CFT8634

Emerging Target Families: Intractable Targets

The druggability of a target is defined by the likelihood of a certain target binding site to be amendable to functional modulation by a small molecule in vivo. Thus, druggability depends on the ability of the developed small molecule to reach the target site, the properties of the ligand binding pocket and our ability to develop chemical matter that efficiently interact with the drug binding site of interest. Historically enzymes have been the main drug targets because the inhibition of their activity can be easily assayed and catalytic centres are often attractive drug binding sites. However, despite considerable effort, a number of classical enzyme families have not been successfully targeted. More recently protein–protein interactions received considerable attention and several clinical inhibitors have now been developed. Despite the considerable progress made expanding target space, a large number of targets with a very strong rationale for targeting remain intractable. In the following chapter I will sum- marize progress made in developing inhibitors for challenging drug binding sites and emerging target families.

1 The Concept of Druggability and Properties of Drug-Like Molecules

Lipinski et al. noted in 1997 a number of shared physicochemical properties of drug candidates successfully entering phase II clinical trials (Lipinski et al. 2001). These parameters defined in this study are now known as the ‘rule of 5’ (Ro5). Based on the initially defined Ro5 criteria, a pharmacological small molecule is more likely to be active when administered orally if it has no more than one violation of the following criteria: It should not have a) more than 5H-bond donors, b) a molecular weight larger than 500, c) a cLogP (calculated octanol/water partition coefficient) larger than 5 and d) no more than 10 hydrogen bond acceptors. In a subsequent study, the molecular weight constraint has been questioned, and this parameter is nowadays often replaced with the related polar surface area criteria, which should ideally less than 140 A˚ 2.

In addition, the number of rotatable bonds (less than 10) has been identified as an important predictor for compounds that are orally active (Veber et al. 2002). During lead development the molecular weight and lipophilicity are usually increased by medicinal chemists in order to increase potency and target specificity. As a conse- quence, stricter requirements are usually applied to lead compounds (rule of three, Ro3) which are defined by an octanol/water partition coefficient log P not greater than 3, by a molecular mass less than 300 Da and by the presence of not more than three hydrogen bond donors and not more than three hydrogen bond acceptors as well as not more than three rotatable bonds (Congreve et al. 2003). There are however many exceptions to these rules, in particular for drugs that act at protein interfaces such as taxanes that target tubulin or rapamycin, targeting the interaction of the kinase mTOR/FRAP with FKPB12 (FK506 binding protein). These very successful approved drugs largely exceed the molecular weight/polar surface area constraint as well as the recommended number of hydrogen bond donor/acceptors that would be expected to result in a bioactive drug. However, both rapamycin and paclitaxel are natural products and may have been adapted through a natural selection process to function in vivo despite their poor drug-like properties (Fig. 1). The physicochemical constraints of drug-like molecules naturally constrain the type of binding sites that can be targeted. Thus, large and shallow surfaces are not likely to interact with drug-like small molecules with sufficient potency and highly polar surfaces will result in interacting ligands with poor cellular activity. Several prediction tools have been developed to assess the druggability (likelihood of developing compounds with appropriate in vivo bioavailability, efficacy and safety profiles) or chemical tractability (likelihood developing a potent in vitro inhibitor) of target binding sites (Halgren 2009; Keller et al. 2006). The developed tools include methodologies estimating the ‘maximum achievable affinity’ (Cheng et al. 2007) or are based on experimental techniques that correlated with pocket properties, for instance, NMR screening hit rates (Hajduk et al. 2005).

