Progress toward JAK1-selective inhibitors.

Future

Review

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Medicinal Chemistry

Progress toward JAK1-selective inhibitors

The discovery of the JAK-STAT pathway was a landmark in cell biology. The identification of these pathways has changed the landscape of treatment of rheumatoid arthritis and other autoimmune diseases. The two first (unselective) JAK inhibitors have recently been approved by the US FDA for the treatment of myelofibrosis and rheumatoid arthritis and many other JAK inhibitors are currently in clinical development or at the discovery stage. Research groups have demonstrated the different roles of JAK member and the therapeutic potential of targeting them selectively. JAK1 plays a critical and potentially dominant role in the transduction of γc cytokine (γc = common γ chain) and in IL-6 signaling. In this review, we will discuss the state-of-the-art research that evokes JAK1 selective inhibition.

Kinases

Kinases constitute a large family of enzymes within the human genome, with individual members playing key roles in numerous cellular signaling pathways. As a matter of fact, protein phosphorylation is fundamental to all aspects of cell organization and behavior. The interplay of kinases and the phosphatase family creates a signaling network that controls most intracellular signaling processes through reversible phosphorylation [1] . To date, it is estimated that only a small fraction of the kinome has been mined as a source of therapeutic targets [2] . There are actually 518 protein kinase genes known and it is thought that of these, 256 are disease related [3,4] . Since the launch of imatinib, a large fraction of the discovery research of the pharma industry has focused on kinases. Consequent to the increase in the search for kinase drugs, more and more kinase inhibitors (24 to date) have now been approved by the US FDA. While the majority of these are (like imatinib) still directed at oncology indications, recent approval of tofacitinib, 1 (Figure 1) for rheumatoid arthritis (RA), illustrates the potential importance of this class of targets for other disease indications [3] . For a long time it was thought that selectivity of the

10.4155/FMC.14.149 © 2015 Future Science Ltd

Christel J Menet*,1, Oscar Mammoliti1 & Miriam López-Ramos2 1 Department of Medicinal Chemistry, Generaal de Wittelaan L11A3, 2800 Mechelen, Belgium 2 Department of Computational Chemistry & Biology, Galapagos SASU, 102 av. Gaston Roussel, 93230 Romainville, France *Author for correspondence: Tel.: +32 15 342 938 [email protected]

inhibitors may not be achievable between kinase families, but tofacitinib, 1 demonstrates that a single family of kinases can be inhibited selectively [5–7] . These successes have led to unprecedented growth in the field of kinase drug discovery and significant effort has been devoted toward the identification, optimization and pharmacological characterization of novel inhibitors. JAK biology

Janus kinases (JAKs) are cytoplasmic tyrosine kinases that transduce cytokine signaling from membrane receptors to signal transducers and activators of transcription (STAT) factors. Four JAK family members are described, JAK1, JAK2, JAK3 and TYK2. JAKs are constitutively bound to the cytoplasmic tail of cell surface cytokine receptors. Upon binding of the cytokine to the receptor, JAK family members autoand/or transphosphorylate each other, and subsequently phosphorylate the cytoplasmic tails of the cytokine receptors at specific tyrosine (Tyr) residues. These p-Tyr residues form docking sites for STAT proteins that bind to the receptor which in turn are then phosphorylated and activated by the JAK kinases. Such activated STATs then migrate to the

Future Med. Chem. (2015) 7(2), 203–235

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ISSN 1756-8919

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Review Menet, Mammoliti & López-Ramos

Key terms Kinases: Enzymes that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation. Cytokines: Wide variety of proteins that play an important role in intercellular cell signaling.

cell nucleus and modulate transcription (Figure 2) . JAK-STAT intracellular signal transduction serves as the intracellular signaling pathway for the IFNs α,β,γ (IFN), most IL, as well as a variety of cytokines and endocrine factors such as erythropoietin (EPO), thrombopoietin, growth hormone (GH), oncostatin M, leukemia inhibitory factor, ciliary neurotrophic factor, granulocyte monocyte colony-stimulating factor (GM-CSF) and prolactin [8] . A combination of genetic knock-down and knock-in models and small molecule JAK inhibitor research has revealed the therapeutic potential of modulating the individual JAKs in different diseases. JAK3 has been validated by mouse and human genetics as an immune-suppression target [10] . The most common form of Severe Combined Immunodeficiency (SCID) is X-Linked Severe Combined Immunodeficiency (X-SCID) which is attributable to mutations of γc, and which results in impaired signaling via all the cytokines (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21) that utilize this γcreceptor subunit. In addition, as JAK3 associates with the γc receptor subunit, the N N N N

N

N

O

N N

N

N H

N H

N

1

2 N O N S O

N N

N N

N H

3 Figure 1. Tofacitinib (1), Ruxolitinib (2) and baricitinib (3).

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possibility that JAK3 mutations may also be responsible for X-SCID has been investigated in selected SCID patients. It has been demonstrated that mutation of either γc or JAK3 leads to the same functional defects. JAK3 inhibitors have been successfully taken into clinical development, initially for organ transplant rejection, but later also in other immuno-inflammatory indications such as rheumatoid arthritis (RA), psoriasis and Crohn’s disease (CD) [11] . Pfizer’s JAK1/JAK3 mixed inhibitor tofacitinib, 1 was indeed launched in 2012 and Vertex has just released the Phase II clinical trial data of the JAK3 inhibitor VX-509 (decernotinib) for rheumatoid arthritis. In contrast with JAK3, JAK2 knockout animals die in embryo because JAK2 is essential implication in some signaling. Since JAK2 is critical for erythropoiesis and thrombopoiesis (Figure 2), JAK2-/- mice have a defect in red blood cells. Contrary to the other JAKs, JAK2 is the single player for receptors for hormone-like cytokines such as GH, prolactin (Prl), Epo, thrombopoietin (Tpo), and cytokine receptors involved in hematopoietic cell development, such as IL-3 or granulocyte macrophage colony-stimulating factor (GM-CSF). Moreover, activating mutations of JAK2 contribute significantly to the development of myeloproliferative disorders. Some JAK2 inhibitors are currently tested in clinical trials to treat these disorders, and a mixed JAK1/JAK2 has already been approved (INCB18424/ruxolitinib, 2, Figure 1) [12] . TYK2 has been shown to play an important role on the IL-12/IL-23 signaling pathway in vitro using human cells and in in vivo studies in mice (Figure 2) [13,14] . It has been validated using in vitro assays for inflammatory diseases [13] and by human genetics and mouse knock-out studies for conditions related to the innate and acquired immune response [12] . JAK1 deficient mice (like JAK2 -/- mice) die in embryo, but in this case the cause is neurological defects. JAK1 knock-out studies in cells revealed a critical role in signal transduction mediated by class II cytokine receptors (e.g., IFNα/β, IFN-γ, IL-10), the γc receptor subunit (e.g., IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21), the gp130 subunit (e.g., IL-6, IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor) and G-CSF(granulocyte colony-stimulating factor) (Figure 2) [15] . Moreover, recent work suggests that JAK1 rather than JAK3 kinase function is dominant in driving the activity of the immune-relevant γccytokines [16] . Another very interesting result obtained by Sohn et al. demonstrated the essential role of JAK1 inhibitor on IL-6, IL-22 and INF-α pathway [13] . Consequently selective inhibition of JAK1 is expected to be of therapeutic benefit for a range of inflammatory conditions including RA, as well as for noninflammatory diseases

