TARGET PRODUCT PROFILE

Target product profiles (TPP) define the requirements for a drug molecule to be clinically applicable. These include features such as the acceptable route for dosing the compound, the duration of treatment, the patient population, cost and acceptable range of toxicity. These are defined by clinicians and public health agencies who understand the clinical needs of the patients (Wyatt et al. Reference Wyatt, Gilbert, Read and Fairlamb2011). An example of a TPP for HAT, defined by DNDi is shown in Table 1. The TPP needs to be regularly reviewed, as it may well change due to other treatment options becoming available, changes in the clinical situation or drug resistance to existing compounds becoming a significant issue.

Table 1. Example of a new stage 1+2 target product profile (produced by DNDi)

http://www.dndi.org/diseases-projects/diseases/hat/target-product-profile.html

The target product profile will inform drug discovery projects from the very start. This helps to define both the molecular target and chemical matter that is developed during a drug discovery programme.

Thus, the molecular target must be able to fulfil the TPP. For example, in the case of HAT, the molecular target must: (1) be essential for viability of the parasite in the human host; (2) have a rapid cidal effect when inhibited and (3) be amenable to inhibition by small drug-like molecules that have the correct physiochemical properties to be orally bioavailable and cross the blood-brain barrier.

Similarly, the molecules that are for screening must be capable of fulfilling the TPP once optimized through the hits to lead and lead optimization phase. In the case of HAT for example, there is a need for orally bioavailable compounds; thus compounds that are selected should have the appropriate physicochemical and pharmacokinetic properties for oral bioavailability.

Therefore in target-based drug discovery, in order to fulfil the TPP, both the molecular target and the chemical matter must be carefully selected, and their selection actually go hand-in-hand.

SELECTION OF DRUG TARGETS

The selection of a target for drug discovery is of key importance. A target is considered truly validated when it is in the clinic for treating human diseases. In the case of HAT (and the other kinetoplastid diseases), there is really only one target fully validated to this extent, ornithine decarboxylase. This is the target of the drug eflornithine (DFMO), which is used for the treatment of HAT. There are other drug targets which have significant levels of validation though; for example, protein farnesyltransferase (Eastman et al. Reference Eastman, Buckner, Yokoyama, Gelb and Van Voorhis2006; Gillespie et al. Reference Gillespie, Yokoyama, Lu, Eastman, Bollinger, Van Voorhis, Gelb and Buckner2007) and S-adenosylmethionine decarboxylase (Jacobs et al. Reference Jacobs, Nare and Phillips2011; Velez et al. Reference Velez, Brautigam and Phillips2013). Another target for HAT is N-myristoyltransferase (NMT) that we have pursued at the Drug Discovery Unit (DDU) at the University of Dundee (Frearson et al. Reference Frearson, Brand, McElroy, Cleghorn, Smid, Stojanovski, Price, Guther, Torrie, Robinson, Hallyburton, Mpamhanga, Brannigan, Wilkinson, Hodgkinson, Hui, Qiu, Raimi, Van Aalten, Brenk, Gilbert, Read, Fairlamb, Ferguson, Smith and Wyatt2010; Brand et al. Reference Brand, Cleghorn, McElroy, Robinson, Smith, Hallyburton, Harrison, Norcross, Spinks, Bayliss, Norval, Stojanovski, Torrie, Frearson, Brenk, Fairlamb, Ferguson, Read, Wyatt and Gilbert2012).

Current methods for target-based drug discovery usually involve screening diverse or focused libraries against the molecular target. The hits are then characterized and optimized through rounds of design, synthesis and testing against protein and cell. The testing usually includes assays for potency, selectivity and pharmacokinetic properties. In terms of target validation, it is important to prove that the compounds are acting on-target, and that it is inhibition (or modulation) of the target that is causing the death of the parasite. The compounds are then assayed in animal models of infection. It is only at this stage that it becomes clear if the proposed drug target has a reasonable degree of validation. In other words, a very considerable amount of time and resource will be consumed before there is reasonable evidence that the target chosen is a valid drug target. Therefore it is important to choose drug targets very carefully at the outset of a drug discovery programme.

