designing innovative therapies for neuropathic pain: pros and cons of target-based drug discovery

Inglese Journal of the Peripheral Nervous System 19(Supplement):S2–S9 (2014)

Designing innovative therapies for neuropathic pain:pros and cons of target-based drug discovery

J Inglese1,2

1National Center for Advancing Translational Sciences; and 2National Human Genome Research Institute, National Institutesof Health, Rockville, MD

Among the many challenges facing academic transla-tional research and the pharmaceutical industry is theneed for better pre-clinical models to validate drug tar-gets (Plenge et al., 2013). Data have shown that mostfailures during phase II trials are due to a lack of efficacy(50%) or noted toxicity (25%) (Kola and Landis, 2004;Arrowsmith, 2011). Targeting a specific protein’s func-tion is generally the most effective means to approachwhen developing a pharmacological basis to modify adisease. Establishing a therapeutic hypothesis utilizinga target-based approach ideally requires that the targetis clinically validated, mechanistically and structurallyelucidated, assayable, pharmacologically tractable, andfunctionally indistinguishable from the cellular context.The number of these validation criteria required in aprogram is dependent on what are considered accept-able levels of program risk proportionate to the overallstrategy (Table 1).

Clearly the establishment of a dose–response rela-tionship between the target and efficacy in humans,also known as “clinical validation,” is a highly desir-able piece of evidence to have though often difficultto obtain. Evaluating the relevance of a specific tar-get can be estimated in some cases by examiningthe relationship or direct involvement in rare inheriteddiseases. For example, cystic fibrosis (CF) is due tomutations in the CF transmembrane conductance reg-ulator (CFTR) protein. In one form of the disease amissense mutation (G551D) results in a dysfunctionalCFTR protein that is expressed on the epithelial cellsurface. Ivacaftor, a CFTR potentiator and approved

Table 1. Molecular target validation criteria.

Clinically validated Satisfies the therapeutic hypothesis from sources of evidence that caninclude approved drugs, epidemiology, in vivo biomarkers, and genetics

Mechanistically and structurallyelucidated

Sufficient knowledge underlying the reaction and/or binding interactionmediated by the target

Assayable/pharmacologically tractable Target’s function/binding interactions can be modulated by an agentdisplaying a structure-activity relationship (SAR)

Functionally indistinguishable from thecellular context

The measured (assayed) target activity or binding properties have afunctional link to relevant cell-based or in vivo activity

drug developed by Vertex and the CF Foundation,partially restores the chloride transport activity of theimpaired protein resulting in clinical benefit.

On the other hand, while a target protein maybe highly correlated to disease through mutation orderegulated expression, this protein may be refrac-tory toward direct pharmacological intervention. Forexample, type 1A Charcot-Marie-Tooth peripheral neu-ropathy occurs in individuals harboring a duplicationof the gene for the peripheral myelin protein 22(Pmp22), and haploinsufficiency of this gene results inHNPP (hereditary neuropathy with liability to pressurepalsies). While in this case a target is clearly linked todisease and the nature of the disease is dependenton gene dosage, the unknown structure and functionof this membrane-spanning protein precludes it frommany of the current approaches used to identify a phar-macological means to directly affect the function ofPMP22. Other sources of evidence that may be usefulwhen attempting to establish clinical validity are listedin Table 1 (Dietz, 2010; Fishman, 2013).

Rational drug design

In many cases, mechanistic and structural knowl-edge underlying the enzymology or binding interactionmediated by the target are sufficient to enable com-pound ligands to be rationally designed and optimiza-tion through medicinal chemistry (Marmorstein, 2001).Such pharmacological tractability needs to be accom-panied by a robust structure–activity relationship(SAR) where ligand structures display a quantifiable

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1. Saquinavir (1995) 2. Indinavir (1996)

catalytic mechanism x-ray structure resistance mechanism

3. Darunavir (2006)

improved potency improved effectiveness1st in class

Figure 1. Rational drug design.

relationship to target activity and/or binding affin-ity. Families of compounds having a high degree ofstructural similarity are known as a chemotype. For asubset of cases, a chemotype can become the basisfor a clinically useful therapeutic agent. For example,development of human immunodeficiency virus (HIV)protease inhibitors based on the peptidomimetichydroxyethylamine transition state analog chemotypewere the first drugs to treat HIV/acquired immunode-ficiency syndrome (AIDS) and represents a triumph ofrational drug design (Fig. 1).