future science group


Review

A B

TYK2

JAK2

Il-12, IL-23

JAK2

JAK2

EPO

JAK2

JAK1

TYK2

INF

JAK2

JAK1

JAK3

JAK1

JA K

JA K

JAK

JAK

P

IL6

TYK2

IL2

Cytokines

P

STATs

C P

Cell membrane

P

Gene transcription Nucleus

JAK1 1

34

JH7–JH5

JH4–JH3

JH2

JH1

FERM

SH2

Pseudokinase

Kinase

439

583

875

1154 Amino acids

Figure 2. JAK signaling. (A) JAK members play a central role in cytokine pathway. (B) JAK and cytokine receptor signaling. (C) Domain structure of JAK1. Reproduced with permission from [9] © Elsevier (2013).

driven by JAK1-mediated signal transduction. JAK1 has also been involved in hyperproliferative disorders as it was found to be mutated in acute myeloid leukemia, acute lymphoblastic leukemia (ALL) and in uterine leiomyosarcoma. Overactive mutants were found in 18% of adult T-cell ALLs (48 cases studied) [17,18] . On the basis of all these results, it can be considered that a selective JAK1 inhibitor would be sufficient, for treatment of a number of diseases. In particular, safe therapy should minimize inhibition of JAK2, as JAK2 plays an integral role in the EPO signaling pathway. This statement is reinforced by the effects observed for tofacitinib at high dose (>10 mg b.i.d.), hemoglobin reductions [19] . Even modest selectivity for JAK1 over JAK2 could widen the therapeutic index for JAK1mediated effects relative to JAK2-dependent anemia. Other adverse events that seemed to be associated with tofacitinib include: infections; a decrease in neutrophil counts, with one patient developing moderate neutropenia but no patient developing severe neutropenia. One might expect it to be consequences of blocking several cytokine signals such as granulocyte colonystimulating factor (G-CSF) that play an important role in the proliferation and differentiation of neutrophils. Both GSCF and GMCSF are important players in neutrophil differentiation. As JAK1 selective inhibition is known to leave GM-CSF signaling unaffected, one might expect the impact of such selective inhibitors on neutrophil counts to be lower compared with unselective JAK inhibitors. The future development of JAK1 selective inhibitors will allow to understand the balance of efficacy and safety of a selective inhibition compared with PAN JAK inhibition.


Inhibition of JAK1 & selectivity

The structural similarity between differing proteinkinases initially cast doubt on the notion that safe kinase inhibitors could be generated. As there are more than 500 human kinases, many of which serve critical cellular functions, would it really be possible to attain the specificity needed? Now that, some 20 years later, a number of protein tyrosine kinase inhibitors have been approved by the FDA and many more agents are close to market, these fears have disappeared. The selectivity characterization of kinase inhibitors exclusively utilized most of the time the respective Ki values. These were measured using enzymatic assays performed with an ATP concentration equal to the enzyme/ATP Km value, producing the intrinsic affinity of the inhibitors (Ki). It has long been recognized that the cellular concentration of ATP is in the 1–5 mM range, whereas the enzyme/ATP Km value is highly different between kinases and ranges from nanomolar to millimolar [20] . Therefore, IC50 measurements of inhibitors measured at the ATP Km are not a good descriptor of the true relative cellular activity for ATP competitive inhibitors. Such biochemical data require supplementary cellular assay data to generate a more realistic selectivity profile. Discrepancies between enzyme and cellular data for inhibitors of JAK kinases have been recognized for some time but only recently have potential explanations supported by experimentation been published to address these inconsistencies [13,16] . Indeed new data cites the IC50 evaluation against each JAK protein when carried out at 1 mM ATP concentration

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Review Menet, Mammoliti & López-Ramos independently of the ATP Km of the kinase, to quickly have an estimation of the selectivity [21–25] . The second factor to be taken into account to determine the selectivity of inhibitors is the role of JAKs on the cytokine receptors, and in particular the importance of the various individual JAKs in cytokine signaling. It is indeed known that, except for JAK2 in some signaling pathways, JAKs exist as heterodimeric complexes. These recent years, a number of research groups have discovered a different hierarchical role of each JAK within the cytokine pathways of the cell, impacting the selectivity of the compounds on these signalings. Due to all these observations, careful analysis of JAK inhibitors has demonstrated that their selectivity for one JAK enzyme compared with other JAK enzymes is often different in a cellular setting from biochemical assays [26] . Retrospective comparison of compound series has shown that the potency of compounds in some cellular assays is strongly determined by the JAK1 inhibition component even if the other JAKs are known to be heterodimers with JAK1 [13,16,27] . In this review, the selectivity mentioned will refer almost always to the relative biochemical activities; sometimes, however, this biochemical selectivity has also been confirmed in cellular assays.

