Inhibition of Hypoxia-Inducible Factor Prolyl Hydroxylase Domain

Perspective pubs.acs.org/jmc

Inhibition of Hypoxia-Inducible Factor Prolyl Hydroxylase Domain Oxygen Sensors: Tricking the Body into Mounting Orchestrated Survival and Repair Responses Michael H. Rabinowitz* Janssen Pharmaceutical Research & Development, LLC, 3210 Merryfield Row, San Diego, California 92121, United States ABSTRACT: Hypoxia-inducible factor (HIF) is an oxygen-sensitive dimeric transcription factor that responds to pathophysiologically low O2 tensions via up-regulation, which leads to an orchestrated biological response to hypoxia. The HIF prolyl hydroxylase domain (PHD) enzymes are non-heme, ironcontaining dioxygenases requiring for activity both molecular oxygen and 2oxoglutarate that, under normoxia, selectively hydroxylate proline residues of HIF, initiating proteosomal degradation of the latter. The dependence of HIF protein levels on the concentration of O2 present, mediated by the PHD enzymes, forms the basis for one of the most significant biological sensor systems of tissue oxygenation in response to ischemic and inflammatory events. Consequently, pharmacological inhibition of PHD enzymes, leading to stabilization of HIF, may be of considerable therapeutic potential in treating conditions of tissue stress and injury. This Perspective reviews the PHDs and small molecule drug discovery efforts. A critical view of this challenging field is offered, which addresses potential concerns and highlights exciting possibilities for the future.

■

A BRIEF HISTORY OF OXYGEN Oxygen is the most abundant element on the planet, making up nearly 90% of the mass of our waters and nearly 50% of the mass of the earth’s crust. However, dioxygen (molecular oxygen, O2) was never a significant component of our atmosphere until roughly 2.4 billion years ago when the inability of seas and minerals to further absorb photosynthetically produced molecular oxygen led to an explosion of O2 levels in the atmosphere known as the great oxidation event or great oxidation catastrophe, depending on whether you were an aerobe or anaerobe at the time. The increase of atmospheric O2 brought on the extinction of countless anaerobic organisms and a fundamental shift of the terrestrial biome favoring species that could not only tolerate this newly abundant “toxic” byproduct but also thrive on its potential for generating energy through oxidation. Although O2 levels have fluctuated greatly over the past 500 million years, today O2 in the atmosphere at sea level makes up 20.8% by volume, which decreases in partial pressure (but remains constant in proportion) in a predictable way with increasing altitude. Eukaryotic organisms have evolved to adapt to changing O2 levels in their environments to maintain a metabolic homeostasis and are capable of surviving habitually at high altitude (∼10 000 feet) and acutely at extreme altitudes (29 029 feet, demonstrated dramatically by Messner and Habeler’s ascent of Everest without supplemental oxygen). Consequently, the ability of the body to sense, regulate, and respond to changing O2 concentrations in the environment must be tightly controlled and must respond to both acute and chronic changes in O2 concentrations with an orchestrated response at the cellular, tissue, and organ levels. This tight © XXXX American Chemical Society

sensing and response to changing O2 levels goes well beyond sensing changes in ambient O2; it forms the very basis by which animals respond to tissue injury, which often involves loss of blood or blood supply to affected tissues. Outside the normal ranges of O2 pressures on the terrestrial planet (∼160 mmHg at sea level, ∼110 mmHg at 10 000 feet) the body suffers from either too much oxygen (hyperoxia) or too little (hypoxia). Hyperoxia is typically a concern with an ambient pO2 above 225 mmHg (30 kPa) and leads to the formation of reactive oxygen species (ROS) such as peroxide, superoxide, etc., which can cause damaging oxidation to nucleic acids, proteins, and lipids and result in vision and hearing loss, renal and hepatic failure, neural damage, and ultimately death.1,2 Severe hypoxia on the other hand is most commonly associated ischemic disease, vascular disease, and chronic inflammation and may be lethal at arterial O2 pressures below 50 mmHg, half of the physiological arterial pressure of 100− 120 mmHg.

■

OXYGEN IN THE BODY Oxygen is the ultimate electron sink for eukaryotic oxidation of carbohydrates and fats and is the terminal electron acceptor in the electron transport chain of oxidative phosphorylation that aerobes use to efficiently produce ATP. Respired atmospheric O2 is diffused into the blood, whose carrying capacity of O2 is increased greatly by the presence of hemoglobin, and circulated throughout the body by the heart and the vasculature where Received: March 21, 2013

A

dx.doi.org/10.1021/jm400386j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Perspective

Table 1. Some Gene Products Thought to Be Controlled by HIFa erythropoiesis and Fe metabolism erythropoietin transferrin transferrin receptor cerruloplasmin ferrochelase cytochrome b divalent metal transporter hepcidin (neg) MDR p-glycoprotein

glucose metabolism pyruvate kinase M phosphoglycerate kinase 1 phosphofruktokinase liver isoenzyme hexokinase 1 hexokinase 2 glyceraldehyde 3-phosphate dehydrogenase aldehyde dehydrogenase A aldehyde dehydrogenase C enolase 1 carbonic anhydrase 9 pyruvate dehydrogenase kinase 1 PFKFB3 GLUT-1 GLUT-3 lactase lactate dehydrogenase A phosphoglycerate mutase B triphosphate isomerase

angiogenesis/vacular tone

immunity/cell viability and proliferation

matrix and barrier

FGF-2 epidermal growth factor platelet-derived growth factor β VEGF-A VEGFR1 VEGFR2

IL1-β TGF-β IGF-2

procollagen prolyl hydroxylase α1 intestinal trefoil factor ecto-5′-nucleotidase

IGF-BP1 IGF-BP3 p21

placental growth factor stromal-derived growth factor 1 angiopoietin 1 angiopoietin 2 angiopoietin 4 response gene to complement 32 CXCR4 plasminogen activator inhibitor 1 cyclooyxgenase-2 thrombospondin plasminogen-activator receptor 1 i-NOS heme oxygenase 1 adrenomedulin α-adrenergic receptor 1b Tie-2 eNOS leptin endothelin 1 endothelin 2 tyrosine hydroxylase atrial naturetic peptide

p35srj NIX

stromal derived factor 1 c-Met CXC chemokine receptor 4 (SDF-1 receptor) β2-integrin HSP90

NDRG TGF-α FADD (neg) GMCSF

SCF mucin netrin 1

HGH endoglin cyclin D1 ROR-γt NIP-3 Noxa adenylate kinase α-fetoprotein (neg) calcitonin-receptor-like receptor IKK-β IFN-γ CD-73

a

Data are from the following: (1) Mole, D. R.; Blancher, C.; Copley, R. R.; Pollard, P. J.; Gleadle, J. M.; Ragoussis, J.; Ratcliffe, P. J. J. Biol. Chem. 2009, 284, 16767. (2) Kim, J.-w.; Tchernyshyov, I.; Semenza, G. L.; Dang, C. V. Cell Metab. 2006, 3, 177. (3) Papandreou, I.; Cairns, R. A.; Fontana, L.; Lim, A. L.; Denko, N. C. Cell Metab. 2006, 3, 187. (4) Ortiz-Barahona, A.; Villar, D.; Pescador, N.; Amigo, J.; del Peso, L. Nucleic Acids Res. 2010, 38, 2332. (5) Gaber, T.; Dziurla, R.; Tripmacher, R.; Burmester, G. R.; Buttgereit, F. Ann. Rheum. Dis. 2005, 64, 971. (6) Schofield, C. J.; Ratcliffe, P. J. Nat. Rev. Mol. Cell Biol. 2004, 5, 343. (7) Andrikopoulou, E.; Zhang, X.; Sebastian, R.; Marti, G.; Liu, L.; Milner, S. M.; Harmon, J. W. Curr. Mol. Med. 2011, 11, 218.

angiogenesis, vasomotor tone, cell viability and proliferation, inhibition of apoptosis, matrix and barrier function, production of pro- and anti-inflammatory cytokines, hormone regulation, host defense, cell migration, transcriptional regulation, and transport (Table 1).9−11 That there should exist one or more molecular mechanism by which changing O2 levels can be sensed in aerobic species makes a good deal of sense, but not until 1992 was a single factor, now known as hypoxia inducible factor (HIF), identified and characterized.12 This ubiquitous heterodimeric transcription factor accumulates in response to hypoxia and binds the HRE consensus motif in DNA promotor regions, controlling gene transcription.

concentrations drop with each passage through distal tissues. Physiological levels of O2 are roughly 160 mmHg (sea level air), 100 mmHg (arterial), 50 mmHg (venous), 30 mmHg (interstitial tissues), and 20 mmHg (cytosol).3 It was originally believed that O2 was sensed in mammals by very select tissues in the pulmonary arteries and neuroepithelial cells, and glomus cells of the carotid body, to name a few.4 The linkage between erythropoietin (EPO, the body’s master hormone controlling erythropoiesis) protein and message upregulation and hypoxia or anemia has been studied as well.5 It is now known that all nucleated cells in the body are able to detect changes in O2 levels and mount a physiological response when O2 drops to pathophysiological levels.6 These responses come in the form of changes in hundreds of mRNAs, both via up-regulation and down-regulation.7,8 A so-called hypoxic response element (HRE, a hypoxia-responsive transcriptional regulatory motif usually found in or near the promoter of hypoxia responsive genes) has been found both 5′ and 3′ to hundreds of genes in nearly all tissue types.9 Activation or blocking of these HREs is known to lead to changes in erythropoiesis and iron metabolism, energy metabolism,

■

HIF BIOLOGY The transcription factor HIF was discovered in 1992 by Semenza and Wang who identified it as a multimeric factor binding directly to a 50-base-pair hypoxia-inducible enhancer 3′ to the EPO gene and that this protein appears in response to low oxygen.12 (Later work showed that it is the degradation of this protein, not its synthesis, that is controlled by O2 levels; see B



Perspective

Figure 1. Reprinted with permission from Journal of Medicinal Chemistry: cover artwork of April 2012 issues of Volume 55 (issues 7 and 8) by Vachal et al.160 Copyright 2012 American Chemical Society.

