On the paradigm shift from rational to random design

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THEO CHEM

Journal of Molecular Structure (Theochem) 398-399 (I 997) 467-473

On the paradigm shift from rational to random design’

Klaus Miiller

Abstract

Lead identification and optimization are key elements of the drug discovery process, in which “rational molecular design” plays an important role. Its ultimate goal, the successful de novo design of high-affinity ligands based on three-dimensional structure information, is still only partially reached due to our limited understanding of nonbonding interactions and solvation effects. A plethora of highly sophisticated molecular modelling, biocomputing and graphics methods are available, providing powerful interactive environments for the semiquantitative molecular design. This structure-based approach is complemented by efforts of random screening of large compound repositories without preconceived notions on structure-activity relationships. This dual approach of “rational design and random screening” has established itself as an essential element in the overall drug discovery process. An interesting shift of paradigm has taken place towards “random design and rational screening”. Random design entails diverse methods of combinatorial modelling, fuzzy or sketchy design, and nonstructural design concepts. Screening efforts are no longer performed on extensive compound repositories comprising hundreds of thousands of compounds, but rather on biased subsets, rationally selected by substructural or pharmacophore hypothesis-based database searches. These efforts are complemented by new methodologies of macro- and microsynthetic combinatorial chemistry approaches with novel tools for structure diversity planning and assessment. 0 1997 Elsevier Science B.V.

Keywords: Drug discovery; Rational: Random; Design; Screening

Drug discovery is a complex process typically starting at the early investigation of a disease at the mole-

cular level and eventually leading to the identification of a drug candidate for clinical development. This

process is highly interdisciplinary involving most areas of the life sciences. While there is not a single

recipe of success, one can identify a sequence of key stages with an intrinsic logic of mandatory activities and prerequisites. The so-called rational molecular design plays an important element in this process. However, it has to be seen in the context of the whole process containing many essential elements both before, during and after the actual molecular design step.

’ Presented at WATOC ‘96, Jerusalem, Israel, 7- 12 July 1996.

The increased requirements for highly innovative drugs as well as the pressures to shorten the time of discovery call for novel methods and technologies and their continuous refinements in all areas of

the discovery process. We witness dramatic meth- odological developments in virtually all relevant disciplines, even in those that have been considered essentially mature, such as synthetic organic chemistry or standard analytics by physicochemical methods.

A first stage in drug discovery involves research activities directed towards the understanding of molecular mechanisms underlying a disease with the goal to identify, characterize and validate potential targets for medical intervention. This research phase has

Ol66-1280/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved P/I SOl66- 1280(96)05000-2

gained dramatic momentum through enhanced capabilities of fast sequencing of both human and non- human genomes, novel miniaturized high-capacity gene expression display technologies, refined molecular genomics and gene technologies, (sub)picomolar protein analytics and biochemistry, second-generation bioinformatics and protein modelling, and synchro- tron-radiation-enhanced biostructural analytics that provide biofunctional and biostructural insights at much accelerated rates.

A second stage entails activities directed towards the discovery and optimization of highly potent and selective ligands against a given target. Quite often exploratory research on this target is producing suffi- cient information for mechanism-based or even structure-based ligand design efforts, particularly if a suitable structural model of the target or a fragment thereof can be produced at an early phase of this second stage of drug discovery. Such “rational” ligand design approaches are generally paralleled by broad screening efforts of compound collections or natural

products, which improve the chances of early identification of potential leads and often result in a widen- ing of the structural scope of ligand design and the discovery of new binding motifs. It is important to note that an early identification of potential lead structures is a prerequisite not only for accelerating the discovery process overall, but also for a further characterization and validation of a selected target. Thus, the inability to identify a suitable lead compound for a

chemical lead optimization program in due course or the discovery of undesired, but inherent, biofunctional properties of a selected target may lead to an early termination of a project. While during the past fifteen years ligand design has been increasingly assisted by

sophisticated computer graphics and computational methods exploiting biostructural information, this second stage of drug discovery is experiencing a dramatic boost due to the advent of various combinatorial chemistry and high-throughput assay technologies, miniaturized high-capacity analytics, novel technologies for smart handling of large compound

repositories and concomitant developments of novel combinatorial design, diversity planning and virtual library handling methodologies.

A third stage in drug discovery entails the multi- disciplinary activities of in vivo investigation of selected lead compounds, nonclinical development

of few selected candidates, and may extend into the clinical characterization of selected drug candidates up to proof-of-concept in man. It is evident that in reality the third stage strongly overlaps the second. The early inclusion of pharmacological properties into the lead optimization process is vital for an accelerated drug discovery process. While the methodologies in the areas of drug formulation,

pharmacokinetics, metabolism and toxicology are continuously being refined through improved analytics and monitoring devices, as well as complementary cell- and enzyme-based assay systems, we witness dramatic changes by the emergence of new concepts to describe and experimentally determine in vitro compound properties that have significant predictive power for relevant pharmacological properties. The concomitant development of novel measuring techniques in miniaturized high-throughput formats provides opportunities for improvement

and acceleration of lead optimization processes in an unprecedented fashion.

