Targeting Protein–Protein Interactions in the HIF System

Abstract Animals respond to chronic hypoxia by increasing the levels of a transcription factor known as the hypoxia‐inducible factor (HIF). HIF upregulates multiple genes, the products of which work to ameliorate the effects of limited oxygen at cellular and systemic levels. Hypoxia sensing by the HIF system involves hydroxylase‐catalysed post‐translational modifications of the HIF α‐subunits, which 1) signal for degradation of HIF‐α and 2) limit binding of HIF to transcriptional coactivator proteins. Because the hypoxic response is relevant to multiple disease states, therapeutic manipulation of the HIF‐mediated response has considerable medicinal potential. In addition to modulation of catalysis by the HIF hydroxylases, the HIF system manifests other possibilities for therapeutic intervention involving protein–protein and protein–nucleic acid interactions. Recent advances in our understanding of the structural biology and biochemistry of the HIF system are facilitating medicinal chemistry efforts. Herein we give an overview of the HIF system, focusing on structural knowledge of protein–protein interactions and how this might be used to modulate the hypoxic response for therapeutic benefit.


Introduction
The chronic response to hypoxia( limited oxygen availability) in humans ando ther animalsi ss ubstantially mediatedb yt he a,b-heterodimeric hypoxia-inducible factor (HIF) transcription factor. [1] HIF-a protein levels are increased in hypoxia;i tt ravels to the nucleus, dimerises with HIF-b and binds to hypoxia response elements (HREs) in the promoter regions of HIF target genes. To gether with transcriptional coactivatorp roteins, HIF promotes the context-dependente xpression of multiple genes that work to counteractt he effects of hypoxia at ac ellular,a nd subsequently,s ystemic level. [2] Although there are other mechanismso fh ypoxic adaptation,i ncluding those acting on as horter time-scale than the HIF system, the extent of the effects of the HIF system has led HIF to be characteriseda s am aster regulator of the hypoxic response.
Many HIF target genes are of medical importance, especially in relationt oc ancer and ischaemic diseases. HIF target genes include those encoding erythropoietin (EPO) and vascular endothelial growth factor (VEGF), which inducet he production of red blood cells and blood vessels,r espectively,a sw ell as many other proteins involved in metabolic and physiological adaptations to hypoxia. Modulation of the HIF system for therapeutic benefiti sh ence of considerable interest. Major efforts to date have focused on 1) the upregulation of HIF target genes (e.g., epo)f or the treatment of anaemia and 2) the inhibition of HIF transcriptional activity as ac ancer therapy.H owever,m ultiple other therapeutica pplicationso ft he HIF system can be envisaged, such as in woundh ealing or for the treatment of stroke. Further,b ecause HIF is ap leiotropic transcription factor that is rapidly and efficiently induced by ag aseous small molecule, it is an attractive model system for basic studies on the control of gene expression.
The regulation of protein-protein interactions by oxygen-dependent post-translational modifications is central to the hypoxia-sensing capacity of the HIF system. Crucially,i th as been found that hydroxylation of HIF a-subunits signals for their proteolytic degradation and regulates the transcriptional activity of HIF. [3] The discovery of these modifications and the hydroxylases that catalyset hem haso pened up an ew vista in oxygen-dependent signalling, the relevance of which extends far beyond the HIF system. However,o ther protein-protein and protein-nucleic acid interactions play central roles in the HIF system and offer therapeuticp ossibilities. The purpose of this review is to give an overview of the HIF system,f ocusing on knowledge of the oligomeric interactions involved, and outlining how this knowledge might be exploited fort herapeutic benefit.

The HIF transcription factors
Active HIF transcription factorsa re comprised of an oxygenregulated a-subunit and ac onstitutive b-subunit. [4] In humans there are three HIF-a isoforms, of which HIF-1a andH IF-2a are the bestc haracterised; together with HIF-b (also known as ARNT)t hey form active transcription factorst ermedH IF-1 and HIF-2,r espectively ( Figure 1). HIF-1 and HIF-2 are closely related, but upregulate distinct (ands ometimes overlapping) sets of genes in hypoxia. [5] HIF a-a nd b-subunits belong to the bHLH/PAS (basic helix-loop-helix/Per-ARNT-Sim homology) family of transcription factors( Figure 1). [4] The bHLH and PAS domainsm ediate a,b-dimerisation and DNA binding, while Nand C-terminal transcriptional activation domains (NAD and CAD, respectively) recruit coactivator proteinst of orm active transcriptional complexes on DNA. [6] HIF a-subunits also contain an oxygen-dependent degradation (ODD) domain, the hydroxylation of which renders them labile in oxygenated conditions. [7]

Regulation of HIF-a by hydroxylation
The HIF a-subunits have as hort half-life in normoxia due to their rapid turnoverb yt he ubiquitin-proteasome system. [7,8] An E3 ubiquitin ligase complex composed of ElonginC,E longin Ba nd the von Hippel Lindaut umour suppressor protein Animals respondt oc hronic hypoxia by increasing the levels of at ranscription factor known as the hypoxia-inducible factor (HIF). HIF upregulates multiple genes, the products of which work to ameliorate the effects of limited oxygen at cellular and systemic levels. Hypoxia sensing by the HIF system involves hydroxylase-catalysedp ost-translational modifications of the HIF a-subunits, which 1) signal for degradation of HIF-a and 2) limit binding of HIF to transcriptionalc oactivator proteins. Because the hypoxicr esponse is relevant to multiple disease states, therapeutic manipulation of the HIF-mediated response has considerable medicinal potential. In addition to modulation of catalysis by the HIF hydroxylases,t he HIFs ystem manifests other possibilities for therapeutic intervention involving protein-protein and protein-nucleic acid interactions. Recent advances in our understanding of the structural biologya nd biochemistry of the HIF system are facilitating medicinal chemistry efforts. Herein we give an overview of the HIF system,f ocusingo ns tructuralk nowledge of protein-protein interactions and how this might be used to modulate the hypoxic response for therapeutic benefit.