data reflect the relative inhibition of a JAK1-dependent cytokine signaling (IL-6) and a JAK2-dependent cytokine signaling (GMCSF). Clinical proof of concept and Phase IIA data were reported in 2012. Data from two 4-week studies have been reported: one study with 36 patients who were methotrexate–inadequate responders comparing 4 at 100 mg b.i.d. and 200 mg q.d. (quaque die = once a day), and a second study in which 96 methotrexate insensitive patients received compound 4 at four different doses, 30, 75, 150, 200 mg, all once a day. In summary, for the second study little effect was seen at the lowest dose and from 75 mg q.d., effect was seen in a dose-range manner. No safety events and, importantly, no consistent increase in LDL cholesterol were reported [29] . A Phase IIB study on 4 started in 2013, evaluating the effect of 4 on a larger number of RA patients (875 moderate-to-severe RA patients refractory to methotrexate) and over a longer period of 24 weeks. Galapagos has recently reported that 4 is metabolized to a JAK1 selective inhibitor that is 10- to 20-fold less potent in human whole blood, but has a compensating increase in exposure, which drives prolonged inhibition [30] . INCB39110, 5 & INCB47986, 6

Global portfolio of clinical candidates The following table presents the progress of JAK1 inhibitors that have entered clinical trials over the last 10 years (Table 1) . GSK2586184 (GLPG0778)

Galapagos has entered the JAK field by focusing its efforts on the discovery of JAK1 selective inhibitors. Publicly available data show that it has taken two selective JAK1 inhibitors in development within Galapagos’ inflammation alliance with GlaxoSmithKline ([GSK], London, UK). The first compound to reach clinical trials was GLPG0555 [28] but only Phase I trial data are available; no other progress on the compound has been reported. GSK exercised its option to develop both GLPG0555 and a second compound, GLPG0778. The latter compound has been designated GSK2586184. Filgotinib (GLPG0634), 4

A third compound from the Galapagos stable is GLPG0634 (Filgotinib), 4, which is now in Phase IIB study in RA within the Galapagos/AbbVie alliance. Preclinical data have been published, showing that the compound is a JAK1 selective inhibitor in whole blood (WB) assays (about 30-fold) but not in biochemical assays where it is almost JAK1/JAK2 equipotent (JAK1 IC50 : 10 nM, JAK2 IC50 : 28 nM, JAK3 IC50 : 810 nM, TYK2 IC50 : 110 nM). The WB

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After the discovery of ruxolitinib, 2 and baricitinib 3 (Figure 1), two JAK1/JAK2 inhibitors, Incyte appears to have commenced research to design JAK1 selective inhibitors, as can be concluded from patent publications on JAK1 inhibitors recently disclosed (see section Incyte: Pyrrolopyrimidines). This has resulted in two JAK1 selective inhibitors in clinical trials [31] . For strategic reasons, Incyte eventually targeted INCB39110, 5, into clinical studies in oncology after a first evaluation in an RA trial. The data on INCB39110 have been reported in the 2013 ACR symposium (Supplementary Table 1) [32] . Compound 5 is a potent and selective inhibitor of JAK1, with greater than 20 and greater than 200-fold selectivity over JAK2 and JAK3, respectively. It is efficacious in the rat adjuvant-induced arthritis model at exposures that do not affect EPO-induced stimulation of reticulocytes. Compound 5 showed evidence of benefit with all doses tested ranging from 200 to 600 mg total daily dose, with the highest dose being the most effective dose. Benefit was observed as early as after 1 week of treatment. The treatment was generally well tolerated, with no evidence of myelosuppression or immunosuppression at the doses tested. A larger patient population and longer term exposure will be needed to fully explore the safety profile. INCYTE is advancing its second JAK1 inhibitor (INCB47986) into inflammation (RA) and it plans to initiate a Phase II trial in 2014.



ABT-494

Review

and EPO-stimulated UT7epo cells (JAK2). Selectivity for JAK1 against JAK2 in these cellular assays was reported to be 74-fold, whereas tofacitinib, 1 in the same assay was only 24-fold selective (cell assay: IL6-stimulated TF1 cells/EPO-stimulated UT7epo cells (JAK2)). Selectivity against JAK3 was reported to be 58-fold for ABT-494 compared with only twofold for 1 in the same assay (biochemical assay). Therefore ABT494 showed less effects on peripheral NK cell (natural killer cell) counts than 1 over the efficacious exposure range in a preclinical in vivo model. In Phase I clinical trials, 6 mg ABT-494 dosed two-times a day for 14 days in healthy human subjects did not affect reticulocyte, NK- or NKT-cell counts (per milliliter), confirming the cellular in vitro selectivity. Single oral doses of 5 mg of compound 1 and 3 mg ABT-494 have similar pharmacodynamic effects on JAK1 signaling in healthy human subjects, looking at STAT3 phosphorylation

ABT-494 (structure undisclosed at date of publication) is a JAK1 selective inhibitor developed by AbbVie. From this JAK1 discovery program, a spin-off program on JAK3 covalent inhibitors was initiated [33,34] (see section ‘AbbVie’). At the 2013 ACR symposium, AbbVie described preclinical and early clinical data [35] on ABT-494. The hypothesis proposed is that JAK1 inhibition drives efficacy in inflammation, and that JAK2 and JAK3 inhibition drives the side effects from EPO and common γ-chain signaling. For this reason, ABT-494 was designed to have selectivity for JAK1 inhibition versus JAK2 inhibition, using structural predictions that indicated the potential for differential binding interactions outside the ATP-binding sites of the two enzymes [35] . The efficacy and selectivity of ABT-494 were tested in a battery of relevant cellular assays including IL6-stimulated TF1 cells (JAK1), Table 1. JAK1 inhibitors currently in clinical trials. Company

Name

Galapogos

GLPG0634, Filgotinib

Structure N N

NH N

Compund

JAK profile

Phase

Indication

4

JAK1

Phase IIB

RA/Crohn’s disease

O

N S O O

Galapogos/ GlaxoSmithKline

GLPG0555

Not disclosed

JAK1

Phase I

Not disclosed

Galapogos/ GlaxoSmithKline

GSK2586184 (GLPG0778)

Not disclosed

JAK1

Phase II

Psoriasis/ ulcerative colitis

Incyte

INCB039110

5

JAK1

On hold

RA

6†

JAK1

Phase II

RA

JAK1

Phase II

RA

NC N

N N

O

N

F N N

Incyte

INCB47986

NC N

N N

O

N

N N

AbbVie

ABT-494

N

F3C

N H

N H

Not disclosed

F3C

N

N H

Structure published recently [27], although no reference was available in this reference, and no similar structure was identified in Incyte’s patent RA: Rheumatoid arthritis. †



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Review Menet, Mammoliti & López-Ramos triggered by IL-6 ex vivo. To date, there are only a few patent applications described by AbbVie on JAK inhibitors (see section ‘Incyte: Pyrropyrimidines’). Structural characterization of JAK1 General structure of the kinase domain