While the so-called rational molecular design con- tinues to play its important role during the second stage of drug discovery, it has to be seen in this context of many key processes both before, during and after the actual design phase. The increased requirements for highly innovative drugs as well as the pressures to shorten the time of discovery call for a relentless search, integration and refinement of novel methods and technologies in all areas of the drug discovery process.

The ultimate of “rational design” is still considered to be the successful de novo design of a

high-affinity ligand based on the three-dimensional structure of a given target molecule, usually a pharmacologically relevant enzyme or a receptor fragment. This goal is still only partially reached due to our limited understanding of many aspects of nonbonding interactions and solvation effects. Never- theless, a multitude of highly sophisticated computer methods in molecular modelling, biocomputing and graphic display have been developed and are being further refined. They provide powerful interactive environments for molecular design at least at a semiquantitative level. At the other end of the spectrum is random screening in which large sets of compounds are assayed for a desired biological activity without any preconceived notions on structure-activity

K. Miiller/Journal of Molecular Structure (Theochem) 398-399 (1997) 467-471 469

relationships. Both approaches are important in their own right and may be individually successful. How-

ever, they are complementary and should therefore be used in combination rather than to the exclusion of each other.

Rational design would typically start with a close examination of the structural characteristics of the target or a ligand complex with the target. Careful modelling of novel structures into the binding site, semiquantitative assessments of their relative binding

energies, followed by synthesis and affinity testing of most promising prototype or lead structures, possibly complemented by the experimental determination of ligand binding modes, would represent a typical cycle in an iterative process to turn the original lead structure into a high-affinity and eventually selective ligand. The initial lead structure in this process may originate from de novo design, from a screening program, or from an already known ligand, e.g. a natural substrate or inhibitor. Their binding modes may be totally unknown, guessed by analogy or mechanistic reasoning, or already known through experimental

three-dimensional structure determinations of target-ligand complexes.

Ideally, this iterative process of design, synthesis

and testing, complemented by structure determinations, would result in a refined understanding of ligand binding modes and an assessment of important molecular interaction patterns. However, despite all the advances in computational chemistry over the last two decades, this process is still beset with many dif-

ficulties and leaves much room for the unexpected. The latter may frustrate the newcomer, but is gener-

ally the source of inspiration to the expert. Despite all its shortcomings, the dual approach of

“rational design and random screening” has established itself as an essential element in the overall

drug discovery process. While design methodologies are still being further refined, particularly with respect to a better description of polarization effects in nonbonding interactions and solvation energy contri- butions, an interesting shift of paradigm has taken

place towards ’ ‘random design and rational

screening”. Random design entails new tools of combinatorial

design as well as methodologies based on fuzzy structural or even nonstructural concepts. Combinatorial tools allow us to create large virtual structure libraries

and to handle whole sets of potential ligands simul-

taneously, rather than individual ligand entities at a time. Ligand clusters typically emerge from computer screening of large structural databases and require special tools for efficient handling, manipulation and examination of whole structure sets in order to gen- erate useful patterns for ligand binding. Structural databases may be computer generated based on exist-

ing compound repositories, or may be generated by computer-automated combinatorial building block assembly strategies, or by computer-assisted combinatorial side-chain attachments to a given central tem-

plate with or without structural constraints derived from a pharmacophore hypothesis.

Alternatively, multiple docking of small fragment molecules, representing potential substructural elements of a ligand, may provide insights into patterns of preferred binding sites for such fragments. Select- ing the most promising clusters of favourably bound fragments, possible strategies for linking fragments

may be explored by computer-assisted linker design tools using atom- or fragment-based structure build-

up procedures or by approximate matching of pre- modelled linker moieties taken from specially designed databases containing a large collection of synthetically feasible linker fragments modelled in all possible low-energy conformations. The latter approach is particularly efficient, typically producing a multitude of sketchy ligand structures. These may require limited refinement and a crude ranking using novel tolerant energy functions, and finally clustering into classes of ligand motifs. Such combinatorial and fuzzy structure set generation and analysis provide a rich source of inspiration even at a rather unrefined

structural level. It is then only at a relatively advanced stage of this “random design” phase that suitable representatives of promising structural motifs are individually examined and eventually further elabo- rated in the more traditional way of computer-assisted modelling.

These developments of a new generation of design tools and methodologies have been paralleled by complementary changes in screening philosophy.

Although the throughput capabilities of compound screening have been increased dramatically over the past few years by miniaturization, robotics and novel assay technologies, making large-scale random screening efforts even more feasible within

470 K. Miillrr/Jounml of Moleculnr Srruciure (Throchrm) 39X-399 (1997) 467-471

reasonable amounts of time, we witness a tendency to Low-energy binding modes of individual or replace such screening efforts by more focused selected sets of fragments can be examined inter- screening of subsets of available compound reposi- actively on the screen. The user may dynamically tories, selected by mechanism- or structure-based change the ranges of relative binding energies so criteria, or by prior searching structural databases that multiple fragment copies pop up in different using three-dimensional pharmacophore hypotheses locations of the binding site as colour-coded clusters, generated from known bioactive compounds. Hence, providing valuable insights into binding patterns for elements of “rational design” are becoming an individual fragments or combinations of selected frag- integral part of screening activities. ment sets.