than HIF-1 but is thought to be the primary regulator of renal EPO production in response to low O2 20,21 and is the main mediator of iron metabolism,22 although HIF-1 does play a small role.23 HIF-1β, also known as ARNT, the aryl hydrocarbon receptor nuclear translocator, is an 87 kDa, 789 amino acid24 member of the basic helix−loop−helix (bHLH) family of proteins containing a PAS (Per-ARNT-Sim) signaling/heterodimerization domains25 and an N-terminal DNA binding region bHLH domain that specifically binds the HRE 5′-G/ACGTG-3′ motif and also contributes to heterodimerization.26 HIF-1β is found in the nucleus of all nucleated cells, and its concentrations are constant and insensitive to O2. What made the identification of HIF as an O2 sensor challenging is the nature of the second partner of the dimer, HIF-α. Unlike HIF-1β, HIF-1α is sensitive to O2. It is an 826 amino acid, 120 kDa protein that, like HIF-1β, is a PAS domain containing bHLH family peptide whose mRNA has also been detected in all nucleated cells. Steady state levels of HIF-1α and HIF-1β mRNA appear to be fairly insensitive to biological O2 levels. HIF-1α protein product, however, exists with a half-life of approximately 5 min, which masks its presence under normoxia.27 Under normoxia, HIF-α is ubiquitinated at one (or

below.) The identity of the exact HRE motif was subsequently narrowed to the 5 nucleotide sequence 5′-G/ACGTG-3′ now thought to be present in all DNA HIF binding sites.13 The biology described in this section and the next is shown pictorially in Figure 1. HIF is present in cells almost exclusively in two forms: HIF-1 and HIF-2. They are heterodimeric transcription factors consisting of a constitutively produced highly abundant HIFβ subunit and either a HIF-1α or HIF-2α partner, in the case of HIF-1 and HIF-2, respectively, sharing 48% sequence homology. Expression of HIF-1α and -1β mRNA had been detected in all human tissues.14 A HIF-3 has been identified but has been less well studied. HIF-1 and HIF-2 have largely overlapping but some nonredundant functions. HIF-1 is frequently associated with metabolic and vascular responses to hypoxia, whereas HIF-2 is associated with vascular systems but also somewhat more with erythropoiesis.7,15 Glycolytic enzymes, particularly pyruvate dehydrogenase kinase (PDK), are under the control of HIF-1 primarily, mediating the inhibition of entry of glycolysis pathway products into the TCA cycle under O2-limiting conditions.13,16,17 HIF-1 is also associated with the angiogenic response to local tissue ischemia.18,19 HIF-2 on the other hand is less broadly expressed C



Perspective

Figure 2. Proposed catalytic cycle of the HIF PHD enzymes.

both HIF (C4 hydroxylation) and 2-OG (to succinate and CO2). The term dioxygenase refers to the family of enzymes that reduces O2 and incorporates each atom of oxygen into an organic molecule (succinate and CO2 in the current case), as opposed to water. It is the combination of the discovery of the PHD class of HIF hydroxylation enzymes, the understanding of their functional dependence on biological O2 concentrations, and the susceptibility of oxidized HIF to degradation that form the basis for our current understanding of metazoan O2 sensing and the hypoxic response.

more) of three Lys residues (K532, K538, and K547) by an E3ubiquitin ligase consisting of elongin B, elongin C, cullin 2, and ring-box 1 and mediated by von Hippel−Lindau protein (pVHL), targeting HIF-α for proteosomal degradation by the 26S proteosome.28−30 High resolution X-ray structures of the pVHL−elongin B−elongin C (VBC) complex bound to a hydroxylated HIF-α analogue has been solved vividly demonstrating the important role of proline hydroxylation in this protein−protein interaction.31,32 In addition to bHLH-PAS domains complementary to HIF-1β, HIF-1α contains both Nand C-terminal transactivation domains (N-TAD and C-TAD). Importantly, there are also present two oxygen dependent degradation domains, NODDD and CODDD, named for their positions relative to the N and C termini of the peptide. The latter is located on or near the N-TAD. It is only via specific trans C4 hydroxylation of either of two proline residues (Pro402 and Pro564) in NODDD and CODDD, respectively, that pVHL is able to recognize HIF-α and mediate its ubiquitination, displaying at least a 2000× greater affinity for the hydroxylated form.31 Erythropoietin protein expression is known to be inversely regulated by O2 concentrations in the kidney. The main sensor of O2 in the regulation of EPO was originally postulated to be a heme protein because of gene induction by traditional blockers of heme oxygenation (low pO2, by CO, Ni2+, and Co2+).33 It is now known that cellular HIF is degraded rapidly in the presence of physiological concentrations of O2 and that this degradation is controlled not by heme proteins but by a class of dioxygenases known as the HIF prolyl-4-hydroxylase domain enzymes, referred to here as PHD enzymes. PHDs are non-heme, iron-containing dioxygenases that require molecular oxygen (O2), 2-oxoglutarate (2-OG), and a bioreductant (usually ascorbate) in order to catalyze the concerted four-electron reduction of O2 and the oxidation of

■

HIF PROLYL HYDROXYLASE DOMAIN ENZYMES Roughly 10 years after the discovery of HIF-α the PHD enzymes were first described.34−37 They are members of the dioxygenase family that requires O2, Fe, and the TCA cycle intermediate 2-oxoglutarate for their catalytic activity, which is the largest family of non-heme containing iron-dependent enzymes.38 The active site is characterized as containing octahedral ferrous ion (Fe(II)) bound by the highly conserved facial triad of His313, Asp315, and His374 (PHD2 numbering) with the remaining three coordination sites presumably occupied by labile water molecules in the resting state (Figure 2). It is believed that these loosely bound water molecules are important for the stabilization of this complex in the absence of HIF substrate.39 2-OG may then displace two of the water ligands on Fe(II) in a bidentate chelation mode through the 2OG C1 carboxylate and C2 carbonyl, with the C5 carboxylate group making a strong ionic bond with the highly conserved Arg383 in the back of the active site pocket. Displacement of the final water molecule by O2 at the apical position and directly adjacent to the HIF substrate binding site is followed by internal redox reorganization, formally resulting in oxidation of Fe(II) to Fe(IV) with concomitant C−O bond formation D



Perspective

with the α-keto group of 2-OG followed by C1−C2 bond cleavage and the formation of bound succinate and CO2 and a reactive high-spin Fe(IV)-oxo species.40−42 In this sense, both Fe(II) and 2-OG act as two-electron reductants to convert O2 into 2O2‑. The binding of substrate (HIF in this case) prior to the formation of the Fe(IV)-oxo species is considered obligatory because the highly reactive metal-oxo containing protein, in the absence of substrate, would be expected to autoxidize active site residues, leading to inactivation. High spin Fe(IV)-oxo complexes have varying reactivities toward hydrocarbon oxidation and may be either octahedral or trigonal bipyramidal in geometry.43,44 It is tempting to envision a change in geometry of the metal center upon oxidation, promoting active site reorganization allowing movement of a HIF proline residue into proximity for hydroxylation. An X-ray cocrystal structure of a CODDD HIF fragment bound to PHD2 catalytic domain shows Pro564 positioned away from the presumed apical metal-oxo center, suggesting that a conformation shift would be required for hydroxylation at the pro(R) position of the C−H bond at C4.45 Km values for 2-OG are ∼1 μM for the purified enzymes in vitro,46,47 which are considerably lower than those of crude enzyme preparations.48 The difference may be due to the presence of high endogenous concentrations of 2-OG.47 Even lower Km values for 2-OG have been found using pseudo fulllength HIF constructs, and the authors suggest that high measured Km values for 2-OG may arise from the presence of endogenous 2-OG, iron, or TCA cycle intermediates.49 PHD enzymes show a low affinity for O2 (Km of 230−250 μM),48 which is about 2× to 10× above the various O2 concentrations observed physiologically.3 This important observation is critical in understanding the HIF PHDs to be true O2 sensors: the location of tissue O2 concentrations on the steepest part of the PHD velocity/O2 concentration curve means that enzyme turnover is exquisitely sensitive to changes in local O2 concentrations. Factor inhibiting HIF, or FIH, is another important dioxygenase that controls HIF activity, but not abundance, via specific hydroxylation of an Asn residue at the CODDD of HIF. It has low sequence homology with the PHDs and thus may be the target of selective drugs. FIH has been well reviewed elsewhere.50,51

pathway, and other isoforms may be more important in alternative hypoxia-responsive pathways (e.g., PHD1 and NFκB). It is present, mainly in the cytoplasm, in all tissues and is abundant in the heart. It has a substrate preference for HIF-1α over HIF-2α and is induced by HIF (negative feedback). PHD2 knockout mice are not viable and die between embryonic days 12.5 and 14.5 because of placental and cardiac defects.54 However, PHD2 conditional knockouts show a clear phenotype of increased angiogenesis, increased serum VEGF-A, and increased serum EPO and are polycythemic.56 Like PHD1, PHD2 hydroxylates both Pro402 and Pro562 of HIF. The third isoform, 27.3 kDa (239 amino acid) PHD3, is also induced by hypoxia and hypoxia mimetics.57 It acts equally on HIF-1α and HIF-2α under normoxia, but preferentially HIF-2α in hypoxic conditions, and is believed to be present in roughly equal amounts in both the nucleus and cytoplasm. PHD3 knockout mice are viable but show distinct CNS changes in innervation and reduced resting blood pressure. PHD3 hydroxylates Pro564 of HIF-α exclusively. A fourth isoform has been described but is much less well characterized.58 By use of a HIF protein construct containing the NODDD, CODDD, and CAD domains, it has been shown, via proteolysis linked with LC−MS/MS techniques, that the three PHD isozymes have a preference for CODDD hydroxylation (Pro564) over NODDD (Pro402) hydroxylation for both HIF-1α and HIF-2α constructs, with PHD3 favoring reaction at CODDD almost exclusively.49

■

HIF TARGET GENES The response to hypoxia differs substantially by cell type. In an integrative approach to identifying HIF target genes, hypoxic array data were combined with mathematical modeling. The result was the identification of over 6000 genes that are hypoxia responsive. In ∼70% of the promoter regions of those genes, however, an HRE was not identified, suggesting that if HIF is involved, its control is indirect.59 A selection of known HIFregulated genes is listed in Table 1.