JAKs share seven regions of homology, termed the JAK homology (JH) domains (Figure 2C) . For JAK2, Ungureanu et al. showed that the JAK2 JH2 domain possesses a dual kinase specificity critical to control the activity of the JH1 kinase domain [36] . The other JH domains do not encode functional domains but overlap with a FERM (a band four point one, ezrin, radixin, moesin) domain (JH5–7), mediating binding to the receptor and an SH2 (Src homology 2)-like domain (JH3–4). The structures of the various JAK family kinase domains have been intensively studied, reflecting growing interest from large and small pharmaceutical companies as well as academia for this therapeutic target class. The first structures of the JH1 kinase domain appeared in 2005 (JAK3, PDB 1YVJ [37]) and 2006 (JAK2, PDB 2B7A [38]), but contained flat ATP analogs or staurosporine derivatives as ligands, and were not suitable for selectivity exploration. From 2008 onward, a considerable number of JAK2 structures were released, followed by JAK1 (2009) and later on JAK3 and TYK2 (2010). To date, 78 structures have been deposited in the Protein DataBank for the whole JAK family, 14 of which are of the JAK1 kinase domain and two of the JAK1 JH2 domain. Many more proprietary structures are likely to exist, not yet released into the public domain. Structural differences between proteins of the JAK family

The first JAK1 structures, in complex with tofacitinib 1 (Figure 3) and CMP6 compound (Supplementary Figure 1) were published by a Monash University-Cytopia collaboration in 2009 (PDB 3EYG and 3EYH [39]). A JAK2 structure in combination with compound 1 was released simultaneously (3FUP), and a Pfizer group completed the set of 1- and CMP6 compound-bound structures by disclosing JAK3 (3LXK and 3LXL) and TYK2 (3LXN and 3LXP) structures in 2010 [40] . These two sets of structures bound to the same ligands allowed selectivity analyses between the various JAK enzymes to be performed on a proper structural basis. Extremely high structural conservation of the binding sites was reported by all authors. Indeed, only a few structural differences were found around the ATP-binding site occupied by the inhibitors (Figure 3) . Furthermore, the available JAK x-ray structures display little or no ligand-induced side-chain

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mobility (with two exceptions: a Genentech thiadiazolo compound bound to TYK2 (PDB 3NYX) and a Novartis compound bound to JAK2 in a DFG-out conformation (PDB 3UGC)). This underlines the challenges of developing truly isotype-selective JAK inhibitors. Subtle differences present in the active site have, however, been exploited to provide molecules with various inhibition profiles. Highlighted below are key amino-acid residue differences that have been used to leverage selectivity. • Gly-rich loop (aka P-loop): inhibitors with substituents that extend under the P-loop are in some cases able to achieve selectivity: –– Glu883/905 (JAK1/TYK2) versus Lys857/830 (JAK2/JAK3). Although the residue side-chains do not point toward the ligand, they could induce a different electrostatic environment, negatively or positively charged, respectively. This difference has been invoked as an explanation for the selectivity of some Genentech’s tricyclic imidazopyrrolopyridine compounds for example (see section ‘Imidazopyrrolopyridines’); –– His885/907 (JAK1/TYK2) versus Asn859/832 (JAK2/JAK3). Pi-stacking with the aromatic sidechain of His is only possible in JAK1 and TYK2, which could be the reason for the selectivity of some Incyte and Pfizer inhibitors that bear an aromatic ring at the end of a long substituent tail (compound 5, see section ‘Incyte: pyrrolopyrimidines’ and compounds 7 to 8, see section ‘Pfizer’). • Arg879/901 (JAK1/TYK2) versus Gln853/Ser826 (JAK2/JAK3). Some substituents linked to the inhibitor core can be oriented to interact selectively with Arg879 in JAK1 and TYK2, whereas this interaction would be absent in JAK2 and JAK3. This has been proposed as the origin of the selectivity of some inhibitors with a JAK1/TYK2 profile, not described in this review [41] . Cellzome’s and Galapagos’ inhibitors might also exploit this mechanism to achieve selectivity (see sections ‘Galapagos’ JAK1 inhibitor discovery’ & ‘Cellzome’); • Glu966 (JAK1) versus Asp (JAK2/JAK3/ TYK2): some ligands are able to take advantage of a longer side chain in JAK1 to establish additional interactions that cannot exist in the other JAK proteins. This is the case of the C-2



Leu891 Met865 Leu838 Leu913

Review

Asp880 Gln854 Gln827 Asp902

Arg879 Gln853 Ser826 Arg901

Glu883 Lys857 Lys830

Phe958 Tyr931 Tyr904 Tyr980

Glu905

Leu959 Leu932 Leu905 Val981

Val938 Val911 Val884 Ile960

Gly1020 Gly993 Ala966 Gly1040 Ser963 Ser936

Glu966 Asp939 Asp912 Asp988

His885 Asn859 Asn832 His907

Cys909 Ser985

Ser961 Tyr934 Ser907 Leu983

Lys965 Arg938 Arg911 Arg987

Figure 3. X-ray structure of 1 in JAK1 JH1 domain (PDB ID: 3EYG), highlighting residue differences among JAK family members in the ATP-binding site. At each nonconserved position, respective sequences are indicated for JAK1 (top), JAK2, JAK3 and TYK2 (bottom). Amino acid compositions deviating from consensus are shown in bold font. Reproduced with permission from [9] © Elsevier (2013).

substituted tricyclic imidazo-pyrrolopyridine family for example (see section ‘C2-substituted imidazo-pyrrolopyridines’);

Although very small, these differences have allowed the design of selective inhibitors, in particular targeting JAK1.

• Ala966 (JAK3) versus Gly (JAK1/JAK2/TYK2): situated at the beginning of the activation loop, the flexibility of the glycine residue of JAK1, JAK2 and TYK2 allows its backbone to adopt a ‘carbonyl-up’ or a ‘carbonyl-down’ conformation, as well as intermediate positions. The Ala966-Asp967 sequence in JAK3 requires on the contrary a ‘carbonyl-down’ conformation, which limits the flexibility of this region of the protein and its ability to adapt to the ligand, as compared with the other JAK isoforms. This difference has been used by Vertex in the development of JAK2 inhibitors [42] , but could also be applied to obtain JAK1 selectivity against JAK3.

