-7µM). Blocks the activity of MEK thereby inhibiting the phosphorylation and activation of MAP kinase.LIT: PD 098059 is a specific inhibitor of the activation of mitogenactivated protein kinase kinase in vitro and in vivo: D.R. Alessi, et al.; J. Biol. Chem. 270, 27489 (1995)

Raf1 Kinase Inhibitor[5-Iodo-3-[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-indolinone]ALX-270-324-M001 1 mgPotent cRAF1 kinase inhibitor (IC50 = 9nM). Shows >100-fold selectivity for Raf kinase versus CDK1, CDK2, c-Src, ERK2, MEK, p38, Tie2, VEGFR2, and c-Fms.LIT: The discovery of potent cRaf1 kinase inhibitors: K. Lackey, et al.; Bioorg. Med. Chem. Lett. 10, 223 (2000)

RapamycinALX-380-004-C100 100 µgALX-380-004-5100 5 x 100 µgALX-380-004-M001 1 mgALX-380-004-5001 5 x 1 mgSpecifi c inhibitor of mTOR.

SB203580ALX-270-179-M001 1 mgALX-270-179-M005 5 mgALX-270-179-M050 50 mgCell permeable, specifi c inhibitor of p38 (SAPK2a) (IC50 = 0.3-0.5 µM).LIT: The pyridinyl imidazole inhibitor SB203580 blocks phospho-inositide-dependent protein kinase activity, protein kinase B phos-phorylation, and retinoblastoma hyperphosphorylation in inter-leukin-2-stimulated T cells independently of p38 mitog: F.V. Lali, et al.; J. Biol. Chem. 275, 7395 (2000)

SB220025ALX-270-325-C500 500 µgPotent and specifi c inhibitor of human p38 (SAPK2a; IC50 = 60nM).LIT: Pharmacological effects of SB 220025, a selective inhibitor of P38 mitogen-activated protein kinase, in angiogenesis and chronic inflammatory disease models: J.R. Jackson, et al.; J. Pharmacol. Exp. Ther. 284, 687 (1998)

SH-5ALX-270-349-MC05 0.5 mgALX-270-349-M001 1 mgInhibitor of Akt (PKB) activation without affecting activation of the upstream kinase PDK-1, or other kinases downstream of Ras such as MAPK. LIT: Novel PI analogues selectively block activation of the pro-sur-vival serine/threonine kinase, Akt.: A.P. Kozikowski, et al.; JACS 125, 1144 (2003)

SH-6ALX-270-350-MC05 0.5 mgALX-270-350-M001 1 mgInhibitor of Akt (PKB) activation without affecting activation of the upstream kinase PDK-1, or other kinases downstream of Ras such as MAPK. LIT: Novel PI analogues selectively block activation of the pro-sur-vival serine/threonine kinase, Akt.: A.P. Kozikowski, et al.; JACS 125, 1144 (2003)

SP600125ALX-270-339-M005 5 mgALX-270-339-M025 25 mgPotent, cell permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK) (IC50 = 40nM for JNK-1 and JNK-2 and 90nM for JNK-3).LIT: SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase: B.L. Bennett, et al.; PNAS 98, 13681 (2001)

TDZD-8[4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione]ALX-270-354-M005 5 mgHighly selective, non-ATP competitive inhibitor of GSK-3β (IC50 = 2µM).LIT: First non-ATP competitive glycogen synthase kinase 3 beta (GSK-3beta) inhibitors: thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer ‘s disease: A. Martinez, et al.; J. Med. Chem. 45, 1292 (2002)

TIBPO[2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole]ALX-270-358-M005 5 mgPotent inhibitor of glycogen synthase kinase-3β (GSK-3β) (IC50 = 390nM).LIT: Scaffold hopping and optimization towards libraries of glyco-gen synthase kinase-3 inhibitors: L. Naerum, et al.; Bioorg. Med. Chem. Lett. 12, 1525 (2002)

U0126ALX-270-237-M001 1 mgALX-270-237-M005 5 mgPotent inhibitor of MEK1 (IC50 = 0.07 µM) and MEK2 (IC50 = 0.06 µM).LIT: MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products: J.V. Duncia, et al.; Bioorg. Med. Chem. Lett. 8, 2839 (1998)

WortmanninALX-350-020-M001 1 mgALX-350-020-M005 5 mgALX-350-020-M025 25 mgPotent and specifi c inhibitor of phosphatidylinositol-3-kinase (PI(3)K) (IC50 = 5nM)LIT: Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses: A. Arcaro & M.P. Wyman; Biochem. J. 296, 297 (1993)

SMALL MOLECULE INHIBITORS MANUFACTURED BY ALEXIS® BIOCHEMICALS

ALSO AVAILABLE

Phosphoinositide 3-kinase p110γ (human) (rec.) ALX-201-055-C010 10 µg

Phosphoinositide 3-kinase p110α/p85 (bovine) (rec.) ALX-201-119-C005 5 µg

PTEN (human) (rec.) ALX-201-079-C020 20 µg


hypoxia & hif-1

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