■

PHD TARGET PROTEINS In the past few years it has become apparent that PHD enzymes interact with more than just HIF, although these interactions are far from well understood. Most importantly, there is evidence that supports PHD1 likely playing a role in directly controlling NF-κB protein levels. Hypoxia is known to increase NF-κB-dependent protein transcription, and it has been suggested that PHD1 acts directly on the conserved LXXLAP motif of the inhibitor of NF-κB kinase-β (IKKβ) to inactivate it through hydroxylation. Inactivation of PHD1 (via siRNA or the small molecule pan-PHD inhibitor dimethyloxalylglycine, DMOG) leads to increased IKKβ activity, releasing NF-κB from its inhibitory comnponent, IκB, via phosphorylation-dependent targeting to ubiquitination and subsequent proteosomal degradation. Mutation of the key Pro residue in the PHD recognition motif of IKKβ (P191A) resulted in a loss of hypoxia induced expression of IKKβ. Proof of the action of PHD1 on IKKβ remains to be demonstrated (possibly by mass spectrometry).60 Activating transcription factor 4 (ATF4) is a protein that regulates genes involved in oxidative stress, amino acid biosynthesis, cell differentiation, metastasis, hematopoiesis, and angiogenesis among other things.61 It has been shown that ATF4 is up-regulated under hypoxia and that its up-

■

PHD ISOFORMS There are three known isoforms of the HIF PHD enzyme, PHD1, PHD2, and PHD3, (or EGLN-2, EGLN-1, and EGLN3, respectively), named after their cloning in C. elegans from the egg-laying defective 9 gene.36,37,52 The three isoforms share high sequence homology in their C-terminal domains but not in their N-termini. Their localization in cells has been investigated by fluorescent tagging.53 PHD1 is a constitutively produced nuclear form of 43.9 kDa (407 amino acids) and has limited tissue distribution, present mainly in testes but also in brain, kidney, heart, and liver. Under normoxia, it shows a preference for HIF-2α over HIF-1α and has equal affinity for hydroxylation of both Pro402 and Pro564. Knockout of PHD1 produces viable offspring that show a phenotype of tolerance to ischemia but with a reduced exercise tolerance. This phenotype is associated with a shift from TCA cycle (oxidative) to glycolytic cellular energetics,54 and it has been commented that PHD1 knockout mice experience an “oxygen saving” phenotype.55 In contrast, PHD2 (46 kDa, 426 amino acids), considered the most important of the three for the HIF E



Perspective

the fast-twitch phenotype in mouse muscle78 and an increase in autophagy in mouse chondrocytes.79 Postnatal ablation of the HIF-2α gene results in profound anemia, and HIF-2 is considered to be the primary isoform responsible for EPO production.80 One review of the literature indicated that at least 35 polymorphisms of HIF-1α have been identified in various populations; however, their association with disease remains complex and variable.81 The P582S and A588T single nucleotide polymorphisms (SNPs) show an association with non-small-cell lung carcinoma in 285 Taiwanese patients.82 The former SNP has been the only polymorph identified with a gain of HIF-1α function.83,84 This same mutation (P582S) was also associated with protection in diabetics from nephropathy.85 These germline SNPs have also been associated with disease stage, progression, and prognosis in certain cancers.86 However, in a study of Korean colorectal cancer patients, these polymorphisms were not found to associate with disease prognosis.87 This same polymorphism has been associated with increased risk for cervical and endometrial cancers in a Turkish population; however, two other HIF-1α polymorphisms did not associate with disease in the same population.88 Another study concluded that a V116E HIF-1α loss of function variant was not associated with renal cell carcinoma.89 Gain of function HIF-2α mutations in individuals have been reviewed as well.90 Although these polymorphisms are presented as case histories rather than large population associations, all of the individuals carrying these five gain of function mutations (P534L, M535V, M535I, G537W, G537R) presented either frank eythrocytosis or elevated hemoglobin with cardiovascular or pulmonary disease. No instances of cancer were noted. The biochemistry of some of these gain of function mutants was examined, and it was found that they resisted normal HIF-pathway degradation because of reduced binding to PHD enzymes, to pVHL, or to both.91 In vitro, the behavior of SW480 colon cancer cells was investigated in the presence of siRNA to both HIF-1α and HIF2α. The researchers found that knockdown of HIF-1α led to no change in cell proliferation, reduced cell migration and, when injected sc into mice, resulted in smaller tumors than with SW480 cells in the absence of the siRNA. Conversely, knockdown of HIF-2α led to an increase in cell proliferation and no change in cell migration in vitro and larger tumors in vivo with respect to cells absent the siRNA. The authors conclude that HIF-1α and -2α have opposing roles in tumor maintenance due to their effects on different panels of target genes.92

regulation is dependent on PHD3 in a non-VHL/ubiquitin dependent manner, with a half-life under normoxia of ∼13 min. Identification of an oxygen dependent degradation domain in ATF4 and mutation of its five proline residues to alanines resulted in stabilization of ATF4 toward normoxia. Both siRNA to PHD3 and DMOG inhibition led to up-regulation of ATF4 protein, lending support for direct PHD oxygen sensor control of this stress pathway.62 PHD enzymes may interact with other target proteins in a nonhydroxylation dependent manner, and some of these have been reviewed.63

■

MODERATE HYPOXIA AND HEALTH It has been known for a long time that weight loss in humans increases with increasing altitude and, while beneficial at moderate elevations, is pathophysiological at extreme altitudes. Indeed, weight loss is an important factor considered by mountain climbers when scaling the world’s highest peaks. Epidemiological studies have shown that residents of Colorado, the U.S. state with the highest average altitude, have the lowest levels of obesity (15−20% prevalence) in an increasingly obese nation.64 Furthermore, epidemiological studies have suggested either no correlation or increased life expectancy of those living at or above 4500 feet with respect to living at sea level. They have also shown an association between increasing altitude and lower levels of coronary heart disease and possibly increased rates of chronic obstructive pulmonary disease (COPD). There is no association between altitude and statistical changes in incidences of cancer and stroke.65−67

■

HIF KNOCKOUTS AND MUTATIONS As mentioned previously, HIF plays an important part in tissue survival in the face of injury and stress, and it is not surprising therefore that gross deregulation of HIF has significant biological consequences. HIF-1β, -1α, and -2α are required for normal development.14 There is a remarkable conservation of the HIF system across eukaryotic organisms. HIF-1α homozygous knockout mice die during gestation (E11), suffering from decrease in VEGF levels, neural tube defects, and cardiovascular defects.68,69 Isolated cells lacking HIF-1α have been shown to have reduced levels of glycolysis pathway proteins and are not capable of switching from oxidative to glycolytic metabolism under hypoxic conditions via the down-regulation of COX enzymes PDK1 and c-Myc and mitochondrial autophagy.68,70 HIF-1α heterozygous knockout mice, carrying just one functional copy of the HIF-1α gene, develop normally but have poor ventilation response to hypoxia, traced to changes in the carotid body.71 These mice are also no longer conferred cardiac muscle protection from ischemia reperfusion injury72 and have reduced pulmonary hypertension under conditions of low oxygen.73 Conditional HIF-1α knockout of neural cells in mice causes a loss in spatial memory and a disruption of normal brain development.74 Conditional knockout of HIF-2α but not HIF1α in the mouse duodenum showed clear effects on iron metabolism and transport.75 Germ-line knockout of HIF-2α is largely embryonic lethal, and HIF-2α−/− mice that are born suffer from severe cardiopulmonary defects.76 Somatic deletion of the HIF-2α gene leads to severe pancytopenia.77,20 The conditional knockout of HIF-2α has been shown to lead to a switch to

■

VHL KNOCKOUTS AND MUTATIONS Certain defects in the gene encoding for von Hippel−Lindau protein (pVHL, considered to be a tumor suppressor gene) led to von Hippel−Lindau syndrome, a rare genetic condition that increases risk for certain malignancies, most commonly clear cell renal carcinomas (CCRC), pheochromocytomas (adrenal gland cancer), and retinal hemangioblastomas.93 Vhl−/− mice are nonviable and die in gestation, whereas germline heterozygotes are predisposed to these associated cancers, especially if the second allele is somatically inactivated.94 Conditional knockout of VHL results in abnormalities of the vasculature and of the testes, leading to infertility.95 In other studies, conditional knockout of vhl led to hepatic hemangiomas in mice.95,96 F



Perspective

As a tumor suppressor gene, vhl appears to follow the “double-hit” hypothesis whereby the VHL phenotype is typically observed in predisposed individuals (i.e., vhl−/+ heterozygotes) upon somatic disregulation of the second allele.97 Because loss of function of pVHL is associated with HIF-α accumulation due to defects in its breakdown pathway (i.e., proteosomal degradation), the phenotypes of loss of pVHL and of up-regulation of HIF-α are associated, and HIF up-regulation has thus been associated with VHL syndrome phenotypes. While this may be true in some instances, the reality is much more complex. The association of elevated HIF levels with tumor survival and poor prognosis in cancer patients has been reviewed carefully.98 As argued forcefully by Kaelin,99 while there is evidence suggesting that pervasive, persistent deregulation of HIF-α via defects in VHL may be causal in certain VHL cancers, there is also evidence suggesting that the connection between the two is one of correlation and that HIFα stabilization may be a necessary but not sufficient condition for maintenance and growth of certain hypoxic tumors. Supporting this is the increasing understanding of HIFindependent functions of VHL (such as microtubule stabilization, regulation of apoptosis, and extracellular matrix formation). Explaining why not all modes of HIF-α upregulation/stabilization are equal, Kaelin suggests the following: “The existence of HIF-independent functions may be one of the reasons why individuals who are chronically hypoxemic do not have the same clinical manifestations as people with VHL disease...”.94,99 Supporting this assertion as well is genetics. There exist a number of hypomorphic variants of pVHL that are not associated with phenotypic neoplasms or VHL syndrome. Chuvash polycythemia is a hereditary autosomal recessive condition found in high prevalence in the Chuvash Republic of Russia in which affected individuals have headaches, fatigue, and elevated hemoglobin resulting from erythrocytosis and cardiovascular disease.100 This condition is associated with the R200W loss of function mutation producing a protein product with reduced affinity for hydroxylated HIF-α, resulting in upregulation of the latter.101 Despite this loss of function VHL mutation, there is no associated increase in neoplasms in the Chuvash polycythemia population and they do not have symptoms of VHL disease. Other VHL mutations have been found in multiple individuals with most (but not all) not having associated malignancies.102