(pVHL) catalyses the poly-ubiquitination of lysine residues that target HIF-a for degradation by the proteasome (Figure 1). pVHL is the substrate recognition component of this complex and binds directly to HIF-a;t his interaction is substantially promoted by the hydroxylation of two proline residues in HIFa (P402 and P564 in HIF-1a), located within N-and C-terminal oxygen-dependent degradation domains (NODD and CODD, respectively). [9] As ingleh ydroxylation at either site (NODD or CODD) is sufficient to target HIF-a to the pVHL ubiquitin ligase complexf or degradation. [9] In some cancers, particularly kidney tumours, pVHL is inactivated by mutations;t he resultant upregulation of HIF-a may serve to promotet umour growth. [10] HIF prolyl hydroxylation is catalysed by as et of non-haem iron-and 2-oxoglutarate (2OG)-dependent prolyl-4-hydroxylases (PHD1-3, also known as EGLN1-3). [11] Various lines of evidence imply that the catalytic activity of these enzymes is decreasedu nder conditions of sub-optimal oxygen availability, leadingt oadecrease in hydroxylation of HIF-a. [12] Because HIFa is not recognised by pVHL in the absence of prolyl hydroxyl-ation, [13] HIF-a accumulates in hypoxia. The catalytic mechanism of the PHDs likelyp roceeds via the consensus for 2OGdependento xygenases,t hat is, binding of 2OG to the active site is followed by that of substrate and finally,o xygen (for review see Ref. [14]). However,k inetic evidence implies that, at least for PHD2,t he reactiono ft he enzyme with oxygen is unusually slow. [15] Although slow reactionw itho xygen is not ap rerequisite property for ac ellular hypoxia sensor, it is proposed to be advantageous in such ar ole.
As econd mechanism of HIF regulation involves hydroxylation of N803 in the HIF-1a CAD (N847 in HIF-2a)b yt he 2OGdependento xygenase FactorI nhibiting HIF (FIH). [16] In contrast to HIF prolyl hydroxylation, which 'makes' ap rotein-protein interaction, asparaginyl hydroxylationo ft he HIF-a CAD 'breaks' interactions between HIF-a and the transcriptional coactivator proteins CREB binding protein (CBP) and p300, [17] whicha re required for transcriptional activation of mostH IF target genes. [18] Like the PHDs, FIH is inactivatedi nh ypoxia, though it retainsa ctivity at lower oxygen tensionst han the PHDs. [12c, f, 19] Sarah Wilkins completed her undergraduate and postgraduate studies at the University of Adelaide in Australia. She received aPhD in biochemistry in 2012, and has since been conducting postdoctoral research at the University of Oxford in the laboratory of Professor Schofield. Her research interests include structural and biochemical characterisation of enzymes involved in cellular oxygen-sensing mechanisms, especially 2-oxoglutarate-dependent oxygenases that catalyse protein hydroxylation.
Martine Abboud graduated with aB Sc in biology and aminor in chemistry from the Lebanese American University.In2013 she came to Oxford University as aB iochemical Society Krebs Memorial Scholar to pursue aPhD in chemical biology under the supervision of Professor Schofield. Her research foci involve biophysical investigations of protein-protein/ligand interactions, with particular emphasis on 2-oxoglutarate-dependent oxygenases and metallo-b-lactamases, using various methodologies, in particular,h igh-field NMR. Christopher Schofield studied for afirst degree in chemistry at the University of Manchester Institute of Science and Te chnology (1979)(1980)(1981)(1982). In 1982 he moved to Oxford to pursue aPhD with Professor Jack Baldwin on the synthesis and biosynthesis of antibiotics. In 1985 he became aDepartmental Demonstrator in the Dyson Perrins Laboratory,Oxford University, followed by his appointment as Lecturer in Chemistry and Fellow of Hertford College in 1990. In 1998 he became Professor of Chemistry, and in 2011w as appointed Head of Organic Chemistry.Hei s aFellow of the Royal Society of Chemistry and of the Royal Society. His research group works at the interface of chemistry,b iology and medicine. His work has opened up new fields in antibiotic research, oxygen sensing and gene regulation. His work has identified new opportunities for medicinal intervention that are being pursued by academic and commercial laboratories.
Thus, the HIF-a CAD is silenced under normalo xygen conditions and transcriptionally active in hypoxia.

HIF-a,b dimerisation
Early studies involvingd eletion analyses of HIF proteins suggested that both the bHLH and PASd omains of HIF subunits contribute to heterodimerisation, [20] an observation that is supported by recent crystallographic analyses of HRE-bound HIF-1a nd HIF-2 bHLH-PAS complexes. [21] As illustrated in Figure 2a,t he dimerisation interface of the HIF-1c omplex is asymmetric; the bHLH, PAS-A and PAS-B domainso fH IF-1a pack together in ac ompactm anner,w hereas HIF-b binds in an extended conformation, wrapping around the outer surface of HIF-1a with relativelyf ew intramolecular contacts. The HIF-1a PAS-B domain appears to be important in 'scaffolding' the complex, making contacts with the adjacent HIF-1a PAS-A domain,asw ell as both PASdomains in HIF-b.