Literature on JAK1 selective inhibitors Structure–activity relationship (SAR) studies directed toward obtaining selective inhibition by small molecules against JAK proteins have lagged behind the elegant insights gained into JAK biology, mechanism of action and potential therapeutic utility [13,16] . This section will provide a review of the discovery of JAK1 selective inhibitors.


Galapagos’ JAK1 inhibitor discovery

Galapagos was the first company to report a JAK1 selective inhibitor to enter clinical trial (GLPG0555) and there are now three selective JAK1 inhibitors discovered


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Review Menet, Mammoliti & López-Ramos at Galapagos which have entered clinical trial. The first series of compounds reported had a triazolopyridine scaffold (9,10,11,12, 13,14,15,4,16, Table 2) and was discovered by a JAK1 biochemical high-throughput screening assay [43] . The initial hit compound displayed potent JAK1 inhibition, IC50 = 60 nM but little selectivity against JAK2. From this triazolopyridine scaffold, two types of substitutions in position 2 could be derived: either acyclopropylamide (10,11,14,15,4,16) [44–50] , which includes GLPG0634, 4 [48] , or an aniline (9,12,13) (Table 2) [51–53] . From the literature and from what Galapagos has disclosed at conferences, the 2-position cyclopropylamide group gives better JAK1 selectivity over JAK2, compared with the aniline moiety [43] . Indeed the 2-position sits in a region where amino acid residues are different between JAK2 and JAK1, offering

selectivity options. This was corroborated by the fact that 2-position anilines substituted by basic amines were shown to have inverse selectivity for JAK2 over JAK1 [57] . Data released on GLPG0634 after screening on a panel of 150 kinases showed that the series has an overall interkinase class good selectivity [43] . Substitution at both positions 5 and 8 of the above triazolopyridine core seems to be tolerated for JAK1 inhibition, as shown by the structures 10 and 14 (Table 2) . These five or eight-positions appear to be open to a wide variety of groups, in particular substituted phenyls or nonaromatic substituted heterocycles [56] which retain JAK1 selectivity and potency. Other related scaffolds like the pyrazolopyridine 17 have also been developed (Table 2) . It should be noted that in all these patents data reported are biochemical data from tests using the Km concentration of ATP, so it is

Table 2. Summary of all Galapagos patent references with a representative compound for each application. Number

Example structure

WO2010010184

N

N

Compound Selectivity

Ref.

9

Only range of potency given, so the selectivity cannot be determined from patent. 9:JAK1 IC50 10-fold selectivity) where the aromatic group takes the place of the ethyl sulfonyl substituent (Table 6) . As pointed out also by others [9,55] , the presence of the aromatic tail is probably a key driver for selectivity. It has been suggested that an aromatic group in this area can interact with His885 at the edge of the P-loop in JAK1, which is absent in JAK2 [9] . The aromatic tail showed consistent substitution patterns for the most representative compounds across different patent applications. Halogens, CF3 and electron-withdrawing groups in general are present in all compounds from Table 6, indicating that an electron-poor aromatic ring can be particularly favorable for selectivity. This is also supported by the presence of heteroatoms in the ring for most of the examples (5, 58, 59, 61, 62, 64, 67, 68 and 69, Table 6). 62

222


is a representative example possessing this substitution pattern. As pointed out by others [64] , 62 is the preferred compound of Incyte’s patent application WO2011112662 [23] and it has been suggested that structure 62 is that of INCB-39110 [82] . Regarding WO2010135650 [22] , it has been suggested elsewhere [64] that 58 is the preferred compound, as it is object of specific claims; however, compound 5 is the most selective compound described in this filing (188-fold selectivity vs JAK2). Regarding WO2012177606 [79] , all compounds are reported with selectivity greater than tenfold, but there is no indication of a preferred molecule. Compound 66 is one of the most potent compounds (IC50 5

WO2011028685

[78]

61

≤0.7

69.2

WO2011112662

[23]

Tail

N N

N

Spacer Core N N H

N

N

F N

N

N

Cl

N N

N N

N H

N

N

S

N N

N N N

N H F

O N N

N

N N N

N N

N H O

N

N

N N N N

CF3 N

N

N N

NH



223


Table 6. Selection of examples extracted from Incyte patent applications (cont.). Structure

Compound JAK1 IC50 (nM) Reported selectivity (JAK2/JAK1) Patent application

N

O

Ref.

62

≤2

49.2 (mono adipate salt)

WO2011112662

[23]

63

≤5

>20

WO2012068440

[25]

64

10

WO2012068450

[78]

65

10

WO2012068450

66

≤1

≥10

WO2012177606

[79]

N N F

N N

N F3C

N N

N H F O

N

N

N

N N

N N H

N N

O N

N

N

N N F N N

N H

N

O N

N

N

N N N N N

N H N HN N

N N

O F

N N

224

N H




Review

Table 6. Selection of examples extracted from Incyte patent applications (cont.). Structure

Compound JAK1 IC50 (nM) Reported selectivity (JAK2/JAK1) Patent application N

CF3

HO N

Ref.

67

20-fold selectivity, JAK1 IC50 ≤5). Other examples to be noticed are compounds 65 and 69. Also in these cases, the compounds with benzylamino substituents are among the most selective. In these latter two cases, however, the predominance over other chemotypes cannot be concluded from the reported data. As mentioned above, the patent applications can be differentiated from one another by the nature of the spacer that connects the propylnitrile group to the aromatic tail. Some filings describe a bicyclic spacer formed by a four-membered saturated ring connected to a six-membered heterocycloalkyl group (62, 65, 67 and 69). The difference among these four patent applications is in the position of the heteroatoms. Two of the patent applications describe a spacer formed by a methylene group connected to a six-membered saturated


heterocycle (60 and 63). However, patent applications WO2010135650 [22] and WO2012177606 [79] describe compounds with a linker formed by a pyrrolidine and an azetidine, respectively (5 and 66). Owing to the lack of an absolute scale of reported activity and selectivity, it is difficult to compare the different linkers with one another in terms of selectivity. It is worth noting the presence of basic groups in many linkers (60, 62, 63, 65, 67 and 69). In conclusion, the JAK1/JAK2 selective inhibitors 3 and 2 were modified with monocyclic or bicyclic linkers terminating with aromatic rings. These changes led to a shift in profile resulting in JAK1 selective compounds. Electron-poor aromatic rings, including pyridines and pyrimidines, seem to favor JAK1 selectivity. The presence of benzylamino groups in the metaposition also appears to favor selectivity, as some of the