Of the 10 PHD2 SNPs studied, none, save for a mutation of residue 374, were associated with a neoplastic phenotype. In this case the H374 residue, part of the highly conserved HisAsp-His triad that binds the catalytic Fe, was replaced by an arginine resulting in a denatured protein that would be expected to not bind iron and hence have no catalytic activity in hydroxylating HIF. Preparation of this mutant in vitro supported this hypothesis, as the resulting protein was found to be unstable and nearly completely inactive in a luciferase reporter assay over a 10-fold concentration range. The presenting individual with this mutation, in addition to erythrocytosis, revealed a phenotype similar to that of von Hippel−Lindau patients: recurrent paraganglioma. Biopsy and genotyping of the tumor revealed a loss of heterozygosity in the neoplastic tissue, supporting the “double hit” hypothesis as discussed above with respect to von Hippel−Lindau disease.106 Because of the appearance of paraganglioma associated with a PHD2 polymorphism in an individual, groups of patients displaying phaeochromocytoma (82 patients) and renal cell carcinoma (RCC, 64 patients) were genotyped for PHD1, -2, and -3 mutations. No association of PHD enzyme mutations and cancer was found. The researchers concluded that while HIF pathway dysregulation is necessary for predisposition to phaeochromocytoma and RCC, it is not sufficient and that it is likely impairment of non-HIF-related functions of pVHL that is required for the neoplastic phenotype.107 Furthermore, inhibition of PHD enzymes through catalytic inactivation with iron chelators such as deferasirox (approved by the U.S. FDA in 2005), leading to the promotion of EPO production and to hematopoiesis in anemia patients, is not associated with increased risk for cancer or VHL-type sequelae. A 104 week rat study revealed no risk for carcinogenicity.108 Kaelin and co-workers have shown that down-regulation of PHD1 using siRNA results in a decrease in cyclin D1 protein levels in a HIF-independent manner and a subsequent suppression of cancer cell proliferation.109 Also, HIF-1α stabilization via PHD inhibition with DMOG reduced the expression of the M-MITF oncogene, leading to melanoma cell growth arrest.110 These studies suggest an antitumor role for PHD inhibition. Taken as a whole, the evidence supports the expectation that moderate, transient pharmacological inhibition of PHD enzymes and the resulting stabilization of HIF are mechanistically distinct from both pVHL dysfunction and the chronic, global HIF elevation that are associated with malignancies.

PHD KNOCKOUTS AND MUTATIONS As mentioned above, PHD2 knockout is embryonic lethal whereas germline phd1 and phd3 knockout mice are viable and appear normal. Somatic deletion of phd2 resulted in polycythemic mice with enlarged spleens and livers with notable vascular phenotypes and premature death.103 McMullin has reviewed 10 human PHD2 mutations discovered since 2006.90 Of these 10 variants, all are loss of function mutations that resulted in erythrocytosis due to inhibition of HIF-α degradation. Studies of some of the mutant enzymes (P317R and R371H) showed either an increase of Km for HIF-1α or for 2-OG or a reduction in kcat for the overall reaction.104,105 Both mutants showed significantly reduced kcat/ Km (25% and 82% for P317R with respect to HIF-1α and HIF2α, respectively, and 30% for R371H for both isoforms), suggesting greatly reduced catalytic efficiency. Both residues 317 and 371 are located in the HIF binding groove.

THERAPEUTIC INDICATIONS FOR PHD INHIBITORS Anemia. The promotion of EPO secretion and thus hematopoiesis through the stabilization of HIF via PHD inhibition is the most established and obvious therapeutic indication for small molecule PHD inhibitors (PHDis). Indeed this mechanism of action has been demonstrated in many species and with multiple inhibitors, ending with the demonstration of the reversing of anemia in rhesus macaques111 and more recently in humans.112,113 The utility of PHDis for this therapeutic indication has recently been carefully reviewed.114 There are currently six PHD inhibitors in various stages of clinical evaluation for the treatment of anemia associated with kidney disease (see below). Ischemic Diseases. HIF stabilization also shows therapeutic potential in treating ischemic diseases. In a rat model of renal failure due to ischemia, CoCl2 was effective in reducing tissue damage when administered both prior to and after

■

■

G



Perspective

ischemic insult.115,116(Cobalt is a known inhibitor of PHD enzymes.) DMOG was also seen to be effective in renal protection from ischemic damage.117−119 A similar effect was seen with the PHDi FG-4487, which was employed as a pharmacological “ischemic preconditioning” agent and prevented subsequent acute ischemic damage due to renal arterial clamping.120 The protective effects of HIF stabilization on renal ischemic damage (such as is associated with diabetes) were also shown genetically in HIF heterozygotes and homozygotes.117 Prolyl hydroxylase inhibition has also proven to be effective in models of ischemic stroke.121 DMOG administration to mice as late as 60 min following occlusion of the middle cerebral artery reduced ischemic infarct volume and improved postischemia cerebral function, effects that were reversed upon subsequent pharmacological down-regulation of HIF-1α with digioxin.122 Revascularization via HIF up-regulation may also be a potential therapy for treating coronary artery disease. Overexpression of HIF-1α in transgenic mice has been shown to lead to reduced infarct size in mice after coronary artery ligation compared with wild-type mice.123 In another transgenic model, cardioprotection in mice was achieved through the generation of PHD2 knockdown. The researchers showed increases in genes associated with glucose metabolism, cardiac function, and blood pressure, and when the cardiac tissue was challenged with ischemia/reperfusion (I/R) injury, overall cardiac function was improved compared with wild-type mice.124 This effect was shown pharmacologically as well. Pretreatment of mice with DMOG prior to I/R injury was found to be as protective for cardiac tissue as was ischemic preconditioning at attenuating myocardial infarct extent due to ischemia.125 In a phase I clinical trial, coronary-artery bypass graft (CABG) patients were treated intramyocardially with adenovirus particles expressing a stable form of HIF-α with the expectation of promoting cardiac angiogenesis. The procedure did not result in any adverse drug related safety concerns and will likely be followed in the clinic by broad efficacy trials.126 While the protective effects on ischemic tissue of intermittent HIF elevation through PHD inhibition is well-established, studies of VHL disease patients and other genetic HIF-1α and HIF-2α gain-of-function mutations show that chronic, pervasive up-regulation of HIF function is associated with cardiopulmonary abnormalities such as pulmonary hypertension and increased heart rate and cardiac output associated with reduced blood pressure.127 This consideration, along with ADME parameters and tissue distribution of small molecule drugs, will be important in designing PHDis for the treatment of ischemic diseases. Arterial Disease. The application of HIF stabilization in the treatment of arterial disease has recently been reviewed.128 As cytokine generation and angiogenesis are impaired in elderly patients with peripheral arterial disease, gene therapy consisting of the administration of an adenovirus encoding constitutively active HIF-1α was used to improve limb perfusion in aged mice in a model of limb ischemia.129 In addition, GSK has investigated its PHDi in the clinic for the treatment of peripheral artery disease (see below). The findings of these trials have not been made public, but positive results will certainly stimulate the interest of other drug makers for this indication. Gastrointestinal Disease. Activating genes involved in tissue growth and repair, HIF plays a complex role in inflammation because it regulates both proinflammatory

cytokines (e.g., IL-1β, TGF-β) and antiinflammatory pathways such as the promotion of adenosine signaling via the upregulation of netrin-1, CD39, adenosine receptors, and CD73. In addition, PHD inhibition can promote tissue repair through reduced cellular apoptosis, possibly by the activation of NF-κB and the down-regulation of FADD (via HIF stabilization), a key adaptor molecule mediating TNF-α signaling. As such, PHD inhibition may play a role in gut repair due to gastroinstestinal disease (Table 1, matrix and barrier function proteins regulated by HIF). Mice with conditional HIF-1α knockdown in the intestinal epithelium showed increased susceptibility to TNBS-induced colitis compared with wild-type litter mates. In the same study, the authors demonstrated that the up-regulation of HIF-α via VHL knockout led to protection from colitis, as measured by extent of weight loss and reduced colon shortening.130 Both DMOG and another FibroGen PHDi (16; see below) were shown to be highly efficacious in models of ulcerative colitis.131,132 It was subsequently shown that gastrointestinal injury induced by a variety of inflammatory stimuli including TNF-overexpression,133 exposure to bacterial pathogens,134 ischemia/reperfusion injury, 135 and NSAIDs136 can be effectively reversed by PHD inhibition. Wound Healing. HIF-driven gene transcription contributes to the normal process of wound healing through the local release of growth and angiogenic factors, suppression of apoptosis, and stimulation of the formation of the extracellular matrix. It is known that HIF expression is reduced in old age, by high blood glucose, and in burns leading to the expectation that PHD inhibitors may be effective topical agents for the treatment of venous and diabetic ulcers as well as burn wounds.137 Both PHD inhibitors DMOG and DFO were found to speed wound healing in diabetic mice.138,139 Adenovirus therapy expressing a stable form of HIF-α has been shown to promote wound healing in diabetic mice and in the clinic.138,140

■

PHD STRUCTURAL BIOCHEMISTRY There are 11 crystal structures of HIF PHD2 constructs that have been deposited in PDB as of the writing of this Perspective. Sequence alignment and modeling studies suggest that the catalytic domains of the PHD1 and PHD3 isoforms share a high degree of homology with PHD2. The first from groups at Oxford University and Amgen (PDB 2G1M) is a 1.70 Å resolution structure of a 246-residue C-terminal catalytic domain of PHD2 bound to iodoisoquinoline 1 (Figure 3).141 The protein is found to contain eight β strands making up a double-stranded β helix fold that largely forms the walls of the active site. Three α-helices brace the exterior of the protein, forming a scaffold holding the β sheets in place. The three conserved iron-binding triad residues (His313-Asp315-His374) are clearly observed making up half of the ligand sphere of the bound Fe (presumably ferrous ion). The remaining three metal coordination sites of the octahedral complex consist of the bidentate binding of the isoquinoline nitrogen atom (trans to His374) and the exocyclic carbonyl oxygen atom (trans to Asp315) of the ligand, as well as a water molecule (not shown). The terminal carboxylate group of the ligand makes an important salt bridge interaction with Arg383 and a hydrogen bonding interaction with Tyr329 in the back of the 2-OG binding pocket. Mutation of Arg383 (R383A) resulted in a protein with a near total loss of catalytic activity, demonstrating the crucial role of this residue in cofactor organization within the active site. The wall of the 2-OG pocket is lined H



Perspective

structure was solved of the same PHD2 domain with Noxalylglycine (NOG), a nonhydrolyzable 2-OG congener, bound in the active site (Figure 4). Importantly, the entire

Figure 3. Structure of a 246-residue C-terminal catalytic domain of PHD2 bound to iodoisoquinoline 1 (PDB 2G1M).