TRAFFIC LIGHT SYSTEM

At the DDU at the University of Dundee, we have carried out target-based drug discovery programmes against HAT. We use 6 criteria to help us select molecular targets to enter our drug discovery pipeline. The criteria are: essentiality, druggability, assayability, resistance potential, toxicity potential and structural information. To help compare between the different targets, we have established a ‘traffic light’ scoring system for the different criteria, as reported (Frearson et al. Reference Frearson, Wyatt, Gilbert and Fairlamb2007; Wyatt et al. Reference Wyatt, Gilbert, Read and Fairlamb2011). Essentially each criterion is given a green, amber or red score, according to a number of definitions, which are summarized in Table 2. This provides a rapid visual method to assess an individual target and to compare between different potential drug targets. The assignment of a ‘red’ score does not necessarily mean that this is a no-go situation; it could just indicate a lack of information. There are also clear cases for a no-go decision – for example, if it is shown that the target is not essential for survival of the parasite.

Table 2. Criteria for molecular targets developed at the DDU (Frearson et al. Reference Frearson, Wyatt, Gilbert and Fairlamb2007; Wyatt et al. Reference Wyatt, Gilbert, Read and Fairlamb2011)

PROPERTIES OF MOLECULES

In terms of drug discovery, although it is important to have a good molecular target, it is equally important to develop the molecules with the correct properties to achieve the TPP. The TPP allows criteria to be set for compounds for the different stages of the drug discovery programme in terms of potency, selectivity, pharmacokinetics and safety pharmacology. Given that during the discovery of a clinical candidate, the molecular weight and lipophilicity are likely to increase (Nadin et al. Reference Nadin, Hattotuwagama and Churcher2012), this should be taken into account when choosing the starting chemical library – compounds must be amenable to increases both in lipophilicity and molecular weight during optimization without compromising developability.

The TPPs for the kinetoplastid diseases generally require oral drugs. Lipinski defined his famous ‘rule of 5 as a guide as to whether a molecule is in the correct chemical space to be an orally bioavailable compound (Lipinski et al. Reference Lipinski, Lombardo, Dominy and Feeney1997). In his rules, compounds should have a molecular weight of less than 500, clogP <5, less than 5 hydrogen bond donors and less than 10 hydrogen bond acceptors (Lipinski et al. Reference Lipinski, Lombardo, Dominy and Feeney1997). Current thinking suggests that we should be even more stringent about the physicochemical properties of molecules. Analysis of drug molecules indicates for oral drugs, the average molecular weight is 333 and the average clogP is 2·5 (Gleeson et al. Reference Gleeson, Hersey, Montanari and Overington2011). LogP in particular appears to be correlated to a number of developability issues, including toxicity, metabolic instability and insolubility (Leeson and Springthorpe, Reference Leeson and Springthorpe2007; Hughes et al. Reference Hughes, Blagg, Price, Bailey, Decrescenzo, Devraj, Ellsworth, Fobian, Gibbs, Gilles, Greene, Huang, Krieger-Burke, Loesel, Wager, Whiteley and Zhang2008; Hann, Reference Hann2011). Recent publications suggest an ideal clogP value lies in the range of 1–3. In addition to clogP and MW, analysis indicates that other criteria are important in prospective drug candidates. (1) The 3-dimensionality is important in drug-molecules. Thus the proportion of sp³ carbon atoms in a drug is 0·47 (Lovering et al. Reference Lovering, Bikker and Humblet2009). Analysis indicates that in early drug discovery, the average proportion of sp³ carbon atoms is 0·36 and this gradually rises along the drug discovery pathway. The 3-dimensionality helps properties of molecules such as solubility by decreasing planarity and gives access to more chemical space. (2) The number of carbocyclic aromatic rings impacts negatively on the developability of molecules (Ritchie and MacDonald, Reference Ritchie and MacDonald2009; Ritchie et al. Reference Ritchie, MacDonald, Young and Pickett2011). The number of carbocyclic aromatic rings is correlated with lower solubility, higher logD, higher plasma protein binding, hERG inhibition and inhibition of some major cytochrome P450s (3A4, 2C9, 2C19). (3) For compounds required to reach the CNS, there are additional criteria. Wager et al. at Pfizer have carried out a thorough analysis of properties of compounds that have CNS penetration (Wager et al. Reference Wager, Chandrasekaran, Hou, Troutman, Verhoest, Villalobos and Will2010a, Reference Wager, Hou, Verhoest and Villalobosb). This suggested a number of ‘optimum’ values for a number of factors – for example: clogP=2·8; MW=305; Polar surface area (PSA)=45; HBD=1; pKa=8·4.

