Bioisosterism has played a central role in thedevelopment of drug molecules almost from theoutset of the pharmaceutical industry. The promise ofbioisosterism is that the properties of a compound canbe fine-tuned without affecting its underlyingbiological activity. This promise is not howeverwithout its challenges. Successfully applyingbioisosterism to achieve the desired molecularoutcome is difficult because of the fundamentalproblem that chemical structure is an unreliableindicator of biological activity. Small changes in amolecule can have a profound impact on acompound’s activity, specificity and toxicity, whilstcompletely different chemotypes may have nearidentical biological activity profiles. More rigorousand reproducible methods for suggesting relevant,

non-obvious and yet synthetically intuitivebioisosteres would have wide applicability.

BIOISOSTERES IN DRUG DISCOVERY

Bioisosteres are used by researchers throughout thepharmaceutical industry to find new hits and leads bymodifying known actives or substrates, to develop leadsby modifying physicochemical properties and protectingtheir knowledge using patents.

Traditional drug discovery had bioisosterism at the coreof its strategy for finding hits and leads. Havingidentified an interesting target, researchers often hadlittle choice in finding an active inhibitor or antagonist,except through bioisosteric modification of the natural

Molecular fields give a richer, more intuitive view of active molecules.Application of this technology to the search for bioisosteres results inrelevant, non-obvious suggestions that make a significant impact on the drug development process.

By Tim Cheeseright at Cresset BioMolecularDiscovery

The Identification of Bioisosteres as Drug Development Candidates

Figure 1: Nicotine (shown in green)bioisosteres (3) shownusing ‘field points’. The field point patternsfor these single digitnanomolar actives at α4β2 neuronal nicotinicreceptor are clearly highly similar

ligand in a systematic and thoughtful manner. Themodern HTS era has provided plenty of potential leads,but the need for bioisosteres remains. Actives foundthrough HTS can have undesirable properties (eitherphysical or biological) and often lack novelty.

Once hits are found, the principle of bioisosterism iscentral to converting the hit into a more useful lead ordrug. Medicinal chemists make biologically relevantchanges to the active structure that result in betterphysicochemical properties or that move the hits intodesirable intellectual property (IP) space, facilitatingtheir move from hits to leads. During lead optimisation,the lead molecule may be ‘tweaked’ to introduce thecorrect activity/selectivity profile combined with goodphysicochemical properties. More radically, replacementof core groups in the lead series with new scaffolds thatintroduce better selectivity or physical properties canrescue what would otherwise be a failed project.

Having successfully negotiated the maze of drugdiscovery, companies often need to find a backup seriesthat uses all the knowledge gained in a successful projectbut has unique IP and/or better off-target effects. Thechallenge is to introduce significant structural diversitywhilst at the same time retaining as much of theinformation gained as possible. Bioisostere substitution isan obvious route to achieving this.

The requirement to protect research positions throughpatent applications is critical for the development of newmedicines. In this respect, IP protection is probably themost important use of bioisosteres in the modern drugdiscovery project. Chemical structures derived from acommon scaffold are often used as surrogates for truebioisosteres to define a range of chemical space that isdeemed to have a common biological activity. However,due in large part to the inadequacy of structure todescribe biological activity, there are numerous exampleswhere the definitions in a patent are not sufficient toprevent competitors from finding closely related drugdevelopment candidates. This has led on many occasionsto significant reductions in the value of the originalapplication. Clearly, more automated methods foridentifying biologically relevant bioisosteres would be ofsignificant value in the definition of IP space.

EXISTING APPROACHES TO FINDING NOVEL BIOISOSTERES

The application of bioisosterism in the current drugdevelopment process is widespread but also somewhat ad hoc, relying on the experience of medicinal chemists

to invent new molecules. It is consequently firmlygrounded in the chemical structure descriptions of activemolecules, resulting in only small evolutionary jumps in the underlying chemistry. This limits the testedchemical space to closely related structures, potentiallysynthesising and testing inactive structures and, moreimportantly, missing a range of interesting candidateswhose structures are not closely related to the lead.

A method for describing molecules in a manner morerelated to their biological activity would have thepotential to explore much broader sets of chemical space,suggest a wider range of modifications and enableresearch projects to progress faster. There has beenincreasing interest in methods that can suggest relevant,non-obvious, accurate bioisosteres to project teams. Thesemethods broadly fall into two categories: knowledge-based approaches and computational techniques.