predominantly with hydrophobic residues: tyrosines 310, 303, 329, isoleucines 327 and 256, methionine 299, leucine 343, tryptophan 389, alanines 301 and 365, and valine 376. Another key interaction between inhibitor and protein is a hydrogen bond between the phenolic OH of the inhibitor and Tyr303. This interaction is preserved in the structures of all potent bound small molecules either through a direct contact or via the intermediacy of a structural water molecule. Greater proof that this pocket is in fact the 2-OG binding pocket came later from the solved cocrystal structure of PHD2 catalytic domain and 2-OG itself (PDB 3OUJ; see below). It was suggested that the H bond interaction with Tyr303 is not critical for cofactor binding and that mutants lacking this residue are catalytically active and are inhibited by small molecule PHDis. The role of the phenolic OH therefore in 1 may include other functions such as increasing the strength of chelation with Fe through electron density donation to the ring nitrogen atom and/or reinforcing the required planar bidentate chelate geometry via an intramolecular hydrogen bond with the NH of the exocyclic side chain. Structures 2HGBT and 2HGBU (deposited by a group from Procter and Gamble), 247 amino acid C-terminal catalytic domain constructs bearing a His6 tag, show similar secondary and tertiary structure to PDB 2G1M. In this case though, isoquinoline 2, one of FibroGen’s clinical candidates (see below),142 is bound in the enzyme active site. The details of this work are briefly mentioned in a publication but have not yet been published independently.143 In this structure though, isoquinoline 2 is seen to bind identically to 1. Binding affinity is given as 73 nM compared with 1400 nM for 1. Assuming comparability of assay conditions, the SAR difference between the two may be due to effects of the Cl atom on the isoquinoline ring system, including reducing electron density on the all-carbon ring and improving its π-stacking hydrophobic interaction with Tyr310 and acidifying the phenolic OH to increase the strength of the direct H bond to Tyr303. An important addition to the body of structural data also came from Oxford. The structure of a 246-residue C-terminal catalytic domain of PHD2 was solved with bound Fe, isoquinoline 2, and a HIF fragment containing hydroxyproline (hyPro564). The 2.30 Å structure, however, failed to resolve all but seven of the HIF residues, including hyPro564 (PDB 3HQU). However, after careful experimentation involving mutagenesis and replacement of Fe with Mn, a second

Figure 4. X-ray crystal structure of a PHD2 domain with a HIF CODDD fragment (HIF 558−574) and N-oxalylglycine (NOG) bound (PDB 3HQR).

HIF CODDD fragment (HIF 558−574, not containing hyPro) was observed at 2.00 Å resolution (PDB 3HQR). As expected from the hydrophobic nature of this domain and the highly conserved LXXLAP sequence, bonding interactions with PHD2 are found to be largely hydrophobic in nature. HIF Pro564 can be clearly observed adjacent to the metal center. Comparison of this structure with those of HIF-free complexes suggests conformational change involved in the β-helices allowing not only HIF to bind but also O2 access to the stable and deeply buried metal center. With respect to drug design, the authors suggest a divergence in molecular mechanisms of inhibition between small 2-OG analogues such as NOG and larger heterocyclic inhibitors such as 2, the former allowing for HIF binding but blocking the formation of Fe(IV)O complexes, while the latter, in addition to blocking Fe oxidation, also acted to stabilize a closed conformation preventing HIF substrate binding.45 The cocrystal structure of 2-OG itself bound to the catalytic domain of PHD2 has been solved (PDB 2OUJ) and served to confirm the theorized binding of this endogenous cofactor (Figure 5). In addition to the bidentate chelation to the Fe center via the C1 and C2 sp2 oxygen atoms, and salt bridge between the C5 carboxylate and Arg383, H bonding to Tyr303 is observed but via a cascade to a structural water molecule in the active site (also observed in the NOG cocrystal, 3HQR).144 Guided by the structural information provided by bound 2OG, we at Janssen designed small molecule mimetics of 2-OG, taking advantage of the bidentate chelation of Fe via a 1,4diimino ligand in the form of a 2-pyrazolobenzimidazole isostere of the α-ketoacid group of 2-OG. The diimine moiety in this series has been calculated to provide more favorable binding energy to Fe(II) center than the more common 1,4oxoimine chelation motif seen in Gly amides such as 2. A crystal structure of compound 78 (discussed below) bound to PHD2cat (PDB 3OUI) reveals that the absence of the phenolic OH group, found in many Gly amide inhibitors and responsible for directly H-bonding to Tyr303, is compensated for by the NH group of the benzimidazole ring. While too distant from the OH group of Tyr303 to make a direct H-bond, this I



Perspective

with streptavidin beads.36,155 This SPA format was later adapted by Lundbeck for high throughput screening.47 After these initial disclosures, literature began to appear describing assays in a more robust, high throughput format, suitable for broad screening of corporate compound libraries. By immobilizing biotinylated HIF onto streptavidin beads, workers at Bayer could measure the degree of proline hydroxylation by PHD enzymes and cofactors using a thioredoxin-fused VBC, which was in turn detected by an anti-thioredoxin antibody and peroxidase-linked anti-mouse IgG, all in an ELISA format, free of radioisotopes.156 A fluorescence polarization assay has also appeared that takes advantage of the increase in fluorescent readout of a fluorescein-labeled HIF upon binding to VBC complex in vitro, after incubation with PHD.157 By adaptation of yeast twohybrid technology to the problem, an assay was developed involving linking of a HIF construct to a Gal4 activation domain and detection via interaction with a pVHL-linked binding domain and measuring dye released from XGal hydrolysis by transcribed galactosidase upon HIF hydroxylation.158 A group at Amgen described both electrochemiluminescence (ECL) and time-resolved fluorescence resonance energy transfer assays (TR-FRET or HTRF). The former was created using a ruthenium chelated tris-bypyridyl ligand attached to VBC, mixed with biotinylated HIF after exposure to PHD, and quantified on streptavidin coated paramagnetic beads to which current was applied producing fluorescence in the detection buffer from the bound Ru. The TR-FRET assay is based on the extent of hydroxylation of a biotinylated HIF fragment binding to a europium-labeled VBC followed by capture with an allophycocyanin (APC) tagged streptavidin, measuring fluorescence transfer from the excited Eu to the APC fluor. This latter assay format was employed in the high throughput screening of the Amgen compound library and led to the discovery of 64.159 Merck has also disclosed their version of the HTRF assay using AF647 bound streptavidin beads to capture biotinylated HIF which in turn was recognized (in hydroxylated form) by (His)6-tagged pVHL and an anti-(His)6 chelated Eu (LANCE) reagent. This assay was used to develop their primary PHD1, -2, and -3 SAR for small molecule drug discovery.160 Boeringer Ingelheim have applied for a patent using similar methodology. In their system, VBC complex was labeled with glutathione S-transferase (GST). Binding to biotinylated HIF after PHD treatment resulted in a complex that was captured by APC coated streptavidin on the HIF side and a europiumlabeled anti-GST antibody on the VBC end, with Eu-to-APC fluorescence transfer being measured.161 An in vitro luciferase readout assay has also been developed that relies on the detection of luciferase activity from luciferaseligated pVHL β-domain fragment (not the full VBC complex) binding to biotinylated HIF captured on streptavidin beads. The authors claim that the IC50 values obtained in this assay for known PHD inhibitors are in agreement with those obtained using a more standard FLAG-tagged VBC assay format.162 Cell-based screening is, in principle, straightforward and may be as simple as measuring EPO release into media from EPOproducing cell lines such as Hep3B. Whole cell screening has also been accomplished using a neuroblastoma cell line stably transfected with a gene encoding and constitutively producing a protein consisting of a CODDD HIF fragment ligated to luciferase. The action of endogenous PHD enzymes, in a

Figure 5. X-ray cocrystal structure of 2-OG bound to the catalytic domain of PHD2 (PDB 2OUJ).

important binding determinant is observed to be bridged, like in the case of 2-OG, by a structural water, which explains the potency of this class.

■

ASSAYS FOR ASSESSING THE ACTIVITIES OF PHD INHIBITORS Assays for the enzymatic activity of 2-OG dependent dioxygenases in the past have, in their simplest form, taken advantage of the conversion of [14C]2-OG to succinate with the release of [14C]CO2 gas or the production of [14C]succinate, depending on the location of the radiolabel.48,145−150 This approach has been adapted to the PHDs employing [5-14C]2oxoglutaric acid and separation of residual [14C]2-OG from [ 14 C]succinate by precipitation of the former as its dinitrophenylhydrazone.151 The measurement of O2 consumption using a fiber optic oxygen sensor in the reaction medium has also been described.152 The role of added reductant was studied on the reaction of the purified enzyme.153 It was shown that the addition of ascorbic acid is important for optimal enzyme turnover and that other bioreductants (glutathione, dithiothreitol) are inferior to ascorbate in promoting the reaction in vitro. However, it remains unclear as to whether or not the reductant interacts specifically with the protein (SAR studies suggest that this is not the case) or more generally in solution to keep the ratio of Fe(II)/Fe(III) high. That glutathione and dithiothreitol are reported to have more negative reduction potentials than ascorbate may in fact be inconsequential in the absence of a viable kinetic pathway for the reduction by these thiols of Fe(III). Along with the disclosure that HIF degradation is mediated by dioxygenases, the Oxford group described both a [35S]HIF/ hemaglutinin-tagged pVHL interaction assay and a [35S]pVHL/ Gal-fused-HIF assay as means by which to measure the extent of HIF proline hydroxylation,34 and the use of monitoring of the conversion of 2-OG to CO2 and succinate is specifically claimed by this group in a patent application.154 Along with the discovery of HIF PHD enzymes, a University of Texas group described the use of both whole cell reporter assays and cell free assays that measure the binding of the VBC to hyProHIF in a scintillation proximity pull-down assay (SPA), utilizing biotinylated HIF construct and 35S-labeled pVHL where radioactive counts could be measured after solid phase capture J



Perspective

Figure 6. FibroGen compounds.

particular cell line. It has been suggested, however, that this divergence may in fact be associated with unexpectedly high levels of endogenous 2-OG present in cells that shift the apparent EC50 because of competition with inhibitor. This is certainly a factor that researchers will need to consider in their hunt for tissue-selective PHD inhibitors.47 It is clear that with the wide variety of sensitive high throughput in vitro assays and robust cell-based assays described, drug discovery aimed at finding potent, selective, cell-penetrant small molecule PHDis and their optimization is not only possible but has the potential to lead to the identification of a wide variety of novel chemotypes. Eventually, these assays will be used to pave the way to the development of PHD isozyme-selective drugs as well as selectivity for or against the related HIF hydroxylase, FIH.