Fig. 1 Examples of approved bioactive ‘non-Lipinski’ drugs targeting protein interfaces

A remarkable recent study examined potential drug binding pocket in the protein databank (PDB (http://www.rcsb.org/pdb/home/home.do)). The study identified 290,000 suitable binding pockets present in 42,000 crystal structures that were available during the time of analysis (Sheridan et al. 2010), suggesting that there is a large space of potential drug binding sites that has not been explored. Indeed, the currently accessed target space was recently compiled surveying 27,000 documents including patents. This study identified assay data for 1,736 human proteins that were targeted by 823,179 unique chemical structures (Southan et al. 2011). How- ever, the top 278 most actively pursued targets were classical enzyme or membrane protein drug targets covering 90% of the identified compounds, suggesting that protein interaction inhibitors and nonclassical targets represent still a very small niche area. This is particularly evident analysing the current targets of approved drugs. In 2002 an analysis by Hopkins revealed 399 nonredundant protein targets. However, targets of drugs that are actually marketed constituted only 120 proteins (Hopkins and Groom 2002). Nearly half of the targets did fall into six different target families: G-protein-coupled receptors (GPCRs), serine/threonine and tyro- sine protein kinases, zinc metallopeptidases, serine proteases, nuclear hormone receptors and phosphodiesterases. The rate of new target discovery is still slow (Overington et al. 2006). During the past decade, 19 new chemical entities and biologics have been approved on average each year. From this set, only four new drugs are developed against previously unexploited molecular targets (Rask- Andersen et al. 2011). In the following, I have selected a number of challenging target families with strong biological rationale for drug development.

2 Example 1: Phosphatases, Classical Enzyme Targets with Low Druggability

Protein tyrosine phosphatases (PTPs) counterbalance the action of protein kinases and are essential enzymes regulating cell signalling. Similar to protein kinases, many phosphatases are deregulated in disease making a compelling case target these enzymes. There are 107 PTPs encoded in the human genome that can be grouped into four major families: classes I, II and III of cysteine-based PTPs and aspartate-based PTPs. The class I cysteine-based PTP group contains the phosphotyrosine-specific enzymes that are usually referred to as ‘classical PTPs’ (Alonso et al. 2004). Dual-specificity PTPs and low molecular weight PTPs have very shallow and charged binding sites making inhibitor development a challenging task. Within the classical PTP family, binding cavity properties are more favourable but cellular activity and selectivity remain major challenges (Barr 2010).

Two targets received most of the attention developing PTP inhibitors to date: PTP1B and SHP2 (PTPN11). The phenotype of the PTP1B knockout mice sparked drug development interest as these animals were healthy but displayed enhanced sensitivity to insulin and resistance to a high-fat diet-induced obesity, suggesting that selective PTP1B inhibitors could be beneficial treating both type II diabetes and obesity (Elchebly et al. 1999). In addition, possible applications of PTP1B inhibitors in oncology have been suggested by transgenic mouse studies that showed that PTP1B functions as a positive mediator of the ErbB2 tyrosine kinase signalling leading to breast cancer development and tumour metastasis (Julien et al. 2007). However, PTP1B is highly similar to TC-PTP which share 72% sequence identity and 94% identity considering active site residues. In contrast to PTP1B, TC-PTP knockout mice die shortly after birth as a result of anaemia, hypersensitivity and widespread inflammation strongly suggesting that inhibiting this PTP should be avoided (You-Ten et al. 1997).

There has been also significant drug discovery interest in the development of SHP2 inhibitors for the treatment of cancer. Similar to cytoplasmic tyrosine kinases, SHP2 is kept in an inactive state by its N-terminal SH2 domains (Hof et al. 1998). Binding to phosphorylated tyrosines released the SH2 domain block on SHP2 activity, activating this phosphatase. Mutation in SHP2 (PTPN11) has been associated with Noonan and LEOPARD syndrome and development of several cancer types, and most significantly activating SHP2 mutations are found in 35% of patients with juvenile myeloid leukaemia (JML) (Chan et al. 2008). In addition, high SHP2 expression levels have been associated with increased leukaemia and breast cancer risk (Xu et al. 2005; Zhou et al. 2008). Similar to PTP1B, also SHP2 is closely related to an anti-target: SHP1, a highly similar PTP which is mainly expressed in the haematopoietic system. SHP1 loss of function has been associated with severe autoimmune and immunodeficiency syndrome (Shultz et al. 1997).