Theh elix-loop-helixr egions of the HIF-1 complexi ntertwine to formastableh elical bundle that straddles theD NA ;t wo Nterminal helices, one from each of the a-a nd b-subunits,i ntercalate theDNA,binding the major groove on opposite sides of the double-helix.H RE recognitioni sm ediatedb yacluster of basic residues on these N-terminal helices, sucht hat HIF-1a andH IFb contactt he 5'(AC) and3 '(GTG)e ndso ft he HRE, respectively. TheP AS-A domain of HIF-1a also contributes to DNAb inding, with an extended loop that contacts them inor groove 6bp downstream of theHRE. [21,22] Notably, the crystallographically observed conformationo ft he HIF-2a,b dimeri sn ear-identicalt o that of theH IF-1a,b complex( Figure 2b)d espite differencesi n aminoa cids equencec ompositionb etween HIF-1a and HIF-2a (66% identicali nt he bHLH PASr egion).T he mode of HIF-1a nd HIF-2 DNAb inding is also highly similari nt he reported structures( Figure 2a and2 c),r ationalising thea bilityo fH IF-1 and HIF-2 heterodimers to bind the samecoreHRE sequence. [21,23] 2.2. HIF-a ODD domaininteractions:p VHL and the PHDs Crystals tructures have been solved forp VHL in complex with ah ydroxylated HIF-1a CODD peptide. [13,24] The CODD peptide adopts an extended conformation when bound to the surface of a b-sheet in the N-terminal b-domaino fp VHL ( Figure 3a). Strikingly,alarge number of tumour-associated VHL mutations map to this interface. [25] HIF-1a is held in place through extensive backbone and side-chain hydrogen bonds with pVHL, limiting its conformational flexibility.T he C4 hydroxyproline residue in HIF-1a (HyP564) is almost entirely buried at the interface. HyP564 is positioned to hydrogen bond with H115 and S111 in pVHL, rationalising the strict requirement for pVHL binding to ah ydroxylated proline residue ( Figure 3b). Notably, the conformation of the proline ring appears to be an important determinant of pVHL binding, as is the C4 trans stereochemistry of the hydroxy group. [26] The affinity of VHL for hydroxylated versus non-hydroxylated CODD differsb ya lmost three orders of magnitude, leadingt ot he proposal that HIFa prolyl hydroxylationh as as witch-like effect on HIF signalling. [13] As with binding to pVHL, the conformation of the target proline residue is importantf or HIF binding to the PHDs, as shownb yw ork with PHD2. [26] The non-hydroxylated CODD proline adoptst he C4 endo conformationw hen bound to PHD2;o nt he basis of crystallographic analysis, this conformation is proposed to be required for the productiver eactiono f aF e IV =Oi ntermediate with the C4 trans prolyl hydrogen atom. [27] NMR and other biophysical studies reveal that binding of the HIF-a ODDs to the PHDs involves substantial induced fit mechanisms, in particulari nvolving am obile loop regionl ocated between the b2/b3s trands of PHD2 at the C-terminal region ( Figure 3d). [28] The combined structural resultsi mply that in the absence of HIF-a ODD substrate, the b2/b3l oop is mobile andc an be oriented away from the active site. [29] On binding of aC ODD peptide,t he b2/b3l oop folds to entirely encloset he hydroxylation motif (LAPYIP). [28] The importance of conformational changes in PHD catalysis is highlighted by biophysicala nalysiso fahomologue of the human PHDs from Pseudomonas spp. [30] Studieso nt he Pseudomonas hydroxylase (pPHD)i nc omplex with itsi ntact Elongation Factor-Tus ubstrate reveal major conformational changes in both pPHD and EF-Tu, which may be reflectedi na nalyses of the intact PHDs and varied large HIF-a fragments. [27] The NODD is proposed to bind to the PHDs in as imilarm anner to CODD, though details of the interaction must be different. Mutational analysesi ndicate that that L574, located 10 residues downstream of the HIF-1a CODD hydroxyproline, is an importantd eterminant of PHD2 binding; [31] however,aleucine is not present at the equivalent (+ 10) positionr elative to P402 in NODD.A sy et, there are no structures for PHD:NODD complexes.