225

Review Menet, Mammoliti & López-Ramos most selective compounds in different patent applications present such as motif. The presence of a basic group in the linker is a widely represented feature. Pfizer

After the discovery of the pan-JAK inhibitor tofacitinib, 1, the first Pfizer patent application disclosing a JAK1 selective compound was published in 2010 [83] . It is a very narrow patent application, disclosing only three compounds that are very similar in structure to 1: Compound 70 and its ethyl- and cyclobutylsulfonamide analogs (Table 7) . Biochemical IC50 data are only disclosed for 70 (JAK1 IC50 = 9.53 nM, JAK2 IC50 = 17.5 nM) and the cyclobutyl analog (JAK1 IC50 = 45.0 nM, JAK2 IC50 = 101.0 nM), the selectivity versus JAK3 and TYK2 being greater than eightfold. In vitro inhibition of the proliferation of canine and feline lymphomas is also shown for 70 and dosing for in vivo administration is part of the claims. Dog and companion animals are specifically cited in the claims, which suggest a veterinary therapeutic application of 70. A second patent application was published in 2011 [88] , with 141 examples in which the N-substituent of the sulfonamide is part of a cyclic alkyl group, instead of bearing a methyl group as in 70. Pyrrolidines and piperidines like 71 and 72 showed very good biochemical JAK1 potency combined with more than 10-fold JAK2 and 100-fold JAK3 biochemical selectivity (Table 7) . One of the most potent compounds reported, 73, displayed an extraordinary high biochemical activity (JAK1 IC50 = 2 pM) while retaining eightfold selectivity versus JAK2 and 100-fold versus JAK3. A third patent application [89] completes the previous one by disclosing compounds with an alkyl linker or an aryl substituent distant to the sulfonamide. Among the 57 examples given, some (7, 74, 8, Table 7) display very good JAK1 biochemical potency and JAK2 and JAK3 selectivity (eight- to 12- fold and over 90-fold, respectively). Since all the compounds disclosed in Pfizer’s patent applications are analogs of 1, the same binding mode as for 1 may be expected. The observed selectivity probably comes from the elongated substituent, which likely extends under the P-loop and, when an aromatic ring is present at the end of a long tail, is able to selectively interact with His885 in JAK1 (but which is Asn in JAK2 and JAK3), as is the case for some of the reported Incyte compounds. Hutchison Medipharma

Hutchison Medipharma also published a patent application disclosing JAK inhibitors of structure very close

226


to that of 1, in which the piperidine moiety of 1 is replaced by a pyrrolidine situated between the core and the amine linker [90] . In this case, however, the heterocycle is connected to the core by the endocyclic nitrogen and the substituents interacting with the P-loop are connected by an amine linker. Aryl substituents on the amine linker provided potent and selective compounds: 75, for example, has eightfold biochemical selectivity versus JAK2, but the selectivity drops in cellular assays (Table 7) . 76 on the contrary has low biochemical JAK2 selectivity (threefold) but a very high cellular selectivity (>180-fold). Merck

Currently, there is no indication of clinical development programs involving JAK inhibitors at Merck. However, Merck recently published four patent applications describing molecules with JAK1 selectivity over JAK2 [84–87] . A synopsis of the general formulae presented in the patent applications is given in Supplementary Figure 8. An analysis of the content of these patent applications from Merck has been reported elsewhere [91] . All the Merck patent applications are based on a pyrazole carboxamide scaffold substituted with an N-linked aryl or heteroaryl group at the 3-position and a substituted propylnitrile group at position 1 of the pyrazole (see Table 8 ). This propylnitrile motif is functionalized with alkyl, cycloalkyl and hetereocycloalkyl groups. It is actually on this propylnitrile motif where the point of diversity for the different patent applications lies. A pyrazolecarboxamide scaffold was also present in an earlier patent application from Merck claiming JAK inhibitors (Supplementary Figure 8) [91] . Nevertheless, the molecules exemplified there were associated only with JAK2 data, indicating a predominant inhibition of this isoform. In this case, the compounds presented an aryl group in position 1 (R1 in the patent). It seems that the replacement of the aromatic group R1 in compounds with a propylnitrile motif associated with a saturated substituent resulted in compounds for which inhibition of JAK1 is predominant. In all these four patent applications, both JAK1 and JAK2 IC50 values are reported, making it possible to compare compounds from different applications (IC50 data are reported from experiments in which the ATP concentration is equal to the Km for each enzyme). The presence of a carboxylic acidic on the aromatic ring generally increases the selectivity. For instance, replacement of the CF3 group in 77 (JAK1 IC50 = 2 nM, JAK2 IC50 = 19 nM) with the dimethyl acetic acid in 78 (JAK1 IC50 = 0.12 nM, JAK2 IC50 = 7 nM) resulted in a greater than 15-fold increase



Review

Table 7. Example of WO2010020905. Structure

O

S

N

Compound

JAK1 IC50 (nM)

Sel vs JAK2

Sel vs JAK3

Sel vs JAK2

Sel vs JAK2

Sel vs JAK2

Ref.

70

9.53

1.8

10.0

[83]

71

1.44

33.6

159.7

[84]

72

1.7

13.9

119.4

[84]

73

0.00199

8.3

99.5

[84]

7

1.82

8.2

91.2

[85]

74

2.25

12.7

114.2

[85]

O

N N N

N

S O

NH

N

O

O

O

N N N H

N O

N O

S O

N N N

S O

N

N H

OH

O

N N N

N H

S

H N

O O N

O S O

NH2

N N

N H

S

H N

O O

OH

N N N

N H

Structures and activities of selected compounds from WO2011045702 and WO2011075334 [84,85] 78 and 79: Selected compounds from Hutchison Medipharma patent application [86].



227


Table 7. Example of WO2010020905 (cont.). Structure N

Compound

JAK1 IC50 (nM)

Sel vs JAK2

Sel vs JAK3

Sel vs JAK2

Sel vs JAK2

Sel vs JAK2

Ref.