The structure of PTP1B with a dually phosphorylated peptide derived from the insulin receptor identified a secondary phosphotyrosine binding pocket (Salmeen et al. 2000) and a potential binding site that can be targeted by small molecules. This secondary pocket is present in a number of PTPs and may have different physiochemical properties (Barr et al. 2009). A number of inhibitors targeting this secondary binding site and other binding pockets adjacent to the primary phosphotyrosine binding site have been developed (Fig. 2). One of the most potent inhibitors that makes use of a nonhydrolyzable phosphonodifluoromethyl phenyl group (F2pmp group) has a reported Ki value of 2.4 nM, tenfold selectivity over TC-PTP and excellent selectivity of other members of the PTP family (Shen et al. 2001).

Fig. 2 Binding modes of PTP1B inhibitors. (a) Main structural elements of the PTP catalytic domain. The active site cysteine residue (Cys215) is highlighted in cpk representation. An inhibitor targeting the secondary phosphotyrosine binding site (pdb-ID: 2qbp) is shown in ball and stick representation, (b) overlay of a number of PTP1B inhibitors (pdb-IDs: 1pxh, 2vew, 2qbp, 2fjn) targeting also pocket adjacent to the primary phosphotyrosine binding site as outlined in Barr (2010) and (c) chemical structures of two of the co-crystallized inhibitors.

The charged surface of the PTP active sites spurred the search of alternative targeting sites, and a number of allosteric inhibitor development strategies have been reported. Wiesmann et al. reported an allosteric inhibitor that prevents the closure of the WPD loop (Fig. 3). The reported inhibitor targeting this binding site had however only modest activity (IC50 8 μM) and selectivity for TC-PTP (Wiesmann et al. 2004), but it was active in cell-based assays monitoring insulin receptor phosphorylation. In addition, trodusquemine (MSI-1436), a spermine metabolite of cholesterol that was originally isolated from the dogfish shark liver, has been shown to non-competitively inhibit PTP1B but not TC-PTP. MSI-1436 has an IC50 value of about 1 μM (Fig. 3). The compound is active in vivo producing a dose-dependent weight reduction and has insulin-sensitizing properties (Lantz et al. 2010).

Recently further characterization of this interesting compound revealed that MSI-1436 targets the C-terminal, non-catalytic domain of the long isoform of PTP1B. The PTP1B C-terminus is an intrinsically disordered region of the protein that allosterically regulates PTP1B catalytic activity by interaction with the catalytic domains. In breast cancer, MSI-1436 antagonized HER2 signalling and inhibited tumorigenesis and metastasis in xenografts (Krishnan et al. 2014).

Fig. 3 Chemical structure of allosteric PTP1B inhibitors as well as the potent SHP2 inhibitor 11a-1 (Zeng et al. 2014)

Only few inhibitors have been reported for SHP2 so far. One of the most potent compounds targeting this PTP is NSC-87877 that is cell active and inhibits SHP1 and SHP2 with an IC50 value of 300 nM. Recently, a novel hydroxyindole carboxylic acid-based SHP2 inhibitor 11a-1, with an IC50 value of 200 nM and greater than fivefold selectivity against other PTPs, has been reported (Fig. 3) (Zeng et al. 2014).

3 Example 2: GTPases of the RAS Family

The small GTPases of the RAS family (HRAS, NRAS and KRAS) were the first mutated genes discovered in cancer and constitute today the most frequently mutated oncogenes. The high rate of RAS pathway activating mutations that have been detected in the most lethal cancer types has triggered a considerable research interest developing small molecules that interfere with RAS function. However, despite more than 3 decades of intensive efforts, no RAS inhibitor that targets this GTPase directly has reach clinical testing, suggesting poor druggability of these targets (Karnoub and Weinberg 2008; Cox et al. 2014).