It is important to emphasise that pVHL-and hypoxia-independentm echanismso fH IF (de)stabilisation occur.A ntibodybased studies indicatet hat, at least in somec ases, HIF-a is upregulated in cancer cells but still undergoes prolyl hydroxylation. [32] Althought hese observations couldb ed ue to impaired pVHL function, it is likely that other factors can limit HIF-a degradation. Severalr eports have linked heat shock proteins to HIFstability, with both HSP90 and HSP70 being reported to interact with HIF-a. [33] HSP90 is proposed to bind to HIFa in the cytoplasm and protect it from oxygen-independent degradation. [34] Displacement of HIF-a from HSP90 by smallmolecule inhibitors (e.g.,g eldanamycin) enables binding of RACK1 (receptor of activated protein Ck inase 1), which recruits the ubiquitin ligase machinery and potentiates HIF-a degradation. [35] HSP70 and the ubiquitin ligase CHIP (C-terminal Hsp70 Interacting Protein)a re reportedt op romote HIF-1a,b ut not HIF-2a,d egradation, so blocking HIF-1 activity. [33b] Recent interesting workh as also identified ar ole for the MYND (N-terminal Myeloid Nervy and DEAF-1) zinc-finger domain present in PHD2 homologues, which binds to ac onserved motif in cochaperone proteins, including p23 of the HSP90 system. P23i s proposed to recruit PHD2 to the HSP90m achinery to facilitate hydroxylation (and degradation) of HIF-1a. [38] Figure 2. Architecture of HIF bHLH-PAS heterodimers. a) Twov iews from an X-ray crystal structure of the HIF-1a,b bHLH-PASh eterodimer in complex with DNA (PDB ID:4ZPR). Domains in HIF a-a nd b-subunitsa re indicated, and the HRE (hypoxia response element)ish ighlighted in green.b )Superimposed views from crystal structures of HIF-1 and HIF-2 bHLH-PAS complexes (PDB IDs:4ZPR and 4ZPK, respectively). c) View from ac rystals tructureo ft he HIF-2a,b bHLH-PAS heterodimer in complex with DNA (PDB ID:4ZPK). Binding sitesfor the HIF dimerisation inhibitors 0X3 and proflavinea re superimposed on the HIF-2 heterodimer.Magnifiedviews from crystal structures of HIF-2i nc omplex with d) proflavine (magenta, PDB ID:4 ZPH) and e) 0X3 (cyan, PDB ID:4ZQD) are shownint he adjacentp anels.Important residues from HIF-2a (gold) and HIF-b (deepb lue) that line the binding pockets are showninstick representation. All images were generated using coordinates reported in Wu et al. [21] Multiple other interacting partners for the PHDs have been described, including the tumour suppressor protein LIMD1 (LIM domain containing protein 1), which simultaneously binds the PHDs and pVHL in am anner that promotes HIF-a degradation. [39] Many other interacting proteins have been reportedt o be PHD substrates on the basis of antibody and/or proteomic mass spectrometry analyses. The relevance of these interactions to the hypoxic response remains to be validated, though the findings do raise the possibility that competition for binding to the PHDs may be regulatory.S uch competition is more established for FIH, which we focus on in this review (see [40] for ar eview of non-HIF PHD substrates).

HIF-a CAD interactions:F IH and CBP/p300
CBP/p300 interact with both the NAD and CAD in HIF-a, [41] thougho nly the latter of these interactions is known to be regulated by oxygen-dependent hydroxylation and hasb een structurally characterised. [17,42] NMR structures of the CH1 domains of CBP and p300i nc omplex with HIF-1a CAD peptides have been reported. [42] In both cases, the four alpha helices that constitute the CH1 domain form ab undle that is stabilised by coordination with three Zn 2 + ions. The CAD folds aroundt he CH1 domain like ac lamp, adopting two induced ahelices that bind in an almost parallel arrangement on opposite faces of the CH1 domain (Figure4b). N803 in the HIF-1a CAD is located on the N-terminal helix (Helix 1) andi sb uried within the molecular interface. Hydroxylation at the pro-S position of N803,w hich blocks HIF binding to CBP/p300, likely creates ad irect steric clash with the backbonec arbonyl of D799 in HIF-1a,s od isrupting the formation of this helix (Figure 4b). [42,43] The tertiary structure of the HIF-1a CAD when complexed with the CH1 domain of CBP/p300 is determined almoste xclusivelyb yi ntermolecularc ontacts;c irculard ichroism analyses indicate that the isolated HIF-1a (and likely HIF-2a)C AD is intrinsically disordered in solution. [42] The availablee vidence indicates that FIH does not undergo such major conformational changes as the PHDs on substrate binding. [44] When bound to FIH, the HIF-1a CAD adopts an extended conformation that is less enclosedt han that of CODD bindingt oP HD2. [27,44] Multiple hydrogen bonds are involved in binding the CAD to FIH, as well as hydrophobic interactions, including with av aline residue present immediatelyN -terminal to N803 in the HIF-1a CAD hydroxylation motif (CEVNAP);t his valine forms ah ydrophobic interaction with W296 of FIH, which is involvedi na ni nducedf it mechanism. [44] The primary amide of N803 is positioned to form hydrogen bonds with conserved residues in FIH, notably Q239. The interaction of HIF-1a with FIH is likely more complex beyond the immediate vicinity of the active site;asecond binding site is observed in which the HIF-a residues involved form an a-helix, as observed for these same residues bound to the CH1 domain of CBP/ p300 (Figure 4). [42,44] Figure 3. HIF-1a CODD interactions with pVHLand PHD2. a) View from ac rystal structure of pVHL in complex with ah ydroxylated HIF-1a CODD peptide (PDB ID:1LQB [13] ). b) Magnified view from a) showing the orientation of HyP564and its hydrogen bond interactions with residuesinpVHL. c) View from acrystal structure of pVHLi nc omplexw ith Ligand 51 [36] (purple), an inhibitoroft he pVHL:HIF-1a interaction. d) Superimposed views from X-rayc rystal structures of PHD2 alone (green,PDB ID:2G1M [29] )and in complex with aH IF-1a CODD peptide( blue, PDB ID:3HQR; [27] CODDp eptideiss hown in red). e) Binding mode of ad ihydropyrazole inhibitor( yellow)b ound in the active site of PHD2 (PDB ID:5 A3U [37] ).