Determining that a target isolated from its patho-physiological context is functionally indistinguishablefrom that cellular context when incorporated into anassay is an important assessment to make. This canbe challenging for targets which are studied as specificdomains obtained from modular proteins often rep-resented in numerous proteins, for example, amongthose involved in epigenetic regulation (Chung et al.,2011).

Rational lead discovery

There are cases where despite a detailed knowl-edge of the structure, catalytic, and regulatory mech-anisms of a protein target, the design of a smallmolecule to achieve a specific mode of target activitymodulation is not obvious. However, for an assayabletarget the function or a binding interaction can oftenbe measured in a format amenable to high-throughputscreening (HTS) of chemical libraries or other ligand dis-covery approaches such as biophysical or affinity selec-tion methods.

For instance, there is a great deal of structuraland mechanistic knowledge surrounding the potentialanticancer target pyruvate kinase M2 (PKM2). In can-cer, it was discovered by the Cantley lab that PKM2interacts with oncogenic phosphotyrosine-containingproteins which by inhibiting PKM2 activity alters glu-cose metabolism in a manner beneficial to rapidly divid-ing cells. This occurs by promoting an increase in theavailability of glycolytic intermediate metabolites thatsupport cell proliferation, a result of the so-called War-burg effect. Therefore, the idea of finding molecules

that can activate PKM2 might be potentially useful asanticancer agents by restoring normal glycolytic oxi-dation of glucose and depleting the supply of gly-colytic intermediates needed to build cellular proteinsand nucleic acid (Anastasiou et al., 2012). There areknown binding sites for cellular activators of PK, forexample, fructose-1,6-bisphosphate (FBP). However,the problem with this allosteric binding site on PK isits overlap with the site of oncogenic phosphoproteinbinding which suppresses the activating effect of FBP.Therefore, an alternate means of PKM2 activation topromote a constitutively active enzyme state thatis resistant to inhibition by tyrosine-phosphorylatedproteins is needed.

To search for a novel allosteric binding sitethat can induce an activation of PKM2, an unbi-ased HTS approach was taken. From this worktwo new chemotypes, which formed the start-ing points for the optimized compounds TEPP-46(6-((3-Aminophenyl)methyl)-4-methyl-2-methylsulfinylthieno[3,4]pyrrolo[1,3-d]pyridazin-5-one; MW=372.46)and DASA-58 (3-[[4-(2,3-dihydro-1,4-benzodioxin-6-ylsulfonyl)-1,4-diazepan-1-yl]sulfonyl]aniline; MW= 453.53),were identified which subsequently were determinedby x-ray crystallography to bind at a unique subunitinterface of the PKM2 tetrameric protein (Fig. 2).These new activators of PKM2 are not susceptibleto phospho-oncogenic protein displacement and rep-resent novel leads for investigating a new approachto cancer therapy. This chemotype discovery wasaccomplished by a technologically enabled screeningapproach known as quantitative HTS which examinesthe activity of hundreds of thousands of chemicalsubstances assessed over a broad range of concen-trations (Inglese et al., 2006).

Screening of this type requires advanced roboticand computational algorithm automation to generateand analyze millions of experimental data points. Fromthe analysis of the resultant concentration responsecurves pharmacological parameters such as EC50,efficacy, and nascent SAR are derived and used fordelineated chemotype activity and prioritization forfollow-up interrogation in secondary assays. Assays

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N

S

N

H2N

N

O

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O

O

SN

O O

NSOO

NH2

FBP

*

* *

O

OHO

P+ ADP ATP +

O

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TEPP-46

DASA-58

Figure 2. Two new chemotypes, which formed the starting points for the optimized compounds TEPP-46 and DASA-58.