8

6.85

11.1

90.8

[85]

75

5.1

8.1

16.9

0.337

2.5

1.1

[85]

76

14.0

3.5

25.7

0.009

186.7

[86]

N

S O O

N N N

H2N O

S

O

N H

N

N

N N N H

N NC

F

N

N N N

N H

Structures and activities of selected compounds from WO2011045702 and WO2011075334 [84,85] 78 and 79: Selected compounds from Hutchison Medipharma patent application [86].

in potency and a greater than sixfold increase in selectivity (Table 8) . The same boosting effect was observed with the phenyl acetic acid group in 84; it resulted in an eightfold increase in potency (JAK1 IC50 = 0.1 nM) and a fourfold increase in selectivity (100-fold) when compared with the 4-F-phenyl in 83 (JAK1 IC50 = 0.8 nM, JAK2/JAK1 = 26-fold). In other cases, different groups having relatively acidic protons, such as –CHF2OH (82), –SO2CHF2 (86) or H-pyrazole (87) are widely exemplified across the four patent applications and some of them are very potent (JAK1 IC50 = 0.03 and 0.01 nM for 82 and 87, respectively). Modifications of the substitution pattern of the propylnitrile motif have also a deep impact on activity and selectivity. Particularly remarkable are the data associated with 80 (JAK1 IC50 = 0.6 nM, JAK2/JAK1 = 400-fold). In this molecule, the carbon atoms at positions 2 and 3 of the propylnitrile are embedded in a cyclohexyl ring possessing a basic amine at position 4.

228


Somewhat lower selectivity values are reported for the best compounds described in WO2013043962 [86] . This patent application describes compounds where only the carbon atom at position 3 of the propylnitrile motif belongs to the saturated cycle. The best two compounds are 85 (JAK1 IC50 = 0.8 nM, JAK2/JAK1 = 191-fold) and 86 (JAK1 IC50 = 0.2 nM, JAK2/JAK1 = 160-fold). A direct comparison of these two series is difficult as there is little overlap between them and the best combination of substituents found in WO2013040863 [84] was not applied to WO2013043962 [86] . As already suggested elsewhere [91] , high selectivity seems to be associated with the presence of a basic amine at the 4 position of the cyclohexane ring. This is particularly evident by the direct comparison of 77 (JAK2/JAK1 = 9.5-fold) with 79 (JAK2/JAK1 = 60-fold). It is also interesting to compare compounds 81 (JAK1 IC50 = 27 nM, JAK2/JAK1 = 13.5-fold)



with 83 (JAK1 IC50 = 0.8 nM, JAK2/JAK1 = 26-fold), which constitute a matched pair from different series. Compound 83 shows a greater than 30-fold increase

Review

in selectivity. A possible interpretation of these data is that, for these two molecules, in absence of the strongly polar groups described above, the potency may be

Table 8. Examples of compounds from WO2013040863, WO2013041042, WO2013043962 and WO2013043964. Structure O

NH2

H N

O

NH2

H N

JAK1 IC50 (nM)

JAK2 IC50 (nM)

Selectivity (JAK2/JAK1)

Patent application

Ref.

77

2

19

9.5

WO2013040863

[84]

78

0.12

7

58

WO2013040863

[84]

79

0.35

21

60

WO2013040863

[84]

80

0.6

240

400

WO2013040863

[84]

81

27

367

13.5

WO2013041042

[85]

82

0.03

0.2

6.7

WO2013041042

[85]

N

N N

F3C

Compound

N

N N COOH

O

NH2

H N

N

N N

F3C

N O

NH2

H N

N N N

F3C

N

O

NH2

H N

N N N

F N O O O

NH2

H N N N

HO F

N

F O



229


Table 8. Examples of compounds from WO2013040863, WO2013041042, WO2013043962 and WO2013043964 (cont.). Structure O

NH2

H N

Compound

JAK1 IC50 (nM)

JAK2 IC50 (nM)

Selectivity (JAK2/JAK1)

Patent application

Ref.

83

0.8

21

26

WO2013043962

[86]

84

0.1

10

100

WO2013043962

[86]

85

0.8

153

191

WO2013043962

[86]

86

0.2

32

160

WO2013043962

[86]

87

0.01

0.6

60

WO2013043962

[86]

88

7

425

61

WO2013043964

[87]

N N

F

O

N O

N O

NH2

H N N N

COOH N

O O

NH2

H N

F

N N

N

N H

N O

NH2

H N O

O

N N

S

F

O

N

F

N H

N O H N

N HN

NH2

N N

N

CF3

O

N O H N F

O

N

NH2

N N N

O O

N

F3C

driven substantially by the interaction of the nitrile group with the protein. In the case of compound 83, therefore, the greater conformational freedom would allow the nitrile to have more profitable interactions.

230


Compound 88 (JAK1 IC50 = 7 nM, JAK2/JAK1 = 61-fold) is the most selective compound exemplified in WO2013043964 [87] . This patent application describes predominantly compounds where



a saturated cycle is connected to the propylnitrile by a bond, rather than been fused with it, like in WO2013043962 [86] (the cycle and the propylnitrile share one atom) or WO2013040863 [84] and WO2013041042 [85] (the cycle and the propylnitrile have two atoms in common). Compound 88 features a piperidine where the nitrogen is capped by a carbamate. This is a common motif found in the most selective compounds of this application (data not shown). The same moiety can also be found in compounds with high selectivity from other applications, such as 87. The highest reported potency values for JAK1 in WO2013043964 [87] are between 1 and 10 nM, which are inferior to the best values found in the other three Merck patent applications. The information contained in those patent applications does not allow the reader to deduce if any of the described molecules was potentially being considered for more advanced studies. In conclusion, Merck filed four patent applications describing JAK1 selective inhibitors based on a pyrazolecarboxamide scaffold, which was described earlier for molecules designed to be JAK2 inhibitors. The JAK1 selective molecules display a propylnitrile group substituted with a cycloalkyl group in the region previously occupied by an aromatic substituent. Clear elements of selectivity toward JAK1 are an acidic group on N-linked aromatic substituent and the presence of a basic amine in the cycloalkyl group [92] . Exelixis

Exelixis entered the JAK1 field with a single patent application published in 2012 [93] claiming the general structure displayed in Figure 7, although most of the 62 compounds specifically claimed are defined by the substructure 89 [94] . Only JAK1 biochemical activity is provided, and only as activity ranges. From the 66 examples described, only nine belong to the most active class (IC50 ≤150 nM), the first two described being 90 and 91 (Figure 7) . The scaffold claimed in this patent application, which is completely different from other selective JAK1 inhibitors described, had already attracted interest as a scaffold yielding compounds with VEGFR and VEGFR2 inhibition. In particular, two molecules that are explicitly excluded from the patent application actually correspond to structures disclosed by a group at ImClone as VEGFR2 inhibitors [94,97] . Exelixis was the first company to bring a JAK2 inhibitor into clinical trials (XL-019), but its structure is very different from the ones claimed here. It remains to be seen whether further developments will be reported on these newly disclosed molecules.