presumably more physiological manner than with cell-free conditions, on this fusion protein results in degradation through the ubiquitin pathway. Inhibitors of the PHDs (and able to penetrate the cell membrane) block the degradation resulting in a luciferase readout. This assay format was used to screen an 85 000 compound collection resulting in 160 confirmed hits. Unfortunately, most if not all of the hits contained classical divalent metal chelation fragments leading to the suspicion that they may not be acting in the enzyme active site. Structural modification studies in an 8-hydroxyquinoline hit series led to very little discernible SAR.163 The same was found to be true in a related 8-hydroxyquinoline series discovered with a cell-free purified enzyme system.143 When the activities of PHD inhibitors in cell-based and cellfree assays are compared, potency discrepancies are often noted, with EC50 values for target gene protein production, such as EPO or VEGF, being an order of magnitude or more greater than purified enzyme IC50 values. There may be many explanations for this, such as dioxygenases other than the single isolated PHD playing significant roles in HIF modification in a

■

SMALL MOLECULE PHD INHIBITORS It has been known for some time that iron chelators are able to promote erythropoiesis, although the mechanism by which this occurs was not always understood.164 Certainly as we now K



Perspective

erythrocyte production as well as increases in hematocrit and hemoglobin. Data for compound 2 are also presented from healthy human subjects from a phase I trial with dosing of 20 mg/kg 2 either 2 or 3 times a week for 4 weeks, and increases in hematocrit, reticulocyte, and erythrocyte counts are claimed. On the basis of this disclosure, the identity of clinical candidate FG-2216 is 2.142 A subsequent application provides more detail on the clinical effects of 2: EPO was increased in healthy volunteers in a dose dependent manner after oral doses of 3, 6, 10, 15, and 20 mg/kg, measured by both Cmax (12 h) and AUC, with a 5× increase in Cmax for the highest dose.172 The recommended dose of recombinant parenteral EPO for the treatment of anemia (50−100 IU/kg) results in an increase of serum EPO 140× over baseline. It was disclosed in the same patent application that 20 mg/kg (orally dosed presumably but not stated) of 2 administered 3× per week for 4 weeks to predialysis, EPO-naive patients with chronic kidney disease (CKD) resulted in a significant increase in hemoglobin of 1.9 g/dL compared to placebo treated patients who suffered a decrease of 0.35 g/dL. The importance of this finding should not be underestimated: High dose recombinant EPO products carry significant cardiovascular thromboembolic risks. The ability of 2 (and presumably other PHD inhibitors) to promote erythropoiesis via a mechanism involving significantly lower EPO excursions and via the orchestrated promotion of iron transport and metabolism makes a good case that the treatment of anemia with a PHDi will not necessarily carry the same safety and regulatory risks as does recombinant EPO. Publishing on May 7, 2009, FibroGen disclosed the structure of their second clinical candidate 3 (FG-4592), a 1methylisoquinoline bearing a 7-phenoxy substituent.173 The claim in this case was the treatment of hypertension in both anemic and nonanemic CKD patients. In a single dose (dose not given) patient study, 3 reduced mean arterial (MAP), systolic (SBP), and diastolic (DBP) blood pressures from baseline with respect to vehicle treated patients. In a second, repeat dose study, 3 administered 2× to 3× per week (dose not specified) resulted in continued, sustained reduction in MAP, DBP, and SBP over the month long study compared to placebo-administered patients who generally saw increases in these three parameters. While the origins of the FibroGen PHDi program are clearly in new methods of use for known prolyl hydroxylase inhibitors, they followed up this initial focus by applying for patents on a series of novel compounds, largely based on the 4hydroxyisoquinoline-2-carbonylglycine motif. The resulting series of analoging programs appears to be pharmacophore based, guided by SAR developed using purified enzyme in a CO2 capture assay format.145 Starting with known isoquinolines, they initially developed new compounds in a related series, making provisos for known members of the genus.174 Modification of the A ring led to a series of 62 thieno[3,2c]pyridines and thieno[2,3-c]pyridines (4 and 5), with no biological data provided.175 In another disclosure in the isoquinoline series, substitution of CN at the C1 position is claimed to improve in vivo activity (plasma EPO readout) by ∼2.5× to 400× compared with C1 H, Me, and Cl congeners. Although doses are not provided, it is claimed that the presence of the C1-CN group imparts improved pharmacokinetic properties leading to the improved oral potencies. For example, compound 6 is stated as providing plasma EPO levels 362× and 449× higher than for its C1−Br and C1−Cl analogues, respectively.176

know, it likely occurs at least in part via the depletion of local biological iron stores and ultimately via denaturation and inhibition of intracellular PHD enzymes. Some of these have been reviewed previously.165 Nearly all modern, competitive PHD inhibitor programs trace their origins to collagen prolyl hydroxylase (CPH) inhibitor programs that ran in industry and academia prior to the discovery of the HIF PHDs. Upon recognition that HIF PHD enzymes were also non-heme, iron containing dioxygenases, designed CPH inhibitors were screened for PHD activity and subsequently used as molecular blueprints for structure-based design programs.48 In fact 2 was originally described in a Hoechst patent application for the treatment of collagen disorders.166 Seminal contributions to the development of prolyl hydroxylase inhibitors as biological tools came from Hoechst and ICI programs from the late 1980s and 1990s. The tool compound N-oxalylglycine (NOG) was first described as an inhibitor of glutamate synthase167 and then later developed as an inhibitor of hydroxylase enzymes.168 NOG169 and its more druglike dimethyl ester analogue DMOG170 have become the most published tools for the inhibition of HIF PHDs in vitro and in vivo and early on were the preclinical tools of choice that helped elucidate much of the pharmacology of PHD inhibition. The ICI CPH program led to the first appearance of the hydroxyisoquinoline class of prolyl hydroxylase inhibitor, a structural motif relied on, at least initially, by nearly all Pharma companies working in the HIF PHD arena (see below). As the ICI and Hoechst literature, both patent and peer reviewed, in the area of prolyl hydroxylase inhibition in fibrotic diseases is vast and as it largely covers CPH inhibition, it will not be reviewed in detail here. Unfortunately, very little of the chemistry and medicinal chemistry of the development of HIF PHD inhibitors has been published in the peer-reviewed literature, and what follows is an attempt to systematically review, by organization, the current state of small molecule PHD inhibitors using, largely, issued patents and patent applications. Because of its origins in CPH research programs, we see much overlap in inhibitor structures developed in the various PHDi drug discovery programs, at least in the early stages, (i.e., 24 and 57, 67 and 58, 39 and 65)

■

ACTIVE SITE INHIBITORS FibroGen. FibroGen, Inc. (San Francisco, CA, U.S.) was one of the earliest entrants into the field of developing HIF PHD inhibitors as human therapeutics. Initially they disclosed methods of use for known prolyl hydroxylase inhibitors such as 1,10-phenanthrolines, 8-hydroxyquinolines, isoquinolines, pyridines, and hydroxamic acids in treating ischemic diseases.171 They describe the measurement of activity of these compounds using a crude HPLC assay for the isolated enzyme, measuring HIF-α accumulation in whole cells, VEGF production in various cell lines, HIF-regulated mRNA expression in vivo, and positive effects on preventing cardiac, renal, and liver ischemic injury and promoting wound healing. No data on the effects on EPO are given; however, a subsequent broad disclosure characterizes the hematological effects of 2 (Figure 6), among others.142 The authors show that addition of 2 to HEP3B cells in culture causes the secretion of EPO and overcomes the suppression of EPO production by IL-1β. In vivo, compound 2 ameliorated anemia in rats associated with chronic inflammation (caused by PGPS injection) upon 2 week dosing following induction of anemia as measured by reticulocyte and L



Perspective

Figure 7. GSK compounds.

pad weight compared to the same. Because there were no drug effects on organ weight, it leaves the question as to whether or not the lack of weight gain with 14 was due to reduced food intake.181 In separate applications, FibroGen claims the use of PHDis for promoting T-cell differentiation,182 treatment of multiple sclerosis and the promotion of neurogenesis,183,184 upregulation of endothelial precursor cells in bone marrow,185 treatment of chemotherapy-induced anemia,186 and treatment of stroke.187 Support for the last shows that single iv bolus doses of compound 15 are effective in reducing infarct size in a mouse model of middle cerebral artery occlusion at the time of occlusion as well as up to 5 h after occlusion. The use of 16 was claimed by Brigham and Women’s Hospital, MA, for the treatment of inflammatory bowel diseases (IBD) in which the compound showed potent reversal of disease symptoms (colon length, weight loss, systemic inflammation markers) when dosed parenterally in the TNBS mouse model of colitis.188 This work was subsequently published in the peer-reviewed literature claiming the test compound to be FibroGen’s FG-4497, the linkage of patent and paper thus establishing its chemical identity as 16.131 In addition to claiming the treatment of cancer-associated anemia,189 the FibroGen team provides data supporting the use of PHDis in the treatment of cancer itself. Mice with orthotopic human tumor explants showed significant reductions in tumor volume, weight, growth, and invasiveness with a concomitant reduction in death and morbidity after treatment with 2 and analogues. It is suggested that the antitumor effects may be related to altered tumor cell metabolism and a reduction in permeability of solid tumor vasculature.190

Further studies on A-ring modifications led to a series of 178 examples of pyrrolopyridines and thiazolopyridines (e.g., 7 and 8), along with indolopyridines (not shown).177 No biological data were provided. Elaboration of this theme led to the disclosure of a series of 47 exemplified isothiazolopyridinebased inhibitors with only modest activity data disclosed (e.g., 9, 100% inhibition at 22.2 μM in the CO2 capture assay).178 More extensive modifications to the core structure led to a series of 4-hydroxycoumarins (e.g., 10, 100% PHD2 inhibition at 7.4 μM),179 4-hydroxythiocoumarins (e.g., 11, 99% PHD2 inhibition at 7.4 μM),180 and pyrrolopyridazines,177 also claimed as inhibitors of FIH (e.g., 12, 99% PHD2 inhibition at 22 μM). In addition to these compositions of matter applications, FibroGen has applied for a number of methods-of-use patents covering a wide range of disease treatments. The use of PHDis in the treatment of diabetes is supported with animal data showing positive effects on the induction of metabolic genes in the kidney, liver, and lungs (PFK, Eno-1, Glut-1, LDH-1, Aldolase-1, HK-1), reduction of insulin resistance as measured by hyperglycemic−euglycemic clamping and an increase in glucose uptake in DIO rats, reduction in fasting blood glucose with 2, improved glucose tolerance after 2 weeks in rats on high fat diet with 13 (75 mg/kg), a reduction in HgbA1c in db/db mice treated with 2, and a reduction in body weight and serum triglycerides. Treatment of db/db mice with 0.5 mg/kg 14 daily for 8 weeks resulted in a statistically significant reduction in HgbA1c. In a related study in DIO mice fed a high fat diet, 14 (75 mg kg−1 day−1 for 28 days) showed significantly reduced weight gain than did matched vehicle treated mice. Interestingly, while 13 also showed significant reduction in fat pad weight compared to DIO controls, 14 had no effect on fat M



Perspective

Figure 8. GSK compounds.