Knowledge-based approaches tend to use data miningtechniques to find moieties that have previously beensubstituted for each other without a significant change inthe activity under study. Thus the replacement of arginineby benzamidine in the Thrombin and Factor Xa projects ofthe 1990s would register benzamidine and arginine asbioisosteric. This approach has broad appeal; it canhighlight changes that are known to be successful togetherwith detailed examples. However, many replacements arespecific to a particular protein and take no account of whichparts of the moiety to be replaced are most important foractivity. Equally, if the moiety to be replaced is not presentin the literature then no suggestions are possible.

The variety and availability of computer algorithms to suggest bioisosteric replacements has increasedsignificantly in recent years. Most methods attempt toexcise a chosen moiety from a molecule and replace itwith a fragment from a fragment database. Thesefragments are typically scored against the moiety to bereplaced using shape or electrostatic measures ofsimilarity, or by using the presence or absence ofpharmacophore points.

All of these methods seem to suffer the same problem:they rarely suggest truly novel scaffolds to theexperienced medicinal chemist. The reason for this is notclear but the one commonality is that thesecomputational methods, like the literature methods, relyon fragment-to-fragment comparison. In this method,the replacement fragment is scored as an isolatedmolecule in itself, and not in the context of the finalmolecule. This is a subtle but critical problem, whichmeans that there is no possibility for the properties of theIn

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fragment to influence the properties of the finalmolecule. Moreover, as the final molecular context of thefragment is not considered, fragments that mightrepresent only a small change to the final molecule in itsentirety may be scored poorly because they represent alarge change when scored at the fragment level.

NEW APPROACHES

In this journal in 2007, we outlined the Field Pointapproach to describing molecules (1). We showed how,by encoding the electrostatic environment surrounding aligand, it could be viewed in a more insightful andinformative way. We showed how distilling the full fielddown to a series of ‘hot spots’ around the molecule –termed ‘Field Points’ – provided both a powerful insightinto the behaviour of molecules and a mechanism by which molecules could be compared in acomputationally efficient and therefore rapid manner.

Field Point descriptions of molecules have been usedwidely to provide richer, more informative views of theway in which ligands interact with proteins, to inter-relate compounds from different chemical series that actat the same protein site, to find novel chemical seriesthrough virtual screening, and to decode StructureActivity Relationships (SAR) by comparing molecules asproteins ‘see’ them (2). Field Point technology can alsoprovide a much more accurate basis on which to identifynovel, chemically relevant bioisosteres.

Application of Molecular Fields to Bioisostere FindingThe principle behind fragment replacement methods toidentify bioisosteres is simple: remove a portion of anactive molecule, search a fragment database for areplacement moiety that will physically fit into thevacated space, and score the replacement for similarity tothe original. In practice, a number of factors contributeto the effectiveness of the method. Primary amongstthese are the accurate scoring of potential replacements,the relevance of the fragment database, and theoriginality and synthesisability of the suggestedbioisosteres.

In scoring replacement fragments, it is essential toremember that the molecular fields around them are aproperty of the whole molecule and not of the isolatedfragment. Replacement fragments cannot be assessedaccurately in isolation from the whole molecule as theycan have a significant effect on the retained portions ofthe molecule. Not only will fragments that are stronglyelectron donating have different effects from ones thatare electron withdrawing, but the context of themolecule into which they are placed will determine theextent and character of those effects.

To this end, we chose to join replacement fragmentsinto the retained portions of the target, minimisingthe energy of the result to ensure sensible geometry,before scoring the whole of the proposed newmolecule against the original molecule using FieldPoints. This approach, using the fields of the wholemolecule, is only tractable because of the significantcomputational advantages provided by Field Pointrepresentation.

Because the score is based on the whole molecule, anyeffects that the new fragment may have on the originalmolecule are automatically considered. This process givesa results list that is significantly richer in non-obviousbioisoteres than would be the case had we onlyconsidered the isolated fragment (see Figure 2). Usingthe whole molecule has an additional benefit in that themedicinal chemist is presented with a list of potentiallyactive molecules rather than partial fragments. Thisallows them to select molecules for synthesis more easilywithout mentally having to construct and retrosynthesisethe final molecule.