ChemMedChem 2016, 11,773 -786 www.chemmedchem.org FIH is reported to interact with many proteins othert han HIF-a,m ostlyc omingf rom the ankyrinrepeatdomain structural family (see [45] for detailed reviews on FIH-catalyseda nkyrin repeat hydroxylation). There is strongc ellular and biochemical evidencet hat many (but not all) of these are hydroxylation substrates for FIH. From as tructural biology perspective,t he finding that FIH accepts ankyrin repeat domain proteins was surprising, as they must undergo significant unfolding in order to bind productively at the active site. [46] FIH-catalysed ankyrin repeat hydroxylation occursa tc onserved positions and not just on asparagine residues. [47] Because some ankyrins ubstrates are of major biological importance (e.g.,N otch [48] ), there was interest in the possibility that FIH-catalysed ankyrin repeat domain hydroxylation might have 'switch-like' roles in cellular processes,a lthough as yet there is no clear evidence for this. At least in somec ases, hydroxylations tabilises the stereotypical ankyrinf old. [49] Nonetheless, as the extento fh ydroxylation is rarely complete, [50] it would seem unlikely that ankyrinr epeat domain hydroxylation plays ac rucial structural role (in contrast to trans-4-prolyl hydroxylation which stabilises the collagen triple-helix fold [51] ). One possibility is that competition for FIH between the HIF-a CAD anda nkyrinr epeat domains serves to modulate the HIF-mediatedh ypoxic response such that it can functionindifferent environments; [48a, 52] another is that ankyrin repeatd omain hydroxylation can provide am emory of hypoxic events. [53] The discovery of FIH-catalysed hydroxylation of multiple ARDs also raises the unanswered question of whether HIF-a needs to be targeted to FIH. To enablet his, it is possible that at argetingp rotein may bind to one monomer of the FIH dimer,s op romotingb inding of HIFa to the other.
It is importantt oa ppreciate that in addition to the regulatory oligomeric interactions described above (i.e.,H IF:HRE, HIF:PHD/pVHL,H IF:FIH/CBP/p300), many other protein-protein interactions are involved in the regulation of HIF-mediated transcription, as is likely the case for any pleiotropic transcription factor.M odulation of these complexa nd dynamic interactions, which may occur at transcriptional, RNA-processing/splicing, translational and post-translational levels, offers potential for control of the set of HIF target genes that are upregulated. Further, post-translational modifications such as phosphorylation, acetylation and ubiquitylation are very likely to be important factors in HIF regulation. At present,w eh ave ap oor understanding of the role of theseo ther interactions on the kinetics of HIF-mediated transcription. HIF has been reported to interact with other transcription factors, notably Notch [56] and NFkB, [57] and is proposed to regulate, and be regulated by, multiple proteins involved in transcription/chromatin biology. [58] In this regard, the Jumonji-C histone demethylases are notable since they belong to the same structural subfamily of 2OG oxygenases as FIH.

Inhibition of Protein-Protein Interactions in the HIF System
The HIF system is presently perceived to be an attractive pathway for pharmacological intervention.O ne reason for this is that the discovery of HIFa nd its regulatory elements was motivated by ap hysiology-drivenr esearch approach, that is,t ou nderstandt he underlying molecular mechanisms behind the hypoxia-induced upregulation of EPO. [64] As econd, relatedr eason is that there is strong evidence that modulationo ft he activity of al imited number of key players in the HIF system can have profoundp hysiological consequences. Althought he factorsi nvolved in context-dependent regulation of the HIF system are incredibly complex, the 'core' hypoxicr esponse is principally mediated by ar elativelys mall number of players, that is, HIF, PHD2/VHL, FIH/CBP/p300. Thiss ituation was largely unanticipated;e ven after the discovery of the PHDs and FIH, we expected that other direct hypoxia sensors for the HIF system would be discovered. To date this has not been the case, althougho ther oxygenases (e.g.,h istoned emethylases) no doubt play ar ole in transcriptionalr egulation by HIF and might do so in ah ypoxically regulated manner. [59b, c, 65] Ak ey early concern with respectt op harmacological intervention of the HIF system was that its pleiotropic nature might be as afety issue, for example, PHD inhibitors might promote tumourg rowth by promoting VEGF production. However, counter-arguments include knowledge that major drugs do target transcriptional regulation, [66] living at altitude does not apparently cause as ignificantly increased incidence of cancer, [67] and that cobalt ions have been used for treatmento f anaemia in am echanism proposed to involve PHD inhibi-tion. [11b, 68] Perhaps the most important evidence is that PHD small-molecule inhibitors are now in late-stage clinicalt rials for the treatment of anaemia and are being considered for treatment of other hypoxia-related conditions, includings troke and ischaemic diseases. [69] Studies to date show,a taminimum, that it is possible to target the HIF system in the short term without serious (i.e. life-threatening) side effects. An increasing number of small molecules have been reported to modulate the HIF system by alteringH IF mRNA levels,p rotein synthesis/stability,D NA binding and transactivation. [70] Here we focus on molecules that interferew ith key protein-protein interactions-specifically,b inding of the HIF a-subunit to HIF-b, pVHL and CBP/p300.
