Molecular mechanism of ligand recognition by membrane transport protein, Mhp1.

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.

Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness.

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder ß-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.

An in silico structure-based approach to anti-infective drug discovery.

In light of the low success rate of target-based genomics and HTS (High Throughput Screening) approaches in anti-infective drug discovery, in silico structure-based drug design (SBDD) is becoming increasingly prominent at the forefront of drug discovery. In silico SBDD can be used to identify novel enzyme inhibitors rapidly, where the strength of this approach lies with its ability to model and predict the outcome of protein-ligand binding. Over the past 10 years, our group have applied this approach to a diverse number of anti-infective drug targets ranging from bacterial D-ala-D-ala ligase to Plasmodium falciparum DHODH. Our search for new inhibitors has produced lead compounds with both enzyme and whole-cell activity with established on-target mode of action. This has been achieved with greater speed and efficiency compared with the more traditional HTS initiatives and at significantly reduced cost and manpower.

Discovery of biphenylacetamide-derived inhibitors of BACE1 using de novo structure-based molecular design.

ß-Secretase (BACE1), the enzyme responsible for the first and rate-limiting step in the production of amyloid-ß peptides, is an attractive target for the treatment of Alzheimer's disease. In this study, we report the application of the de novo fragment-based molecular design program SPROUT to the discovery of a series of nonpeptide BACE1 inhibitors based upon a biphenylacetamide scaffold. The binding affinity of molecules based upon this designed molecular scaffold was increased from an initial BACE1 IC50 of 323 µM to 27 µM following the synthesis of a library of optimized ligands whose structures were refined using the recently developed SPROUT-HitOpt software. Although a number of inhibitors were found to exhibit cellular toxicity, one compound in the series was found to have useful BACE1 inhibitory activity in a cellular assay with minimal cellular toxicity. This work demonstrates the power of an in silico fragment-based molecular design approach in the discovery of novel BACE1 inhibitors.

A virtual high-throughput screening approach to the discovery of novel inhibitors of the bacterial leucine transporter, LeuT.

Membrane proteins are intrinsically involved in both human and pathogen physiology, and are the target of 60% of all marketed drugs. During the past decade, advances in the studies of membrane proteins using X-ray crystallography, electron microscopy and NMR-based techniques led to the elucidation of over 250 unique membrane protein crystal structures. The aim of the European Drug Initiative for Channels and Transporter (EDICT) project is to use the structures of clinically significant membrane proteins for the development of lead molecules. One of the approaches used to achieve this is a virtual high-throughput screening (vHTS) technique initially developed for soluble proteins. This paper describes application of this technique to the discovery of inhibitors of the leucine transporter (LeuT), a member of the neurotransmitter:sodium symporter (NSS) family.

Factors influencing the specificity of inhibitor binding to the human and malaria parasite dihydroorotate dehydrogenases.

The de novo pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase is an emerging drug target for the treatment of malaria. In this context a key property of Plasmodium falciparum DHODH (PfDHODH) is that it can be selectively inhibited over its human homologue (HsDHODH). However, HsDHODH is also a validated drug target for autoimmune diseases such as arthritis. Here a series of novel inhibitors is described that includes compounds that switch specificity between the two enzymes as a result of small alterations in chemical structure. Structure-activity relationship (SAR), crystallography, docking, and mutagenesis studies are used to examine the binding modes of the compounds within the two enzymes and to reveal structural changes induced by inhibitor binding. Within this series, compounds with therapeutically relevant HsDHODH activity are described and their binding modes characterized using X-ray crystallography, which reveals a novel conformational shift within the inhibitor binding site.

Identification of selective inhibitors of the potassium channel Kv1.1-1.2((3)) by high-throughput virtual screening and automated patch clamp.