GTPases are activated by GTP and inactivated by binding of GDP. This process is tightly controlled by a number of regulatory proteins such as the GTPase- activating proteins (GAPs) as well as GTP exchange factors (GEFs). Mutationally activated RAS usually show impaired GAP stimulation and the mutations stabilize the persistently GTP-bound active RAS state (Bos et al. 2007). The extremely high affinity of GTPases for GTP precluded unfortunately the development of GTP competitive ligands. Other targeting strategies have been therefore attempted. Among the most advanced strategies targeting RAS regulators has been the devel- opment of farnesyltransferase inhibitors. This enzyme transfers a C15 farnesyl isoprenoid lipid to its carboxy-terminal CAAX motif in RAS resulting in targeting of RAS to the plasma membrane. Several highly potent inhibitors have been developed that showed remarkable efficacy in mouse models, and several inhibitors such as lonafarnib and tipifarnib advanced to phase III clinical trials. Unfortunately, however the developed inhibitors failed to demonstrate efficacy in RAS-driven tumours, a finding that was later rationalized by the compensating action of other lipidation enzymes in particular prenyltransferases (Whyte et al. 1997).

Fig. 4 Fragment binding site identified in RAS. Shown is a superimposition of several fragments co-crystallized with RAS (a) as well as the chemical structures of the co-crystallized compounds (b). The GTP/Mg binding site is indicated as well

Direct targeting strategies for RAS have been hampered by the absence of druggable binding pockets (Buhrman et al. 2011). However, a number of weakly binding fragments have been identified (Wang et al. 2012) (Fig. 4). Structural analysis revealed that all crystallized fragments bind to a pocket adjacent to the switch I/II regions. Indeed, the identified fragments inhibited SOS-mediated nucle- otide exchange and prevented RAS activation by blocking the formation of intermediates of the exchange reaction (Maurer et al. 2012; Sun et al. 2012).
Other inhibitors have been developed based on the nonsteroidal, anti- inflammatory drug sulindac sulphide. Compounds of this class have been reported inhibiting the formation of the RAS–RAF complex and have been shown to inhibit proliferation of RAS-transformed cells. However, this compound class may have other off-target effects that may contribute to the observed phenotypes (Karaguni et al. 2002).
An interesting approach has been reported recently by Shokat and colleagues who recently reported small molecules that covalently and selectively bind to the G12C mutant form of KRAS, a frequent mutation often found in non-small-cell lung cancer. The developed compounds blocked SOS1-mediated nucleotide exchange and decreased the binding of RAS to both BRAF and CRAF in G12C- mutated cell lines (Ostrem et al. 2013).

4 Example 3: Protein–Protein Interactions

Targeting protein has gained popularity after a number of highly potent inhibitors have been developed. The targeted interactions are however typically characterized by a well-defined binding site. For instance, interaction inhibitors that disrupt the binding of MDM2 (murine double minute 2) and MDMX to the tumour suppressor p53 have now entered clinical testing in cancer (Vassilev 2007; Brown et al. 2009). MDM2 binds the p53 tumour suppressor protein with high affinity and negatively modulates its transcriptional activity and stability. Overexpression of MDM2 is frequently found in tumours leading to impairment of p53 function. Thus, it has been hypothesized that inhibition of MDM2–p53 interaction can stabilize p53 and may offer a pharmacological strategy restoring p53 function in MDM2 overexpressing tumours.
Recent structural studies showed that a new class of dual MDM2/MDMX inhibitors block the binding of MDM2 and MDMX to p53 by stabilizing MDM2/ MDMX homo- and heterodimerization occluding the p53 binding pocket (Graves et al. 2012).

A number of protein–protein interaction domains that selectively recognize sequences containing post-translational modifications have recently emerged as interesting targets for the development of inhibitors. The targeted interaction modules comprise in particular members of the so-called epigenetic reader domain family which include acetyl-lysine-dependent bromodomains (Muller et al. 2011; Filippakopoulos and Knapp 2014) as well as readers of methyl-lysine or methyl- arginine containing sequences such as PHD zinc finger domains and the Royal family of reader domains, which is composed of Tudor, MBT, PWWP and chromodomains (Herold et al. 2011a; James et al. 2013a). Several members of this family of protein interaction modules have good predicted druggability, and several inhibitors have been developed in particular for acetyl-lysine-dependent bromodomains (Vidler et al. 2012; Santiago et al. 2011). A shared feature of epigenetic reader domains that makes these protein interaction modules attractive targets is the observation that the interaction with their specific recognition sites is usually weak and localized to a binding pocket of suitable size for inhibitor development. Typically KDs of reader domain interactions are in the low μM regions suggesting that protein interactions mediated by these domains can be easily inhibited by low molecular weight inhibitors. In addition, lysine acetylation neutralizes the charge of the lysine side chain resulting in aromatic and hydropho- bic binding sites. Indeed, fragment-based screening approaches identified several diverse chemotypes suggesting excellent druggability of bromodomains (Vidler et al. 2013).