With the advent of combinatorial chemistry, particularly semi-automated parallel synthesis methodologies, large sets of compounds corresponding to a given ligand motif or pharmacophore hypothesis can be generated and tested quickly. Thus, whenever possible, new ligand concepts are no longer tested by sequential syntheses and assays of individual compounds, but rather in terms of small combinatorial compound sets or biased libraries of single compounds from parallel synthesis, containing sets of

representative compounds with designed structural diversity.

Let us discuss in more detail relevant aspects of the new design philosophy using combinatorial and fuzzy structural tools as they are being implemented in our laboratory. Given the binding site of a selected target molecule, we may start by a set of representative molecular fragments which are rapidly docked in multiple ways into different locations of the selected target binding site. These fragments are taken from a library containing molecular structures of pharmaco-

logically relevant mono- and bis-heterocyclic fragments as well as recurrent functional groups or rigid linker units. The fragment selection can be comple-

mented by additional user-selected fragments. The docking is fully automated. Based on a surface analysis of the given binding site, a representative ensemble of well-separated points in space are generated that serve as starting points for fragment docking. For enzyme binding sites typically ensembles of 100 to 200 surface points are generated which are spaced at least 1 A apart. For each start position, individual fragments are placed in 24 different initial orienta- tions for automated docking by energy minimization to the nearest local energy minimum keeping the target molecule rigid. The manifold of docked fragments are then pruned to eliminate redundant or high-energy binding modes and ranked according to increasing binding energies.

Individually positioned fragments or whole clusters of fragments can be specified for the next (automated) step, exploring ways to link fragments in a pairwise fashion. This linking utility is based on an approximate matching of rigid substructures of potential linker units to pairs of docked fragments, using rough geometrical criteria for the assessment of possibilities for covalent linking. No attempt is

made at this stage to arrive at regularized covalent ligand structures. The virtue of this rough matching

approach is to provide a very fast overview of potential fragment linking solutions, keeping in mind that any subsequent structure regularization will anyhow reposition somewhat the originally docked fragments at their respective binding sites. In this way. relevant solutions will not be lost due to a premature structural pruning by an oversophisticated analysis of covalent linking geometries. However, spatial constraints imposed by the target are taken into account during this matching step.

The linker units are retrieved from a specially designed library holding a large collection of pre- modelled substructures, typically containing single- atom spacers, acyclic two- to six-atom chains in different low-energy conformations, as well as small cyclic and polycyclic units. These linking elements represent synthetically accessible moieties incorpor- ating functionalities that confer high retrosynthetic potential to resulting linked fragment structures. The linker library can be complemented by user-selected fragments or templates to reflect established synthetic expertise available in the laboratory.

At this design stage, the resulting ligand structures

are only sketchy, generally violating standard valence geometries at connecting sites by significant margins. Interestingly, however, interactive graphic examination of these intermediate results can be quite reward- ing often stimulating new ligand structural concepts. Intermediate structure regularization may then be

K. Miiller/Jounml of Molecular Structure (Theochem) 398-399 (1997) 467-471 471

needed for individual candidates or for whole clusters of linked structures. For this purpose fast and generally applicable united-atom force field methods are

extremely beneficial, which are able to handle large sets of candidate structures automatically and in reasonable time. The resulting ligands are then com- patible with the structural requirements of the target binding site, conformationally unstrained and synthetically accessible. New tolerant energy functions for fast and rough estimations of ligand binding energies,

including also aspects of solvation effects, are much needed and currently being developed. Such energy functions will provide the means to rank large numbers of ligand candidates adequately with respect to ranges of relative ligand binding energies. This is important in order to avoid potential combinatorial explosions in the course of repeated linking of frag-

ment clusters. The fragment linking step can be repeated for any

subsequent pair of fragments, fragment clusters or set of intermediately linked ligands. Hence, typically in a

very short period of time and in an interactive step- wise exploratory fashion, several fragment clusters can be transformed into novel interesting ligand structures, providing a plethora of new ideas that can be translated into a number of synthetically amenable prototype ligand concepts based on experimentally

established building block assembly strategies. This may then form the basis for a biased combinatorial diversity design for miniaturized high-capacity

parallel synthesis methodologies to create, within a reasonable amount of time, a representative set of compounds to test experimentally a given pharmaco-

phore hypothesis. The development of such new-generation design

tools is in full swing. Although it may be too early to assess their full impact on the process of drug dis-

covery, there can be no doubt about the enormous potential of these methodologies in speeding up the lead discovery and optimization process. Likewise, it is evident that there is much room for further metho- dological and technological innovations. At the same time, we witness dramatic developments in the areas of combinatorial chemistry, miniaturized and automated analytics, as well as high-throughput assay technologies, providing an environment for efficient management and processing of structures, com-

pounds, assays and results in a highly automated and parallel fashion. With all these technologies at hand and properly integrated, the classical cycle of lead design, synthesis and testing of individual ligand candidates in a sequential fashion is being transformed into a high-capacity parallel lead discovery and optimization procedure.

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On the paradigm shift from rational to random design