Protein-protein interactions are often perceived to be difficult to target with smallm olecules. However,t he biological effects of FIH-and PHD-catalysed hydroxylationh ave been inspirational to efforts targeting protein-protein interactions, revealing how av ery small modification, that is, addition of as inglen eutralo xygen atom, can have profound effects on protein-protein interactions. [13,17] Indeed, the role of HIFa prolyl hydroxylation has led to an ew general approach to targeting proteins for degradation with small molecules. [71] Such efforts are crucially informed by structural analyses, with ar ecent highlight being the report of structures for the HRE-boundH IF-a,b complexes. [21]

Inhibition of the PHDs and FIH
Ta rgeting the PHDs via small-molecule inhibition has been am ajor focus of pharmaceutical efforts on the HIF system to date. Thev ast majority of the reported PHD inhibitors, and all of those in clinical trials, likely work by bindingt he actives ite iron and competing with 2OG (Figure 3e), though there are variations in binding modes, selectivity,a nd the extent to which they inhibitH IF-a binding. Because descriptionso f these approaches to PHD inhibition have been reviewed in detail elsewhere, [69] we do not describe them here;i nstead we focus on the likely influence of protein-protein interactions on the role of the PHDs.
In our view,i ti sp robably am istaket ot hink of the PHDs (and FIH) only as enzymes catalysing post-translationalm odifications.I tm ay well be that the stoichiometric protein-protein interaction between the PHDs and HIF-a plays ar ole in the hypoxia response. At the very least, understanding the details of the PHD:HIF-a interaction may be usefuli no ptimising PHD inhibitors, for example, some inhibitors more efficientlyd isplace HIF-a from the PHDs than others. [37] The development of substrate-selective inhibitors is of particular interestg iven the distinct physiological roles of HIF-1 and HIF-2 target genes. [5a, 72] Further,c ompounds that promote PHD activity are also of interest, in particular from ac ancer pharmaceuticalp erspective. Such compounds might work by strengthening the PHD:HIFa protein-protein interaction or by promoting the rate of its reactionw ith oxygen.A na lternative strategy for the latter would be to directo xygen to the PHDs inside cells. In this regard, it is of interest that am itochondrial respiration targeting compound has been recently reported to promote HIF-ChemMedChem 2016, 11,773 -786 www.chemmedchem.org a degradation, possibly by promoting PHD activity. [73] Reducing agents, such as ascorbate, have also been foundt op romote PHD activity in vitro, as is the case for someo ther 2OG oxygenases,m ost famously the collagen prolyl hydroxylases. [74] Although the effects of ascorbate are very unlikely to be selective for the PHDs, the cellular redox balance is likely to impact on the HIF system. [75] The emerging structural data on PHD:HIF-a interactions should also enable efforts in pharmaceuticalm odulation of PHD activityb eyond simple inhibition by active site binding.
There has been much less work on FIH inhibition than the PHDs. Thism ay be for severalr easons, including:1 )because FIH is of less fundamental importance than the PHDs in the hypoxic response, 2) because the role of FIH is linked to the function of the pleiotropic transcriptional coactivators CBP/p300, and 3) because FIH has multiple other substrates/bindingp artners than the HIF-a CAD (see above). Only al imited number of FIH inhibitors have been reported, [76] thoughe arly work on FIH was important in that it demonstrated that selectivity for specific 2OG-dependent oxygenases couldb ea chieved. [54] In the case of FIH and the PHDs, selectivity can be achieved by exploiting the smaller 2OG binding pocket presenti nt he PHDs relative to FIH. [44] Further,m odulating the complexp roteinprotein interactions involved in the FIH/HIF-a/ARD axis is of potentialt herapeutic interest, for example, blockingA RD, but not HIF-a,b inding to FIH should promote FIH-mediated inactivation of HIF-a activity,w hich might be of use from ac ancer perspective.

Targeting the HIF-a,b dimer
Formation of the a,b-HIF heterodimer is an apparently strict requirementf or HIFt ob ind to DNA andp romote transcription, [20,77] making this protein-protein interaction an appealing target for pharmaceutical intervention. One of the first drugs reported to modulate HIF dimerisation wasa criflavine, which is reported to bind at the interfaceb etween the HIF-a PAS-B and HIF-b PAS-A domains and to destabilise the HIF-a,b heterodimer. [78] Acriflavine is used as atopical antiseptic and is acombination of two structurally related flavins, trypaflavine andp roflavine ( Figure 5), both of which reportedlyb ind to HIF-1 and HIF-2 with low nanomolar affinities in vitro (40 nm and 41 nm for HIF-1 and HIF-2, respectively). [21] Recent crystallographic analyses reveal that proflavine makes contacts with residues R266 and V305 in HIF-b,w hich are critical for maintaining the integrity of the interface with HIF-a (Figure 2c and 2d). [21] The bindings ite for trypaflavine is unknown, but given its structural similarity to proflavine, it is predicted to be comparable. Acriflavinei nhibits HIF-mediated transcription in cultured cells and tumour xenografts, [78] and has been shown to decrease tumour growth and vascularisation, likelyi np art through inhibition of HIF signalling. [79] Several studies have explored ab uried, largely hydrophobic cavity within the HIF-2a PASd omain as ap otential site for binding of allosterici nhibitors. [80] This site was first revealed in crystallographic studies of the isolatedP AS-Bd omain of HIF-2a, [80d] but is also evident in the recently reported structure of the intact bHLH-PAS domain. [21] Work from Scheuermanne tal. has shown that this pocket can accommodate avariety of bicyclic ligands, some of whichp erturb the formation of the active HIF-2 heterodimer. [80a, d] The most potent of these compounds (0X3, Figure5)b inds to the isolated HIF-2a PAS-B domain in vitro with nanomolar affinity, [80b, c] and disrupts HIF-2a/HIF-b dimerisation and transcriptionala ctivity at low micromolar concentrationsi nc ultured cells. [80c] 0X3 and related bicyclic inhibitors are proposed to act via an 'allosteric' mechanism, inducing conformational changes in the isolated HIF-2a PAS-B domain such that it is no longera ble to bind HIF-b PAS-B.