Fig. 5 Examples of benzodiazepine- and thienodiazepine-type BET inhibitors

The first bromodomain inhibitors appeared in the patent literature and were developed targeting the BET family (Miyoshi et al. 2009; Adachi et al. 2006). These inhibitors belong to the thieno-triazolo-1,4-diazepines which showed strong growth inhibitory activity on a panel of cancer cell lines. The disclosure of thieno- triazolo-1,4-diazepines as BET inhibitors led to the development of the thieno- triazolo-1,4-diazepines JQ1 (Filippakopoulos et al. 2010) (Fig. 5). In parallel, GSK discovered the benzo-triazolo-1,4-diazepine class of BET inhibitors (I-BET) using a combination of phenotypic screens and chemoproteomics (Chung et al. 2011; Nicodeme et al. 2010). Selectivity screening showed that benzo- and thienodiazepines are highly selective for BET bromodomains. Interestingly, the introduction of a stereo centre at the diazepine ring yielded a highly potent (S) enantiomer, (+)-JQ1, whereas the (R) enantiomer, ( )-JQ1, is inactive and may be used as a negative control compound. Crystal structures with BET bromodomains showed that the methyl-triazol ring served as an acetyl-lysine mimetic moiety and formed the canonical hydrogen bond with the conserved asparagine (N140 in BRD4(1)) or analogue residues in other BET family members (Filippakopoulos et al. 2010; Matzuk et al. 2012). The strong anti-proliferative effects of JQ1 and I-BET in cancer and the anti-inflammatory properties of these agents prompted the development of a number of similar benzodiazepine and thienodiazepine molecules which all include either modification on the ester/amid linkage (Zhang et al. 2012) or substitutions in the diazepine ring, which led for instance to benzotriazepines (Bzt-7) in which the asymmetric carbon was replaced by a nitrogen (Filippakopoulos and Knapp 2014; Filippakopoulos et al. 2012; Knapp and Weinmann 2013).

Novel acetyl-lysine competitive ligands have been developed based on fragment hits. In particular based on the 4-phenyl 3,5-dimethyl isoxazole fragment, a number of isoxaxoles have been developed as potent BET inhibitors (Hewings et al. 2011),most notably the highly potent panBET inhibitor I-BET151 (Dawson et al. 2011). Quinazolinone scaffolds (2-one or 4-one) have also been developed resulting in the panBET inhibitor PFI-1 (Picaud et al. 2013; Fish et al. 2012). Co-crystal structures confirmed the acetyl-lysine mimetic binding mode of the quinazolinone head group of PFI-1 which forms two hydrogen bonds with the conserved Asn140 in BRD4 (1) as well as a water-mediated hydrogen bond to the conserved tyrosine Tyr97 (Picaud et al. 2013).

Fig. 6 Acetyl-lysine mimetic inhibitors of bromodomains. General scaffolds: (a) isoxazoles, (b) 3-methyl-dihydroquinazolin-2-one, (c) 3-methyl-quinazolin-4-one and (d) 3-methyl-triazolo- phthalazine. Developed high affinity inhibitors: I-BET151 and PFI-1. Acetyl-lysine mimetic moieties are highlighted by a dashed circle

BET bromodomain inhibition has recently been described as a frequent off-target activity of kinase inhibitors (Ciceri et al. 2014; Ember et al. 2014). A screen carried out on clinical kinase inhibitors revealed a number of inhibitors with potent BET activity suggesting that dual kinase/bromodomain inhibitors could be developed. The frequent hit rate in inhibitor screens and the large number of potent inhibitors that have been developed since the discovery of the triazolodiazepine- type inhibitors certify the excellent druggability of BET bromodomains (Fig. 6).