Two voltage-dependent potassium channels, Kv1.1 (KCNA1) and Kv1.2 (KCNA2), are found to co-localize at the juxtaparanodal region of axons throughout the nervous system and are known to co-assemble in heteromultimeric channels, most likely in the form of the concatemer Kv1.1-1.2((3)) . Loss of the myelin sheath, as is observed in multiple sclerosis, uncovers the juxtaparanodal region of nodes of Ranvier in myelinated axons leading to potassium conductance, resulting in loss of nerve conduction. The selective blocking of these Kv channels is therefore a promising approach to restore nerve conduction and function. In the present study, we searched for novel inhibitors of Kv1.1-1.2((3)) by combining a virtual screening protocol and electrophysiological measurements on a concatemer Kv1.1-1.2((3)) stably expressed in Chinese hamster ovary K1 (CHO-K1) cells. The combined use of four popular virtual screening approaches (eHiTS, FlexX, Glide, and Autodock-Vina) led to the identification of several compounds as potential inhibitors of the Kv1.1-1.2((3)) channel. From 89 electrophysiologically evaluated compounds, 14 novel compounds were found to inhibit the current carried by Kv1.1-1.2((3)) channels by more than 80 % at 10 µM. Accordingly, the IC(50) values calculated from concentration-response curve titrations ranged from 0.6 to 6 µM. Two of these compounds exhibited at least 30-fold higher potency in inhibition of Kv1.1-1.2((3)) than they showed in inhibition of a set of cardiac ion channels (hERG, Nav1.5, and Cav1.2), resulting in a profile of selectivity and cardiac safety. The results presented herein provide a promising basis for the development of novel selective ion channel inhibitors, with a dramatically lower demand in terms of experimental time, effort, and cost than a sole high-throughput screening approach of large compound libraries.

Structure-based ligand design of novel bacterial RNA polymerase inhibitors.

Bacterial RNA polymerase (RNAP) is essential for transcription and is an antibacterial target for small molecule inhibitors. The binding region of myxopyronin B (MyxB), a bacterial RNAP inhibitor, offers the possibility of new inhibitor design. The molecular design program SPROUT has been used in conjunction with the X-ray cocrystal structure of Thermus thermophilus RNAP with MyxB to design novel inhibitors based on a substituted pyridyl-benzamide scaffold. A series of molecules, with molecular masses <350 Da, have been prepared using a simple synthetic approach. A number of these compounds inhibited Escherichia coli RNAP.

A study of the effects of substituents on the selectivity of the binding of N-arylaminomethylene malonate inhibitors to DHODH.

A series of mono- and di-substituted N-arylaminomethylene malonates have been used to probe the potential of utilizing additional H-bonding contacts in the ubiquinone binding channel, for selective inhibition between either human or Plasmodium DHODH. Altered 'head' group functionalities have been utilized in order to probe the role of specific functionalities within the inhibitors in terms of enzyme affinity and selectivity.

Discovery of novel non-peptide inhibitors of BACE-1 using virtual high-throughput screening.

A novel series of isatin-based inhibitors of beta-secretase (BACE-1) have been identified using a virtual high-throughput screening approach. Structure-activity relationship studies revealed structural features important for inhibition. Docking studies suggest these inhibitors may bind within the BACE-1 active site through H-bonding interactions involving the catalytic aspartate residues.

Route Designer: a retrosynthetic analysis tool utilizing automated retrosynthetic rule generation.

Route Designer, version 1.0, is a new retrosynthetic analysis package that generates complete synthetic routes for target molecules starting from readily available starting materials. Rules describing retrosynthetic transformations are automatically generated from reaction databases, which ensure that the rules can be easily updated to reflect the latest reaction literature. These rules are used to carry out an exhaustive retrosynthetic analysis of the target molecule, in which heuristics are used to mitigate the combinatorial explosion. Proposed routes are prioritized by an empirical rating algorithm to present a diverse profile of the most promising solutions. The program runs on a server with a web-based user interface. An overview of the system is presented together with examples that illustrate Route Designer's utility.

Structure-based design, synthesis, and characterization of inhibitors of human and Plasmodium falciparum dihydroorotate dehydrogenases.

Pyrimidine biosynthesis is an attractive drug target in a variety of organisms, including humans and the malaria parasite Plasmodium falciparum. Dihydroorotate dehydrogenase, an enzyme catalyzing the only redox reaction of the pyrimidine biosynthesis pathway, is a well-characterized target for chemotherapeutical intervention. In this study, we have applied SPROUT-LeadOpt, a software package for structure-based drug discovery and lead optimization, to improve the binding of the active metabolite of the anti-inflammatory drug leflunomide to the target cavities of the P. falciparum and human dihydroorotate dehydrogenases. Following synthesis of a library of compounds based upon the SPROUT-optimized molecular scaffolds, a series of inhibitors generally showing good inhibitory activity was obtained, in keeping with the SPROUT-LeadOpt predictions. Furthermore, cocrystal structures of five of these SPROUT-designed inhibitors bound in the ubiquinone binding cavity of the human dihydroorotate dehydrogenase are also analyzed.

CLiDE Pro: the latest generation of CLiDE, a tool for optical chemical structure recognition.