In addition to the physicochemical properties of molecules, many research laboratories filter out functional groups known to be chemically reactive or toxic (Brenk et al. Reference Brenk, Schipani, James, Krasowski, Gilbert, Frearson and Wyatt2008). In some cases this is a clear-cut decision. For example, acyl chlorides are too unstable to be developed. In other cases, the decisions about which groups to include and which to exclude are less clear. Therefore, excluding certain functional groups is a careful balancing act; excluding functional groups can make the compounds ‘cleaner’, but reduces the chemotypes covered. In some cases this becomes a judgement call, balancing the risks of certain chemical functionality against the risks of restricting the chemical space covered in a library. For example, nitro groups may be excluded due to potential genotoxicity concerns, although some drugs have aromatic nitro groups. It is important to understand the risk inherent in certain types of functional groups and to address this risk as early as possible in the discovery process, if it is decided to include these.

PROPERTIES OF CURRENT DRUGS USED FOR TREATMENT OF KINETOPLASTID INFECTIONS

Interestingly, if the current drugs used for the treatment of kinetoplastid diseases are analysed, it is clear that not many of them are ideal from a physicochemical perspective, given that the current TPPs for the kinetoplastid infections require oral drugs. Indeed most of the drugs are not orally bioavailable and require parenteral administration, as would be predicted by their physicochemical properties (Table 3 and Fig. 1). The only orally bioavailable drugs are nifurtimox, benznidazole and miltefosine. The former two are compliant with Lipinski's rules and whilst miltefosine has a logP value of >5, miltefosine is zwitterionic and the clogD value is 1·5 which is well within the Lipinski criteria. Eflornithine theoretically fulfils Lipinski's rules; however the compound is so hydrophilic (clogP=−3·7) that it is very unlikely to traverse membranes well by passive diffusion and is actually given by intravenous infusion.

Fig. 1. Current drugs used for the treatment of kinetoplastid infections.

Table 3. Physicochemical properties of currently used drugs for kinetoplastid infections

^a clogP was calculated using StarDrop™.

^b HDB and HBA are given as defined by Lipinski (Lipinski et al. Reference Lipinski, Lombardo, Dominy and Feeney2001).

^c The precise structures of stibogluconate and meglumine antimoniate are not known.

Interestingly melarsoprol, nifurtimox and eflornithine are used for the treatment of stage 2 HAT, suggesting that the compounds are blood-brain barrier penetrants. Melarsoprol and nifurtimox are not predicted to be blood-brain barrier penetrants based on normal criteria as their polar surface areas are too high (Kelder et al. Reference Kelder, Grootenhuis, Bayada, Delbressine and Ploemen1999). Eflornithine has a marginal level of PSA, but the negative clogP and zwitterionic nature of the molecule suggest that it is not likely to be passively permeable. The uptake of these molecules is likely to be due to transporter-mediated uptake, which is very difficult to predict or plan for.

Whilst clearly there are compounds that are suitable as drug candidates that do not meet criteria such as Lipinski's rules, these mark the exception rather than the rule and the exceptions are difficult to predict as they involve factors such as transporter-mediated uptake. Therefore the physicochemical properties of molecules should be taken into account during the design process.

N-MYRISTOYLTRANSFERASE (NMT)

By way of example, in the DDU (University of Dundee), in collaboration with others, we have provided very strong validation of NMT as a drug target for HAT (Frearson et al. Reference Frearson, Brand, McElroy, Cleghorn, Smid, Stojanovski, Price, Guther, Torrie, Robinson, Hallyburton, Mpamhanga, Brannigan, Wilkinson, Hodgkinson, Hui, Qiu, Raimi, Van Aalten, Brenk, Gilbert, Read, Fairlamb, Ferguson, Smith and Wyatt2010; Brand et al. Reference Brand, Cleghorn, McElroy, Robinson, Smith, Hallyburton, Harrison, Norcross, Spinks, Bayliss, Norval, Stojanovski, Torrie, Frearson, Brenk, Fairlamb, Ferguson, Read, Wyatt and Gilbert2012). I will briefly summarize its validation process below.

NMT is an enzyme found in all eukaryotic cells; its function is co-translational modification of a number of proteins, by N-myristoylation of their N-terminal glycine atoms. NMT has been investigated as a potential target for fungal diseases by a number of companies; but, despite discovery of some very potent inhibitors, none of these have been progressed to market.