By increasing endogenous γ-globin and thus fetal hemoglobin, FibroGen has claimed the treatment of thalassemias, including sickle cell syndrome.191 GlaxoSmithKline. Of all known PHDi drug discovery programs, it is hard to match GlaxoSmithKline (GSK) for the magnitude of the ratio of number of novel chemotypes to disclosed biological data. Indeed, most of their patent applications contain just the briefest mentions of ranges of activities for their exemplified compounds. Nevertheless, the creativity of their program remains clearly evident. Whereas the 4-hydroxyisoquinolone framework was the starting point for much of the FibroGen Discovery program, GSK initially focused on the novel 4-hydroxy-2-quinolone chemotype to build upon (e.g., 17, Figure 7). Using both an HTRF assay (europium-VBC binding to cyanine-labeled HIF CODDD) and a whole cell assay measuring EPO release from HEP3B cells, they developed SAR not only around the 4hydroxy-2-quinolone pharmacophore but also empirically discovered HTS hits (see below). Interestingly, the limited biological data provided in their applications are for activity at

PHD3, although this may not have been the primary isotype guiding their SAR. In one of their initial filings, 150 compounds related to 17 were disclosed with PHD3 IC50 values from 20 to 1000 nM.192 Elaborating further via ring A modifications, they disclosed naphthyridine analogues (18−21) and pyrazolo- and thienopyridone analogues (22−24) with similar activities.193,194 Variation of the pyridone core itself of this series provided a broad collection of pyrimidone and pyridazone analogues (25− 30)195−200 From the sparse data disclosed it appears that the monocyclic analogue series (i.e., 25−27) contain members with very potent activities with PHD3 IC50 values as low as 0.8 nM and EPO release EC50 values of 400 nM in cells. Later work produced pyridylpyrimidones (e.g., 31) and ring A deleted analogues of the initial quinolones (e.g., 32) (Figure 8) that, while displaying low nanomolar enzyme inhibition, were not potent in the whole cell assay (EC50 > 10 000 nM).201,202 Introduction of an aminocarbonyl moiety to frameworks such as 32, however, resulted in compounds such as 33 and 34 with somewhat improved cell potencies (EPO EC50 of 1−20 nM).203 N



Perspective

Figure 9. Procter & Gamble compounds.

the absence of the carboxylate group, the HTS-derived compounds have significantly different ADME and biophysical properties. Proctor & Gamble. Using an X-ray cocrystal structure of inhibitor 2 bound to a PHD2 catalytic domain,215 workers at Procter and Gamble (P&G) designed a series of 8hydroxyquinoline 7-carboxamides as PHD inhibitors, relying on the known Fe-chelating properties of the heterocycle. Using a MALDI-TOF MS-based assay and molecular modeling, they prepared a number of inhibitors derived from 46 (IC50 = 5.0 μM) (Figure 9), varying the amide portion, with little discernible SAR development. This is likely due to the potent solution phase metal chelation of the 8-hydroxyquinoline moiety, leading to noncompetitive inhibition of the enzyme. This notion is further supported by the fact that many of the published inhibitors show greater whole cell EC50 potencies for VEGF release than they do activity at the purified enzyme (IC50).143 Cocrystal structural data were also used to develop a series of imidazol[1,2-a]pyridine Gly amide inhibitors (e.g., 47, IC50 = 3.6 μM) predicted to chelate active site Fe(II) through the imidazole N atom and the exocyclic carbonyl group. Derivatization of this core at C5, as was the case for the 8hydroxyquinolines, resulted in little development of SAR, suggesting either that the C5 appendage does not interact with the protein or the inhibitors exhibit noncompetitive enzyme denaturation through Fe extraction.216 A more promising series was designed around a pyridine Gly amide core, substituted at the C5 position. The initial Gly amide analogues showed poor enzyme activity (e.g., 48, IC50 ≥ 100 μM).217 Bioisosteric replacement of the amide bond with small heterocyclic rings (oxazole, imidazole, oxadiazole) did not improve activity greatly; however, they found that pyrazole-4carboxylic acid was unique in producing inhibitors with greatly improved activities (e.g., 49, IC50 = 4.9 μM). Our own quantum mechanical modeling studies have suggested that the 1,4-

In one of the rare instances of nitrogen being completely absent from the core heterocycle of a PHDi, GSK published a series of Meldrum’s acid derivatives, exemplified by 35, showing poor activities in both the enzyme and cell assays presumably due to high acidity and chemical instability.204 GSK has also disclosed a series of interesting PHDis whose common pharmacophore is a 2-aminobenzamidoglycine core, exemplified by 36−39. While the enzyme IC50 values for these compounds range from 1 to 100 nM in general, there are examples in the quinoxaline series (i.e., 37) that have whole cell EC50 values reported as low as 100 nM, which would make them among the most potent PHDis known with respect to activity on EPO producing cells.205−208 Presumably, binding in aminobenzamide series is through the exocyclic carbonyl and the nitrogen atom of the heterocyclic core rather than the phenolic OH, which may be dispensable (e.g., 38). It is interesting to note that three of the disclosed analogue series (i.e., 38, 35, and 25) have the potential to produce symmetrical (or pseudosymmetrical) analogues in which the two chelation binding modes would be degenerate. Using a very different approach to that of modifying known pharmacophores, GSK performed an HTS of their compound collection and subsequently developed a series of quinazoline2,4-diones and 4-oxo-2-thioxo-7-quinazolines209 that contain no carboxylate group (40−45). All compounds in this series contain the pyrimidinedione or ketopyrimidinethione core containing an N3-azaheterocycle.210,211 It is tempting to believe that the compounds bind to active site iron via chelation of the N atom of the heterocycle and either the keto or thiono group for the core; however, there are no published data to support this. As was the case for the 4-hydroxyisoquinolone core, GSK researchers modified the A ring to include sulfur (e.g., 40, 41, 42)212,213 and nitrogen (45) heterocycles.214 Activity ranges for all exemplified compounds in this series show IC50 values (1− 100 nM) and EC50 values (1−20 μM) that are similar to the previous quinolone-derived inhibitors. Presumably, because of O



Perspective

Figure 10. Amgen compounds.

this application suggest that compounds in this series have positive effects on preventing TNBS-induced colitis in mice when dosed (presumably po) in prevention mode as measured by sparing of body weight loss and colon shortening and reduction in disease activity index. Hematology data suggest that the 1 and 5 mg/kg doses of 53, but not the 0.3 mg/kg dose, raise hematocrit by ∼2% over the 7 days of dosing, suggesting systemic exposure. These hydroxypyridone structures are reminiscent of, but distinct from, the iron chelator natural product mimosine (55). It is not clear whether compounds such as 53 and 54 inhibit PHD enzymes via specific interaction with the protein or via a more generic iron chelation effect.223,224 Iron chelators have previously been suggested in the treatment of intestinal inflammatory disorders,226,227 and 3-hydroxypyridin-2-ones are known Fe chelators that have been investigated for the treatment of iron overload disorders and inflammatory diseases, although other, non-PHDi, mechanisms were suggested as the basis for their activity.228,229 Akebia has spun out its HIF stabilizer/PHDi programs to a new venture called Aerpio Therapeutics, focusing on wound healing and IBD with some funding from the U.S. Department of Defense and the NIH.230 Amgen. Like GSK, early Amgen inhibitors were based on the 2-quinolone core231 (cf. 56 and 17 and thienopyridones 57 and 24) (Figure 10).232,233 Like their competitors in these chemical series, Amgen researchers achieved good enzyme potencies (e.g., 57 IC50 = 3 nM, 58 IC50 < 50 nM). They also have described a series of diazaquinol-2-ones (e.g., 59 IC50 = 1.5 nM)234 that are closely related to a genus disclosed by GSK (e.g., 18−21).193 Using their HTRF and ECL assays (see above), Amgen also published a series of inhibitors with nitrogen atom-free cores:

diimino group in compounds such as 49, depending on the steric environment, may have favorable energetics of chelation for iron compared to 1,4-iminooxo groups such as is present in 48.165 Further modeling by P&G suggested that benzyloxymethyl groups at the pyridine C5 position might make favorable interactions with active site residues Tyr310, Arg322, Met299, and Gln239, and a series of 12 analogues in this series showed improved enzyme potencies with compound 50 having the best balance of enzyme and whole cell activities (IC50 = 2.4 μM, EC50 = 1.0 μM).218 A method for improving the potencies of the pyridine based Gly amides (e.g., 48) was through the addition of an OH group at C3 of the pyridine ring, in analogy to the isoquinoline series of PHDis (e.g., 2).219 For example, IC50 values of >500 and 0.017 μM were found for 51 and 52, respectively. This was also recently demonstrated through SAR developed in a dynamic combinatorial chemistry effort.220 Upon termination of research activities at P&G, some or all of the intellectual property for the PHDi program was transferred to an independent company in 2007,221 Akebia Therapeutics, who now is in the clinic with a PHD inhibitor for the treatment of anemia in patients with end stage renal failure.222 Akebia has independently filed for patents for HIF PHD inhibitors around the hydroxypyridone chemotype, e.g., 53, which has an IC50 of 14 μM for PHD2 and is roughly equipotent in whole cells (VEGF release EC50 = 17 μM), along with sulfonamide congeners such as 54. Claims in the application suggest that Akebia is interested in these compounds for promoting wound healing, via both promoting reepithelialization and bolstering host defense to bacterial infection, and in treating inflammatory bowel conditions such as ulcerative colitis.223−225 53 is uniquely claimed as a compound for the treatment of colitis.225 Limited data from P



Perspective

Figure 11. Merck compounds.

