5CINECA Supercomputing Centre. 6University of Modena and Reggio Emilia.7Christian-Albrechts-University Kiel.8Michigan State University. International course and report were conceived by Pietro Cozzini and Glen E. Kellogg. * To whom correspondence should be addressed. For G.E.K.: Department of Medicinal Chemistry, Virginia Commonwealth University, Box 980540, Richmond, VA 23298-0540; (phone) 804-828-6452; (fax) 804-827-3664; (e-mail) glen.kellogg@vcu.edu. For P.C.: Department of General and Inorganic Chemistry, University of Parma, Via G.P. Usberti 17/A 43100, Parma, Italy; (phone) +39-0521-905669; (fax) +39-0521-905556; (e-mail) pietro.cozzini@unipr.it. NIH Public Access IntroductionStructure-based drug discovery has played an important role in medicinal chemistry 1 beginning nearly when the first X-ray crystal structure of the myoglobin and hemoglobin proteins at nearatomic resolution were described by Perutz, Kendrew and colleagues. 2-5 Even though only static structures were (and still generally are) used for most Structure-Based Drug Design (SBDD), and indeed most molecular modeling, the importance of flexibility was recognized immediately: hemoglobin has two rather different structures, "tense" and "relaxed", depending on its oxygenation, although in recent years a family of relaxed hemoglobin structures with different tertiary structure conformations have been reported. 6 In fact, all proteins are inherently flexible systems. This flexibility is frequently essential for function (e.g., as in hemoglobin). Proteins have an intrinsic ability to undergo functionally relevant conformational transitions under native state conditions, 7,8 on a wide range of scales, both in time and space. 9 In adenylate kinase large conformational changes due to movements of the nucleotide 'lids'-rate-limiting for overall catalytic turnover 10,11 -are 'linked' with relatively small-amplitude atomic fluctuations on the ps timescale such that changes in the local backbone conformation are required for lid closure. 12 Nuclear receptors are modular proteins where a significant degree of conformational flexibility is essential to biological function. Most of the pharmacology of nuclear receptor ligands has been discussed on the basis of their ability to stabilize (or displace) a short α-helix segment (known as H12 or AF-2) localized at the carboxy terminus of the receptor in (or from) its conformation in the protein "active" form. 13-15 Available X-ray crystal structures show a surprisingly wide range of structural diversity in ligands binding to, and inhibiting, nuclear receptor proteins such as the farnesoid X-receptor (FXR). 16,17 Protein dynamics is also a key component of intramolecular and intermolecular communication/signaling mechanisms and an essential requirement for the function of Gprotein coupled receptors (GPCRs), which are the largest known superfamily of membrane proteins. GPCRs regulate cell activity by transmitting extracellular signals to the inside of cells and respond to these signals by catalyzing nucleotide e...

Structural water molecules within protein active sites are relevant for ligand-protein recognition because they modify the active site geometry and contribute to binding affinity. In this work an analysis of the interactions between 23 ligands and dimeric HIV-1 protease is reported. The X-ray structures of these complexes show the presence of four types of structural water molecules: water 301 (on the symmetry axis), water 313, water 313bis, and peripheral waters. Except for water 301, these are generally complemented with a symmetry-related set. The GRID program was used both for checking water locations and for placing water molecules that appear to be missing from the complexes due to crystallographic uncertainty. Hydropathic analysis of the energetic contributions using HINT indicates a significant improvement of the correlation between HINT scores and the experimentally determined binding constants when the appropriate bridging water molecules are taken into account. In the absence of water r 2) 0.30 with a standard error of (1.30 kcal mol-1 and when the energetic contributions of the constrained waters are included r 2) 0.61 with a standard error of (0.98 kcal mol-1. HINT was shown to be able to map quantitatively the contribution of individual structural waters to binding energy. The order of relevance for the various types of water is water 301 > water 313 > water 313bis > peripheral waters. Thus, to obtain the most reliable free energy predictions, the contributions of structural water molecules should be included. However, care must be taken to include the effects of water molecules that add information value and not just noise.

The worldwide emergence of New Delhi metallo-β-lactamase-1 (NDM-1) as a carbapenemase able to hydrolyze nearly all available β-lactam antibiotics has characterized the past decade, endangering efficacious antibacterial treatments. No inhibitors for NDM-1 are available in therapy, nor are promising compounds in the pipeline for future NDM-1 inhibitors. We report the studies dedicated to the design and development of effective NDM-1 inhibitors. The discussion for each agent moves from the employed design strategy to the ability of the identified inhibitor to synergize β-lactam antibiotics. A structural analysis of NDM-1 mechanism of action based on selected X-ray complexes is also reported: the intrinsic flexibility of the binding site and the comparison between penicillin/cephalosporin and carbapenem mechanisms of hydrolysis are evaluated. Despite the valuable progress in terms of structural and mechanistic information, the design of a potent NDM-1 inhibitor to be introduced in therapy remains challenging. Certainly, only the deep knowledge of NDM-1 architecture and of the variable mechanism of action that NDM-1 employs against different classes of substrates could orient a successful drug discovery campaign.