Choosing FragmentsCommercial screening collections provide moleculeswith good physicochemical properties, a high degreeof synthetic accessibility and reasonable diversity.

0 400 800 1,200 1,600 2,000 2,400 2,800 3,200 3,600

Figure 2: Comparison of fragment and whole molecule scoring. Each axis showsthe position in the respective results file for 3,600 possible bioisosteres for thelactone group in rofecoxib (top left, red). The highlighted region shows bioisoteresthat score well using whole molecules but poorly when compared at the fragmentlevel. For example, the known COX2 active series containing the thiazolotriazolereplacement for the lactone (top middle, green) appears some 250 places lower inthe results using fragment only scoring compared with whole molecule scoring

Position in results for fragment versus whole molecule scoring

Position using fragments

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Fragmenting 2 million molecules by breaking bondsto heteroatoms and rings resulted in a database of700,000 fragments. To aid the medicinal chemistfurther, we subdivided the database according to thefrequency with which fragments are observed, arguingthat more frequently occurring fragments were moresynthetically accessible. Since Field Point patternsaround fragments are dependent on the exactconformation of the fragment, each fragment is storedas a set of conformations, and the angles and distancesbetween connection points in a fragment are recordedfor each conformation that is stored.

It is important to have control over the nature of theconnection points in the replacement fragment toreflect the types of chemistry that can be performedon the target molecules. To this end, each fragment isannotated with the nature of the connection pointsavailable. The user can simply specify that theconnection point is, for example, an aromatic carbonto significantly restrict the search and chemistry space.

The originality and synthesisability of compoundsproduced using the Field Point method is bestdemonstrated through an example. Using rofecoxib as a starting point, we requested bioisosteres for the lactone moiety. We limited the chemistry spacethat was searched by specifying the replacementcontain an aromatic carbon attached to the remainingphenyl group. The results (see Figure 3) contain animpressive range of bioisosteres from the ‘obvious’ –such as Valdecoxib and Parecoxib – to moreinteresting actives, such as an analogue of Etoricoxib.However, the results also contain ‘non-obvious’bioisosteres such as the 12nM thiazolotriazolecompound shown.

CONCLUSION

The use of Field Points represents a major advance in thecomputational methods available for finding novelbioisosteres. Unlike previous methods, it scores thesuggested bioisosteres in the context of the final moleculerather than as an isolated fragment. The results aresignificantly more diverse than previous methods with bothknown bioisosteres and novel replacements being suggestedin most cases. Application of Field Point based bioisosteretechnology to drug discovery projects has already beenshown to shorten development time and increase novelty.

References

1. Vinter A and Rose S, Molecular Field Technology and

its Applications in Drug Discovery, Innovations in

Pharmaceutical Technology, 23, pp14-18, 2007

2. Bellenie BR, Barton NP, Emmons AJ, et al, Discovery and

optimization of highly ligand-efficient oxytocin receptor

antagonists using structure-based drug, Bioorganic and

Medicinal Chemistry Letters, 19, pp990-994, 2009

3. Dart MJ, Wasicak JT, Ryther KB, et al, Structural

aspects of high affinity ligands for the a4b2 neuronal

nicotinic receptor, Pharmaceutica Acta Hevetiae, 74,

pp115, 2000

Tim Cheeseright is currently Director, Products, atCresset BioMolecular Discovery Ltd. He trained as asynthetic chemist at the University of Oxford, and thenundertook post-doctoral studies at the University ofManchester and the Institute of Cancer Research beforejoining the Combinatorial Chemistry Group of PeptideTherapeutics Ltd (later Acambis plc). Tim moved into

computational chemistry following the acquisition of Peptide’s smallmolecule business by Medivir AB. He joined Cresset as Chief ApplicationScientist in 2002. Email: tim@cresset-bmd.com

Figure 4: FieldStere screenshot showing the COX-2results. Each bioisostere is presented as a 3D alignmentto the original target and also in a table as a 2D picturetogether with calculated LogP and TPSA values

Figure 3: The resultsof a FieldStereexperiment to findbioisosteres for thelactone portion ofrofecoxib (left, red).They include ‘obvious’bioisosteres (Parecoxiband Valdecoxibanalogues, dark blue,top), ‘less obvious’bioisosteres (Etoricoxiband Celecoxibanalogues, middle),and non-obviousbioisosteres such asthe 12nM inhibitortriazolotriazole (green,bottom)