the discovery of N-hydroxythiazoles such as 64 (IC50 = 73 nM).238 The SAR of this series points out that the acetic acid side chain is required for good activity, that the N−OH group is essential for enzyme potency, and that the sulfone moiety is also required to impart good potency (superior to S, O, NH, and CH2 in that position). Modeling suggests that 64 makes a salt bridge interaction with Arg383 through the carboxylate, binds active site iron in a bidenate manner through OH and the exocyclic imine nitrogen atom, and interacts with Arg389 through the SO2 group. Increasing the lipophilicity of the group bound to the sulfone improved potency (e.g., replacement of the phenyl ring of 64 with a 4-t-Bu-benzyl group provides the most potent analogue in the series, IC50 = 3 nM. No data are provided for whole cell or in vivo potencies. The Amgen group initiated a structure based design effort originating from the X-ray cocrystal structure of PHD2 bound to a 4-hydroxyisoquinoline inhibitor.239 Using bioisostere replacement, Amgen produced a series of imidazopyridines in which the carbocyclic ring was eliminated and the 4-hydroxy group of a 4-hydroxyisoquinoline such as 2 was replaced by an imidazole ring. The resulting compounds showed good potency in the PHD2 enzyme assay (e.g., 65, IC50 = 290 nM). Further improvements to potency were realized with the addition of lipophilic groups ortho to the nitrogen atom of the pyridine

naphthalenones (e.g., 60 and 61) and indanones (e.g., 62).235,236 The latter compound, 62, is reported to have good enzyme potency: IC50 between 3 and 22 nM. Like the Meldrum’s acid-based PHDis from GSK (e.g., 35), these series of compounds are expected to be highly acidic and likely dianionic at neutral pH. It is of interest to note that naphthalenones 60 and 61 differ only in that the former is a “traditional” glycine amide while the latter contains a ketone side chain based on succinic acid. The IC50 values for both are given as “40 000 nM and 13 000 nM for CPH1 and CPH2, respectively. The analogous glycine amide 56 is much less selective, with IC50 values for PHD2, CPH1, and CPH2 of 46, 351, and 111 nM, respectively. Compounds in this ketone series were shown to raise systemic EPO when administered po at 50 mg/kg to rats.237 Like Merck and GSK, Amgen performed an HTS (HTRF assay format) of their compound library aimed at identifying novel chemotypes and were quite successful in that regard, with Q



Perspective

disclosed and these three series contain 169 examples that have IC50 values less than 10 nM in the HTRF assay. A very interesting chemical series was disclosed by Merck researchers that bears resemblance both to the more traditional 2-OG-based inhibitors and to HTS-derived hits.246,247 Compounds such as 71 and 72 represent compounds in a 5aminocarbonyl-4-hydroxypyrimidine genus. Most of the exemplified compounds are claimed to have IC50 of 40 000 nM). By use of stable isotope labels in the PHD2 protein (15N) and in the inhibitor (13C), combined with the paramagnetic properties of the active site Fe(II) atom, solution NMR studies were used to correlate X-ray crystallographic determinations of inhibitor binding mode.240 In this case, the inhibitor 66 was found to bind in the active site of PHD2 in two different modes, which is not surprising for 4-hydroxyquinol-2-one inhibitors such as 66 that have the potential to bind Fe through the exocyclic carbonyl and either the C2 or C4 carbonyl of the central ring. Bias between the two binding modes would then have to be achieved via substitution patterns on the fused A ring. It is highly likely that many of the 2-OG derived inhibitors will have the potential for dual binding modes in the enzyme active site. In a unique approach to the discovery of PHD isozymeselective inhibitors, the quinol-2-one Gly amide core was derivatized via a C7 amide linkage with amino acid chains in a solid phase combinatorial fashion (i.e., 56 derivatized at the Brcontaining carbon atom).241 Assumptions made were the following, in analogy to the isoquinoline inhibitors: (1) The binding mode of the quinoline core involves bidentate chelation between the exocyclic carbonyl oxygen atom at C3. (2) The phenolic OH group and the C7 position would allow access of the pendent combinatorial peptide moieties to the HIF substrate binding groove of the PHDs. However, X-ray structures of the isoquinoline inhibitors suggest that the phenolic OH group in fact is not involved in Fe chelation but instead makes an important H-bond interaction with Tyr 303.141 Knowing the exact mode of active site binding in the quinolone series would be important for design of the side chain linkage into the HIF binding groove. Also, considering that all three PHD isozymes bind the same HIF-α substrates, inhibitor selectivity should not rely heavily on mimicking HIF structural features but rather by taking advantage of enzyme nonhomology in the binding grooves. In the end, however, guided by their HTRF primary assay, the effort resulted in the identification of N-acetyldipeptide inhibitors that showed ∼10× selectivity for PHD1 and PHD3 over PHD2. No data were provided on the effects of these compounds in whole-cell assays, as presumably their physicochemical properties prevent cell penetration. Merck. As was the case for GSK and Amgen, the Merck group investigated the 2-quinolone pharmacophore while separately developing HTS-derived lead series using, largely, an HTRF assay to guide SAR development.160 Early disclosures from their laboratories described naphthyridin-2-one analogues that proved particularly potent in their primary assay, with six disclosed compounds displaying subnanomolar IC50 values, the most potent being 67 (Figure 11, IC50 = 0.65 nM).242 Subsequent A-ring modifications led to a series of 5,7dihydrofuro[3,4-b]pyridin-2(1H)-ones, 5,7-dihydrothieno[3,4b]pyridin-2(1H)-ones, and 1,5,6,7-tetrahydro-2H-pyrrolo[3,4b]pyridin-2-one2 (exemplified by 68, 69, and 70, respectively).243−245 With respect to enzyme potency, only ranges are R



Perspective

Figure 12. Janssen compounds.

Figure 13. Bayer compounds.

(Protein Data Base entry 2G19) and 2-OG (via a conserved water molecule). Inclusion of an H-bond donor group in the heterocyclic portion of the Gly amide resulted in benzimidazoles such as 78 (IC50 = 12 μM) whose X-ray cocrystal structure with PHD2 revealed the presence of an H-bond cascade with the NH of the imidazole ring and Tyr303, mediated by a conserved water molecule, in accord with the binding mode of 2-OG.144 Quantum and molecular mechanical calculations suggested that planar chelation of the 1,4-oxoimino Fe binding group was energetically unfavorable because of both steric and electronic factors. A more favorable bidentate interaction with active site Fe(II) was realized through replacement of the amide bond.165 The resulting 2-(1Hpyrazol-1-yl)-1H-benzimidazole analogues showed improved potencies both at the isolated enzyme and in whole cell EPO release (e.g., 79, IC50 = 100 nM, A50 = 50 μM). Compound 79 is nonselective for the three PHD isozymes, shows 2-OGcompetitive binding kinetics with PHD2, and was found to have low affinity for free Fe(II) in solution. Given orally to a mouse genetically engineered to transcribe luciferase under the control of the hypoxia response element (HRE) produced a robust peritoneal bioluminescence in vivo. Upon repeat oral dosing of 79 to both normal and anemic (chronically inflamed) rats, robust and significant increases in reticulocyte number, hemoglobin, and hematocrit were observed.255 The enzyme activity of 79 was independently confirmed by researchers at Lundbeck using an SPA capture assay.47 Expansion of the benzimidazole core of the above series of inhibitors through the insertion of a “CO” group into the aniline C−N bond resulted in a series of quinazolinones that would have the potential to directly form an H-bond interaction with Tyr303.256 The enzyme activities of these compounds, in general, were found to be higher than those of

pyrrolylpyrimidine series (i.e., 71), there appears to be a requirement for the presence of a C2-linked nitrogencontaining heterocycle (2-pyridylmethyl in 75).160,252,253 However, this series suffered from unacceptable activity at the hERG potassium ion channel and poor PK in rodents, showing high levels of oxidative metabolism. Efforts to improve the PK properties of this lead series led to a redesign of the core structure and replacement of the indolone with an Narylhydantoin group. Optimization of activity and PK and minimization of effects on liver enzyme levels led to compound 76, which displays IC50 values for PHD1, -2, and -3 of 0.2, 0.2, and 1.6 nM, respectively, and has minimal inhibition of hERG, acceptable plasma protein binding, and no inhibition of liver enzymes. Compound 76 is stated as having a short rodent in vivo half-life that likely predicts for high clearance in humans, and this is claimed as a favorable property for stabilizing HIF systemically and minimizing the potential adverse effects of continual HIF stabilization. As is described, 76 “represents a very promising clinical candidate because of its short acting PHDi inhibition resulting in robust downstream in vivo efficacy”. This compound is not described as a clinical candidate, however, and competitive database searches reveal no human trials in this area either enrolling or underway at the current time by Merck. Janssen. Using an X-ray cocrystal structure of 2-OG and compound 77 (Figure 12) bound to the catalytic domain of PHD2, our laboratories at Janssen Pharmaceutical Research & Development, LLC (formerly Johnson & Johnson Pharmaceutical Research and Development, LLC) designed a series of benzimidazole-2-glycinecarboxamides.254 The low activity of 77 in our 2-OG depletion assay151 was attributed to the absence of a H-bond interaction with Tyr303 that is observed in the cocrystal structures of a 4-hydroxyisoquinoline inhibitor S



Perspective

Figure 14. CrystalGenomics, Japan Tobacco, Broad−Vanderbilt, and Yale/Cambridge compounds.

CrystalGenomics. CrystalGenomics, Inc., of Seoul, South Korea, has used the crystal structure of 2-OG bound to PHD2 to design active site inhibitors.264 Making great use of wide data ranges, they disclosed a series of benzothienopyridine Gly amides related to the well-known isoquinoline series (i.e., 2). Representative is 86 (Figure 14), which is claimed to have an IC50 value between 0 (sic) and 25 μM in their fluorescence polarization assay. In HEP3B cells, 86 at 100 μM raises EPO 21× to 50× above control and was found to be a potent stimulator of VEGF in cell culture.265 A subsequent filing included aza analogues of the earlier benzothienoyridine (e.g., 87).266 An impressively broad patent application claims “phenol derivatives” as HIF PHD inhibitors. The magnitude of the genus was recognized in the international search report, awarding it with 11 “X” classifications.267 However, a more common motif from this application is the 5-hydroxynicotinitrile substituted at the C6 position with a heterocyclic ring. Compound 88 is one example (IC50 of PHD1 and PHD3: 0− 100 μM). It is not clear if these compounds are active site or allosteric inhibitors or simple iron chelators. Japan Tobacco. Japan Tobacco, Inc., is in the clinic with a PHDi for the treatment of anemia.268 There is scant information on their program apart from a single patent application in Japanese describing the preparation of triazolopyridine compounds as PHDis as EPO production inducers.269 They appear to have used a FRET assay to discover their lead series of Gly amides that show

Inhibition of Hypoxia-Inducible Factor Prolyl Hydroxylase Domain

Recommend Documents