[80c] Crystallographic analyses of 0X3 bound productively to the intact HIF-2 bHLH-PAS dimer (Figure 2c and 2e)r eveal few structural differences between the apo and liganded complexes, [21] suggesting it is the dynamics rather than the structure of the PAS-B domain that is alteredb yl igand binding; [89] if correct,t his proposal raises interesting possibilities for the fine-tuning of HIF transcriptionala ctivity.I nterestingly,c o-and chromatin-immunoprecipitatione xperimentsr evealt hat 0X3 disrupts the formation and HRE-bindingo ft he HIF-2-a,b heterodimer,w ith little effect on HIF-1.
[80c] These observations can be rationalised by structural comparison of the HIF-1a and HIF-2a PAS-Bd omains;a lthough as imilar cavity exists in HIF-1a,iti sconsiderably smaller and differs in the composition of residues that line the binding-pocket. [21, 80c] The discovery of substantial cavities in HIF-1a and HIF-2a raises the intriguing possibility that HIF is regulated by endogenousl igands, thougha sy et, none have been discovered.N evertheless, these pockets appear to be 'hot-spots' for inhibitor development. Cardosoe tal. have shown that the allosteric cavity in HIF-1a is amenable to smallmolecule inhibition. [90] More recently,acyclic peptide inhibitor (cyclo-CLLFVY,F igure 5) that selectively inhibits HIF-1a,b dimerisation has been described. This peptidei sr eported to decrease HIF-1-mediated activity in av ariety of cell lines, apparently withouta ffecting the function of the closely relatedH IF-2i soform. [81] Ta ken together, the combined studies suggest that the HIFa PAS-B domain is an interesting target for the development of inhibitors targeting the HIF-a,b complex. By exploiting binding pockets revealed from structurala nalyses, small-molecule modulators have the potential to enables electivei nhibition of HIF isoforms, and hence control the expression of HIF target genes. In addition to the compounds described above,w hich target HIF dimerisation, smallm olecules have been developed to block the HIF:HRE interaction by binding directly to DNA in as equence-specific fashion. [91] Though beyondt he scope of this review,t his work demonstrates that targeting the HRE is af easible strategy for HIF inhibition. In the longert erm, developing molecules that target defined sets of HIF target genes by selectively binding to HIFs/HREs associated with the promoterr egionso fs pecific genes is of interest, thoughw hether or not this is viable is presently unknown.

Targeting CBP/p300
One strategy for HIF inhibition is to block the interaction between HIF-a and CBP/p300 transcriptional coactivators. CBP ChemMedChem 2016, 11,773 -786 www.chemmedchem.org and p300 are multi-domainp roteinsa nd presentm ultiple therapeutic possibilities, including inhibition of their bromodomain and acetyltransferase domains. [92] Here we focus on attempts to block the interaction of the CBP/p300 CH1 domain with the HIF-a CAD. Pioneering work by Kung et al. led to the development of ah igh throughput screen for molecules blocking the HIF-a CAD:p300 CH1 domain interaction. [84] This work identified the epidithiodiketopiperazine natural product Chetomin ( Figure 5) as ad isruptor of the HIF:CBP/p300 interaction. Chetomin was observed to inhibitH IF-mediated transcription in tumour cells, [84] and likely works by 'ejecting' zinc from the p300/CBPC H1 domain;l oss of zinc disrupts the CH1 fold and hence binding to the HIF-1a CAD. [93] Related mechanisms of action (i.e.,z inc ejection/binding) likelya pply to the mode of action of other epidithiodiketopiperazines [94] and may account for their toxic effect to ruminants, including sheep. [93,95] Ah igh-throughput screen of natural products also led to the identification of quinones and indandiones that cause loss of structural zinc from CH1. [86] Eudistidine A, amarine alkaloid with an unusual tetracycline core comprised of two fused pyrimidine and imidazole rings ( Figure 5) has also been reportedt oi nhibitt he CH1 CAD interaction. [85] Another strategy used to target the HIF:CBP/p300 interaction has been the development of compounds that mimic the conformation of the HIF-a CAD when bound to the CH1 domain.S upport for this approach comesf rom observations that overexpression of CAD polypeptides attenuates HIF transcriptional activity in cells. [96] Twoh elices that are induced in HIF-1a upon binding to CH1 have been af ocus for development of peptidomimetic inhibitors (Figure 4b). An umber of different scaffolds have been used, including hydrogen bond surrogate helices, [88,97] aromatic oligoamides, [87] and oxopipera- Figure 5. Small-molecule inhibitors of HIF protein-protein interactions. As election of compoundsmodulating HIFp rotein-protein interactions are shown, includingt hose targeting 1) the HIF-a,b dimer:acriflavine, [78] 0X3, [80c] and cyclo-CLLFVY; [81] 2) HIF-a:pVHL interactions:Ligand 51 [36] and Ligand 7; [82] 3) HIFa:CBP/p300 interactions:KCN1, [83] Chetomin, [84] Eudistidine A, [85] Quinone 1 [86] and the peptidomimetic inhibitors OHM1, [55] Compound 3, [87] and HBS1. [88] ChemMedChem 2016, 11,773 -786 www.chemmedchem.org zine helix mimetics [55] (Figure 5). These compounds are predicted to bind in an orientation that positions key side-chains in am anner identical to that observed for the native HIF-1a helices in the CAD:CH1 structure. For example, peptidomimetics based on Helix 2m imict he orientation of residues 815-823, [55,87,88] including two conserved leucine residues (L818 and L822 in HIF-1a)t hat bind in ah ydrophobic pocket in CBP/ p300 (Figure 4b). Phage display-based analyses suggest that Helix 2b inds p300 with highera ffinity than any other region of the HIF-1a CAD, [98] which could explain why most of the reported peptidomimetic compounds have targeted this helix.