We present CLiDE Pro, the latest version of the output of the long-term CLiDE project for the development of tools for automatic extraction of chemical information from the literature. CLiDE Pro is concerned with the extraction of chemical structure and generic structure information from electronic images of chemical molecules available online as well as pages of scanned chemical documents. The information is extracted in three phases, first the image is segmented into text and graphical regions, then graphical regions are analyzed and where possible the connection tables are reconstructed, and finally any generic structures are interpreted by matching R-groups found in structure diagrams with the ones located in the text. The program has been tested on a large set of images of chemical structures originating from various sources. The results demonstrate good performance in the reconstruction of connection tables with few errors in the interpretation of the individual drawing features found in the structure diagrams. This full test set is presented for use in the validation of other similar systems.

Design and synthesis of potent inhibitors of the malaria parasite dihydroorotate dehydrogenase.

Pyrimidine biosynthesis presents an attractive drug target in malaria parasites due to the absence of a pyrimidine salvage pathway. A set of compounds designed to inhibit the Plasmodium falciparum pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (PfDHODH) was synthesized. PfDHODH-specific inhibitors with low nanomolar binding affinities were identified that bind in the N-terminal hydrophobic channel of dihydroorotate dehydrogenase, the presumed site of ubiquinone binding during oxidation of dihydroorotate to orotate. These compounds also prevented growth of cultured parasites at low micromolar concentrations. Models that suggest the mode of inhibitor binding is based on shape complementarity, matching hydrophobic regions of inhibitor and enzyme, and interaction of inhibitors with amino acid residues F188, H185, and R265 are supported by mutagenesis data. These results further highlight PfDHODH as a promising new target for chemotherapeutic intervention in prevention of malaria and provide better understanding of the factors that determine specificity over human dihydroorotate dehydrogenase.

eHiTS: a new fast, exhaustive flexible ligand docking system.

The flexible ligand docking problem is divided into two subproblems: pose/conformation search and scoring function. For successful virtual screening the search algorithm must be fast and able to find the optimal binding pose and conformation of the ligand. Statistical analysis of experimental data of bound ligand conformations is presented with conclusions about the sampling requirements for docking algorithms. eHiTS is an exhaustive flexible-docking method that systematically covers the part of the conformational and positional search space that avoids severe steric clashes, producing highly accurate docking poses at a speed practical for virtual high-throughput screening. The customizable scoring function of eHiTS combines novel terms (based on local surface point contact evaluation) with traditional empirical and statistical approaches. Validation results of eHiTS are presented and compared to three other docking software on a set of 91 PDB structures that are common to the validation sets published for the other programs.

eHiTS: an innovative approach to the docking and scoring function problems.

Virtual Ligand Screening (VLS) has become an integral part of the drug design process for many pharmaceutical companies. In protein structure based VLS the aim is to find a ligand that has a high binding affinity to the target receptor whose 3D structure is known. This review will describe the docking tool eHiTS. eHiTS is an exhaustive and systematic docking tool which contains many automated features that simplify the drug design workflow. A description of the unique docking algorithm and novel approach to scoring used within eHiTS is presented. In addition a validation study is presented that demonstrates the accuracy and wide applicability of eHiTS in re-docking bound ligands into their receptors.

Molecular complexity analysis of de novo designed ligands.

The de novo approach to structure-based rational drug design can provide a powerful tool for suggestion of entirely novel potential leads. However, programs for structure generation typically generate large numbers of putative ligands; therefore, various heuristics (such as estimation of binding affinity and synthetic accessibility) have to be adopted to evaluate and prune large answer sets with the goal of suggesting ligands with high binding affinity but low structural complexity. A novel method for complexity analysis is described. This method provides a rapid and effective ranking technique for elimination of structures with complicated molecular motifs. This complexity analysis technique, implemented within the SPROUT de novo design system, is based on the statistical distribution of various cyclic and acyclic topologies and atom substitution patterns in existing drugs or commercially available starting materials. A novel feature of the technique that distinguishes it from other published methods is that the matching takes place at various levels of abstraction, so that it can evaluate complexity scores, even for structures which contain atoms with unspecified atom type, which is sometimes the case with the initial output of de novo structure generation systems.

The first de novo designed inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase.

The de novo molecular design program SPROUT has been applied to the X-ray crystal structures of Plasmodium and human dihydroorotate dehydrogenase, respectively. The resulting design templates were used to prepare a series of molecules which, in keeping with predictions, showed useful levels of species-selective enzyme inhibition.

Macrocyclic inhibitors of the bacterial cell wall biosynthesis enzyme MurD.

Computer-based molecular design has been used to produce a series of new macrocyclic systems targeted against the bacterial cell wall biosynthetic enzyme MurD. Following their preparation, which involved a novel metathesis-based cyclisation as the key step, these systems were found to show good inhibition when assayed against the MurD enzyme.