The multi-copy simultaneous search methodology: a fundamental tool for structure-based drug design Christian R. Schubert Æ Collin M. Stultz Received: 1 January 2009 / Accepted: 20 May 2009 / Published online: 9 June 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Fragment-based ligand design approaches, such as the multi-copy simultaneous search (MCSS) methodol- ogy, have proven to be useful tools in the search for novel therapeutic compounds that bind pre-specified targets of known structure. MCSS offers a variety of advantages over more traditional high-throughput screening methods, and has been applied successfully to challenging targets. The methodology is quite general and can be used to construct functionality maps for proteins, DNA, and RNA. In this review, we describe the main aspects of the MCSS method and outline the general use of the methodology as a fun- damental tool to guide the design of de novo lead com- pounds. We focus our discussion on the evaluation of MCSS results and the incorporation of protein flexibility into the methodology. In addition, we demonstrate on several specific examples how the information arising from the MCSS functionality maps has been successfully used to predict ligand binding to protein targets and RNA. Keywords Structure-based ligand design  Multi-copy simultaneous search (MCSS)  Ligand binding  Molecular drug design Introduction After nearly three decades since its theoretical inception [1], rational drug design remains a challenge. Typically, the design of compounds that bind pre-specified targets of known structure is one of the first steps in the construction of novel therapeutic compounds [1]. Methods that simplify the discovery of such lead compounds can accelerate the rate at which new drugs are discovered. The discovery of ligands that bind targets with known structure can be approached using a series of steps that include: (1) determining optimal positions of small chemical fragments in the binding site; (2) linking the fragments together to form molecules that are complementary to the target; (3) estimating the binding affinity of the resulting molecules; and (4) synthesis and experimental evaluation of the computationally designed ligands (Fig. 1). The multi-copy simultaneous search (MCSS) methodology [2] addresses the first step in the above-mentioned approach: the MCSS method finds ener- getically favorable positions of different functional groups in a pre-specified binding site of interest, and yields so-called functionality maps of the binding site as output. Functional groups positioned in these energetically favorable positions can then be linked together to construct ligands de novo that are complementary to the binding site of the target. Alter- natively, these positions can be used to modify known ligands to improve their binding affinity. The method is quite general and can be used to construct functionality maps for proteins, DNA, and RNA. Moreover, protein flexibility can be incorporated in the process in a straightforward manner. C. R. Schubert Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA C. R. Schubert  C. M. Stultz (&) Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA e-mail: cmstultz@mit.edu C. M. Stultz Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA C. M. Stultz Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 123 J Comput Aided Mol Des (2009) 23:475–489 DOI 10.1007/s10822-009-9287-y