The most promising compounds identified to date bind p300 with sub-micromolar affinities, although the K d values are still an order of magnitude highert han those measured for HIF-1a CAD. [55,88,97] These compounds are reported to exhibit low cytotoxicity,t ob ea ctive in down-regulating HIF target gene expression in cells, and to suppress tumour growth in mouse xenograft models. [55,88,97] Similare ffects on HIF signalling were observed with the inhibitor KCN1, which arose from structure activity relationship studies on sulfonamides blocking HIF:CBP binding. [83,99] Molecular docking studies suggest that KCN1 may bind at one (or both) of the HIF-1a helix interaction sites in the HIF/p300 complex. [100] Peptides that bind to CH1 and displace the HIF-1a CAD have also been identified by phage-display methods; [98] the results of this work may be of interestw ithr espect to developing potent non-peptideb ased inhibitors.

Targeting HIF-a-pVHL Interactions
As outlined above, the binding of prolyl-hydroxylated HIF-a to pVHL (so signalling for HIF degradation) is central to hypoxia sensing by the HIF system.Thus, one strategy to mediate upregulation of HIF target genes is to disrupt the HIF-a:pVHL interaction. Pioneering studies using NODD/CODD peptides indicated that such an approachm ay be viable. [101] Work in recent years has focusedo nt esting the viability of pVHL as at arget for small molecules blocking HIF-a degradation, thoughi t should be noted that pVHL likely has roles outside of the HIF system. [102] Interestingly,t his work led to the idea of targeting proteins for degradation by the ubiquitin-proteasome system, by linking ap VHL bindert oas mall molecule targeting the protein of interest;t his PROTAC (Proteolysis targeting chimeric molecule) strategyh olds promise for research as well as therapeutic purposes. [71] Recently,B ondeson et al. have reportedo n substantially improved PROTACs, which were shown to have potent activity in cells against the serine-threonine kinase, RIPK2. [103] The first non-peptidic smallm olecules targeting the HIFa:pVHL interaction were designed using computational methods to mimic the structure of the HIF-1a peptideb ound to pVHL. [104] These compounds recapitulate key interactions in the HIF-a:pVHL complex,i ncorporating a trans-4-hydroxyproline residue, which is crucial for binding to pVHL, as well as an isoxazole moiety designed to interact with as tructural water molecule at the HIF-a:pVHL interface (Figure 3c). The lead compound from this early work bound to pVHL with a K d of 5.4 mm,a sd etermined by isothermal titrationc alorimetry. [104,105] Structure activity relationship studies were carried out to increase the binding affinity to pVHL, resulting in as eries of compounds that bind pVHL with sub-micromolar affinity (e.g., Compound 51,F igure 5). [36] Further optimisation was carried out using as tructure-and metrics-driven approach to increase binding affinity and lipophilicity,w ith av iew to obtaining tight-binding cell-active chemical probes. [82] The most potent inhibitor of the HIF-a:pVHL interaction identified to date (Compound 7,F igure 5) binds isolated pVHL with a K d of 185 nm, similar to the value observed for a1 0-mer HIF-1a peptide binding to pVHL (K d = 200 nm). [82] Although compounds in this series still contain a trans-4-hydroxyproline,t hey are largely non-peptideb ased. Furtherw ork could be targeted towards the development of completely non-peptidic compounds based on the 4-hydroxyproline analogues.

Summary and Outlook
Work over the last 15 years or so has led to the discovery that specific protein-protein interactions play central roles in hypoxic sensing in humans and other animals. Considerable progressh as been made towards developing chemically useful inhibitors of the sets of enzymes underlying HIF-a hydroxylation, that is, the PHDs,a nd in demonstrating that disrupting the interactions between prolyl hydroxylated HIF-a and pVHL is at ractable target for small molecules. There remains considerable scope for the development of new types of PHD inhibitor and other compounds that bind to pVHL;i nt he latter case, the identification of compoundsthat do not contain ahydroxyproline residue is of interest. It may also be that new challenges in pharmaceutical targeting of pVHL will becomea pparent as compounds are progressed into animal models, especially due to its HIF-independent roles. Interestingly,t his work has helpeds timulate the use of small molecules to rationally targetp roteins for 'catalytic' degradation of protein targets rathert han just inhibiting by tight binding. TheH IF-a:FIH and CBP/p300 interactions are seeminglye ven more challenging due to the apparently HIF-independent roles of FIH and, in particular, of CBP/p300,w hich are involved in the regulationo f many genes unrelated to HIF.F inally,a lthough at an early stage, use of biophysical insights to guidet he development of compounds that bind to and regulate the activity of the intact HIF:HRE complex in order to alter the kinetics of transcription is ap articularly exciting field, especially in light of recent structural information.