A number of potent bromodomain inhibitors have also been developed outside the now well-studied and well-explored BET family. Modification of the benzodi- azepine scaffold led to the development of promiscuous bromodomain inhibitors (Bromosporines) that show broad spectrum activity targeting in particular BET family members, TAF1, CECR2, BRD7 and BRD9 and the BRPF family (Fig. 7). Selective inhibitors have been developed for bromodomains present in the histone acetyl transferases CREBBP/EP300 (I-CBP112 and CBP30) (Hay et al. 2014), BAZ2A/B (GSK2801 and BAZ2-ICR), BRPF1B (PFI-4) and panBRPF (OF1, NI-C-057), BRD7/9 (LP99) and panSMARCA/PB1(5) (PFI-3) (see http://www. thesgc.org/chemical-probes/epigenetics). The currently available probes represent a good coverage of chemical tool compounds for this family of epigenetic effector domains and demonstrate the feasibility targeting these interaction domains that have not been considered as druggable targets a few years ago. However, if any of the published inhibitors will be developed into an approved pharmaceutical remains to be shown. Clinical trials on BET inhibitors have been initiated recently.

Fig. 7 Bromodomain chemical probes. Shown is a phylogenetic tree (centre) and the currently available chemical probes developed by the SGC (http://www.thesgc.org/chemical-probes/ epigenetics). Targets that are inhibited are shown in red. The structure of the BRD9/7 inhibitor LP99 and the panBRPF inhibitors is currently undisclosed.

Methyl-lysine/arginine reader domains have also recently been targeted but the diversity of this large protein interaction module family and the binding site properties of most methyl-lysine/arginine readers render chemical probe/inhibitor development more challenging. One of the first methyl-lysine epigenetic reader domains that have been targeted was L3MBTL3, a member of the malignant brain tumour (MBT) family of chromatin-interacting transcriptional repressors. Optimiza- tion of weaker starting points (Herold et al. 2011b) led to the discovery of UNC1215 as a potent and selective chemical probe for L3MBTL3 (James et al. 2013b). This chemical probe binds with a KD (dissociation constant) of 120 nM and has excellent selectivity for the targeted reader domain. Biophysical analysis revealed that UNC1215 has a methyl-lysine competitive binding mode, effectively displacing dimethyllysine-containing peptides from the L3MBTL3 binding site.

Recently, another potent methyl-lysine reader domain has been reported. OICR- 9429 is a selective chemical probe for WDR5, a protein that is present in several chromatin regulatory complexes including the MLL1 (mixed lineage leukaemia 1) complex. OICR-9429 binds to WDR5 with KD values of 24 nM (Biacore) and 52 nM (ITC) and was found to be more than 100 selective over other chromatin ‘reader’ domains, methyltransferases and other non-epigenetic targets (Fig. 8).

Fig. 8 Chemical probes targeting methyl-lysine reader domains

A recent druggability analysis of the methyl-lysine/arginine family of reader domains suggested that there are many additional opportunities for the development of selective and potent inhibitors. Improved druggability of these domains may be achieved targeting adjacent pockets. For instance, the methyl-lysine binding site present in CBX is largely extended by a channel that harbours flanking peptide sequences. The ankyrin repeat protein GLP is another example for extended binding sites that lead to improved druggability scores. A mono- or dimethyl containing sequence is anchored into a central cavity containing a typical aromatic cage. The duggability of this pocket is poor (Dscore 0.64; PDB code, 3B95); however, considering two adjacent pockets less than 7 A˚ apart improved the
druggability score to Dscore 0.98 (Herold et al. 2011a). Methyl-lysine reader domains are often mutated in cancer and genetic diseases suggesting that targeting these domains may be beneficial for a number of disease applications.

Recent reports strongly suggest that many protein–protein interactions that are mediated by localized interactions are highly druggable and that potent and bioac- tive inhibitors can be identified. Some of the developed inhibitors are now also tested in phase I/II clinical trials, and it is likely that many more protein–protein interaction inhibitors will be identified and developed in the near future. The development of bioactive inhibitors that target larger interfaces remains however very challenging, and this area would require additional intensive research efforts before potent bioactive inhibitors will be identified and tested clinically.

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