Figure 3. Orthogonal pooling of compounds (also known as a self-deconvoluting matrix).

designed for HTS attempt to incorporate biologicalfidelity with high-sensitivity. The balance in the assaydesign is often between the physiological or patho-physiological relevance that can be captured by aparticular assay format and the automation compat-ibility needed to screen hundreds of thousands ofcompounds.

There are a broad range of assays applicable tomodern drug discovery that can be adapted to the lowassay volume (4–8 μl per well) 1,536 well microtiterplate format which is widely employed in HTS (Ingleseet al., 2007). For the accurate delivery of low volumereagents and compounds to these plate formats spe-cialized reagent dispensers and library compoundtransfer technologies are used (Inglese and Auld,2008). Regardless of the complexity of the biology or

the target under interrogation, the protocols have tobe standardized with a minimum number of steps;therefore, considerable familiarity with both the biol-ogy under investigation and the assay developmentand screening process are required. Given the criticalnature of this basic-to-translational research interface,it is important to engage, ideally in the form of collab-oration, experts from both the disease biology and theassay development and screening disciplines.

Variations on HTS

Most HTS uses a one compound, one wellapproach, which tests each compound individually.Another option, however, is orthogonal pooling ofcompounds, also known as a self-deconvolutingmatrix (SDM, Fig. 3) strategy where as many as 10

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or more compounds per well are arrayed (Kainkaryamand Woolf, 2009). This is most useful for biochemi-cal target-based assays when automation, biologicalreagents, and resources are limited. SDM would beless advisable in cell-based assays because of thepotential higher toxicity to cells and variety of activ-ities coming from any one of the compounds in thecomplex environment of a cell (Motlekar et al., 2008).

Fragment-based screening uses low molecularweight fragments to search for low affinity interactionsthat can form the basis of an optimizable chemical scaf-fold. This approach relies heavily on biophysical meth-ods such as nuclear magnetic resonance and x-ray crys-tallography. These are low-throughput approaches, sothe fragment-based libraries are generally significantlysmaller than those used in the example describedabove. If this approach works the way it was orig-inally envisioned, fragments that bind to differentsites could potentially be combined to generate farmore potent molecules based on a decreased entropypenalty for binding of a single agent or cooperativebinding between the components, however, the morecommon practice is to ‘grow’ the low MW ligand toimprove binding by picking up additional protein–ligandinteraction.

A different tack on screening is embodied in affin-ity selection of peptides and modified peptide ligandsprepared as vast libraries using in vitro transcriptionand translation methods. Here, an immobilized targetis used to capture and enrich high affinity peptideswhich, by virtue of their puromycin-tethered encodingmRNA, can be amplified and further enriched (Robertsand Szostak, 1997). Currently the peptidic nature of theligands that are obtained from this approach is consid-ered a limitation to their translational potential, thoughadvances in the incorporation of non-proteinogenicamino acids, macrocyclization, and post-selectionmodifications are being developed to engender thesepeptides with more drug-like character (Hipolito andSuga, 2012).

In conclusion, more than 90% of the compoundsthat enter clinical trials fail to demonstrate efficacyand to gain regulatory approval (Plenge et al., 2013).This suggests systemic problems in the process, oneof which is a failure on the part of pre-clinical mod-els to predict a clinical benefit of new therapeuticagents. Improving the methodology of testing thera-peutic hypotheses and target validation practices mayimprove this process and facilitate a more efficientdrug development strategy. However, a broader real-ization that drug discovery and development remains aresearch-driven enterprise, which along its continuumwould allow and encourage generally applicable solu-tions to many recurring issues in the process.

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designing innovative therapies for neuropathic pain: pros and cons of target-based drug discovery

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