Review

Cellzome

Cellzome described in a patent application, published in 2013, selective JAK inhibitors with chemical structures that are different from those reported by other companies [95] . Most of the compounds exemplified are very similar to each one and have the generic formula 92 (Figure 7) . R1 is an aziridine, pyrrolidine or piperidine ring in which the nitrogen is part of an amide, a sulfonamide or is substituted by an alkyl chain. R2 is most usually methyl, but also hydrogen, chlorine or fluorine and R3 is an alkyl chain. Biochemical activity ranges in the Cellzome patent application are provided for all JAK proteins. Since the compounds are claimed to be JAK1 selective, the fold selectivity versus JAK2 for the most potently inhibited protein is also indicated. The most selective JAK1 inhibitor described is 93 (Figure 7), 197-fold selectivity versus JAK2. These compounds are likely to bind to the hinge through the nitrogen atoms indicated by an arrow in Figure 7. This binding mode would place the pyrrolidine group under the P-loop, with the nitrile pointing upward, and the pyrazole substituent would be directed toward Arg879 (in JAK1), maximizing the interactions with the protein in the regions of the active site in which selectivity could be gained. Konkuk University (Republic of Korea)

Kim et al. recently described that, when aligning the x-ray structures of all JAK proteins bound to compound 7, they could identify a pocket (delineated by Asp1003 and His885 in JAK1) which is more widely open in the other JAK isoforms [96] . They designed a narrow propargyl substituent to fill this pocket and thus selectively target this pocket for JAK1. Compound 94 (Figure 7) showed 67% inhibition of JAK1 biochemical activity when tested at 10 μM, 38% inhibition of JAK3 and 0% inhibition of JAK2 and TYK2. These results are to be taken with care, since they are only based on single-dose measurements. In addition, the propargylic substituent does not seem to be directed toward the targeted residues, which happen to be misassigned in the article (the equivalent of JAK1 His885 in JAK2 is Asn859 and not Phe860). Conclusion & future perspective There are numerous pharmacological approaches to the treatment of RA. Inhibition of members of the JAK family, especially JAK1 is still central for the development of small molecule drugs – no comparable inhibition of any other kinase is equally compelling at the time of writing. Significant progress has been made over the last 10 years in JAK1 selective inhibitors, with compounds now reaching early-stage clinical trials, and some progressing toward later-stage


231


R2 HN X Y

W

Q, W, X, Y and Z are CH or N R1 is oxo, alkyl, ester, ketone, amine R2 is halo, alkyl, ester, ketone, carbamate, amide, sulfone, substituted heteroaryl or heterocyclyl

N Q

Z

R1

CF3 Cl

R2 CF3

HN CF3

HN

CF3

HN

N N

N

N N

N

N R1

O

89

O

O

NH

90

91

O N N

F R2 R1

N H

R3 N N

N N

N H

92

O N H

N

NH2

F

N

N N

NH2

H N

N N H

N NH

HO

94

93

Figure 7. WO2012037132 general structure and examples [93] . 92: Markush structure and 96, an example of Cellzome patent application [95] . 94, example from Konkuk University [96]

trials and registration. It is to be expected that in the next 5 years, the first compounds will reach the market and will show how they differentiate from the first pan-JAK inhibitors approved by the FDA, tofacitinib and ruxolitinib. Moreover, in the next decade, further developments based on the accumulated medicinal chemistry knowledge, together with new approaches, may lead to even more selective and safer compounds for RA and other immunosuppressive conditions – conditions likely to increase as the global population ages. Furthermore, the recent identification of JAK1 as a possible therapeutic target in hepatic cancer will stimulate further development efforts in other diseases.

232


Supplementary data To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/ doi/full/10.4155/FMC.14.149

Financial & competing interests disclosure The authors are employees of Galapagos NV, which has a JAK1 selective inhibitor in Phase IIb clinical trial. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.



Review

Executive summary JAK inhibitors approved by US FDA • Two nonselective inhibitors were already approved by the US FDA, one for myelofibrosis and the second one for rheumatoid arthritis.

Selective JAK1 inhibitors • JAK1 selective inhibitors have been reported to date in clinical trials. Phase IIa data have already been released and show a possibility to differentiate safety profile compared with tofacitinib.

Selectivity within the JAK family • Biochemical assay is not always the best measure of selectivity: JAKs have different Km values for ATP and different involvements within cytokine pathways.

New generation of JAK1 inhibitors • Tricyclic cores feature strongly in obtaining selective JAK1 inhibitors. • A pyrrolopyrimidine core incorporating a spacer terminating in an aromatic tail is also a preferred chemotype for JAK1 selectivity.

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Review

235

Progress toward synthetic cells.

Progress toward universal health coverage in ASEAN.

Progress toward polio eradication--Somalia, 1998-2013.

Progress toward the assessment of health status.

Progress toward a uniform air pollution index.

Progress toward a Respiratory Syncytial Virus Vaccine.

Progress toward polio eradication--Worldwide, 2013-2014.

Progress toward tissue-engineered heart valves.

Progress toward measles elimination in kyrgyzstan.

Progress toward poliomyelitis eradication in Nigeria.

Supporting labor progress toward physiologic birth.

Progress towards direct inhibitors of Stat5 protein.

Latest progress in tyrosine kinase inhibitors.

Recent progress in development of aromatase inhibitors.

Research progress of indoleamine 2,3-dioxygenase inhibitors.

Inhibitors of gastric secretion: current progress.

Retinal prostheses: progress toward the next generation implants.

Progress toward a vaccine for Middle-Eastern respiratory syndrome.

Progress toward Approval of Stents in Coarctation of the Aorta.

Progress toward curing HIV infection with hematopoietic cell transplantation.

Progress toward overcoming hypoxia-induced resistance to solid tumor therapy.

Progress toward laboratory containment of poliovirus after polio eradication.

Progress toward curing HIV infections with hematopoietic stem cell transplantation.

Recent Progress toward Engineering HIV-1-Specific Neutralizing Monoclonal Antibodies.