Within T. brucei, NMT has been genetically validated initially by RNAi experiments by Price et al. (Reference Price, Menon, Panethymitaki, Goulding, McKean and Smith2003), and then kinetically characterized by Panethymitaki et al. (Reference Panethymitaki, Bowyer, Price, Leatherbarrow, Brown and Smith2006) in York. We set about validation of NMT as a target in T. brucei. It was then established that when NMT is knocked-down by RNAi, the parasites are unable to establish an infection in mice, adding further genetic validation (Price et al. Reference Price, Guther, Ferguson and Smith2010). To obtain chemical validation, at the DDU, we profiled a number of reported inhibitors of fungal NMT against T. brucei NMT (Brand et al. Reference Brand, Cleghorn, McElroy, Robinson, Smith, Hallyburton, Harrison, Norcross, Spinks, Bayliss, Norval, Stojanovski, Torrie, Frearson, Brenk, Fairlamb, Ferguson, Read, Wyatt and Gilbert2012); but none of these showed significant inhibition of the T. brucei enzyme. We therefore carried out a high-throughput screen using a 62 000 compound diversity library (Brenk et al. Reference Brenk, Schipani, James, Krasowski, Gilbert, Frearson and Wyatt2008) from the DDU. This led to identification of a series of pyrazole sulphonamides as hit molecules, with the most potent compound having an IC₅₀ value of 1·9 μ m (Fig. 2). These were optimized to a potent inhibitor, DDD85646 with an IC₅₀ value of 0·002 μ m against T. brucei NMT and EC₅₀ value of 0·002 μ m against the parasite. We were then able to show that the compounds were active in rodent models of infection, both for T. b. brucei (curative at 12·5 mg/kg po bid for 4 days) and T. b. rhodesiense (curative at 50 mg/kg po bid for 4 days).

Fig. 2. Compounds active against T. brucei NMT.

In order to validate that these compounds were acting against NMT, a number of experiments were undertaken. These have been reported previously (Frearson et al. Reference Frearson, Brand, McElroy, Cleghorn, Smid, Stojanovski, Price, Guther, Torrie, Robinson, Hallyburton, Mpamhanga, Brannigan, Wilkinson, Hodgkinson, Hui, Qiu, Raimi, Van Aalten, Brenk, Gilbert, Read, Fairlamb, Ferguson, Smith and Wyatt2010), but are summarized here to show how they are important in validation of the target. (1) It was possible to obtain a co-crystal structure of the inhibitor DDD85646 with the T. brucei NMT homologue, Leishmania major NMT. This shows the inhibitor bound in the enzyme active site, occupying the peptide binding pocket. This is evidence that the compound can bind to the enzyme and that it binds in the substrate binding pocket. (2) We looked at a large number of inhibitors of T. brucei NMT and compared them to inhibition of parasite growth. There was a very good correlation between inhibition of the enzyme and inhibition of parasite growth, which is a good indication that inhibition of TbNMT is responsible for the death of the parasites. (3) The gene encoding for NMT was over-expressed within the parasite T. brucei by about 5-fold. This gave rise to an 8-fold change in potency from 2·1 to 17 nm. This indicates that the compounds are inhibiting NMT and that cell death is due to inhibition of NMT. (4) A further experiment was to incubate the parasites with radio-labelled myristic acid. This is incorporated into proteins through myristoylation, which could be checked by SDS-PAGE. When the experiment was repeated in the presence of DDD85646, myristoylation is prevented and no radio-labelled proteins could be detected. This is good evidence that in cells, NMT inhibitors prevent myristoylation of proteins.

Therefore, through a suite of experiments, we have obtained good evidence that NMT is a valid drug target for HAT (Frearson et al. Reference Frearson, Brand, McElroy, Cleghorn, Smid, Stojanovski, Price, Guther, Torrie, Robinson, Hallyburton, Mpamhanga, Brannigan, Wilkinson, Hodgkinson, Hui, Qiu, Raimi, Van Aalten, Brenk, Gilbert, Read, Fairlamb, Ferguson, Smith and Wyatt2010). The target has been genetically validated through RNAi both in cells and in a mouse model of infection. We have demonstrated that our compounds inhibit NMT through binding to the peptide binding pocket of the enzyme. Inhibition of NMT prevents myristoylation of proteins in cells and leads to cell death of T. brucei. When dosed to mice orally, at sufficient levels to give unbound blood levels at EC₉₉, these NMT inhibitors cure animal models of HAT.

In terms of physicochemical properties, these compounds are appropriate for oral bioavailability: i.e. MW=495; cLogP=3·1; HBD=2; HBA=5. However, DDD85646 does not have the correct physicochemical properties for CNS penetration, and indeed it is not active in a CNS model of infection. Interestingly when we started this project, there was a TPP for oral stage 1 compounds for HAT but during the work on this project, the priority was changed to requiring a TPP activity in the CNS stage 2 of infection. This demonstrates how TPPs can alter and the effect that they can have on the types of molecule that are required for the indication.