Structure-aided Drug Design Research Articles

Crystal Structure of Inhibitor-Bound GII.4 Sydney 2012 Norovirus 3C-Like Protease.

Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses.

Structure-Aided Design, Synthesis, and Biological Evaluation of Potent and Selective Non-Nucleoside Inhibitors Targeting Protein Arginine Methyltransferase 5.

PRMT5 is a major type II protein arginine methyltransferase and plays important roles in diverse cellular processes. Overexpression of PRMT5 is implicated in various types of cancer. Many efforts have been made to develop potent and selective PRMT5 inhibitors, the most potent of which is usually derived from nucleoside structures. Here, we designed a novel series of non-nucleoside PRMT5 inhibitors through the structure-aided drug design approach. SAR exploration and metabolic stability optimization led to the discovery of compound 41 as a potent PRMT5 inhibitor with good selectivity. Additionally, compound 41 exerted antiproliferative effects against A375 cells by inducing apoptosis and potently inhibited the methyltransferase activity of PRMT5 in cells. Moreover, it showed attractive pharmacokinetic properties and markedly suppressed the tumor growth in an A375 tumor xenograft model. These results clearly indicate that 41 is a highly potent and selective non-nucleoside PRMT5 inhibitor.

Development and validation of chemical features-based proton-coupled folate transporter/activity and reduced folate carrier/activity models (pharmacophores)

All clinically used antifolates lack transport selectivity for tumors over normal cells resulting in dose-limiting toxicities. There is growing interest in developing novel tumor-targeted cytotoxic antifolates with selective transport into tumors over normal cells via the proton-coupled folate transporter (PCFT) over the ubiquitously expressed reduced folate carrier (RFC). A lack of X-ray crystal structures or predictive models for PCFT or RFC has hindered structure-aided drug design for PCFT-selective therapeutics. Four-point validated models (pharmacophores) were generated for PCFT/Activity (HBA, NI, RA, RA) and RFC/Activity (HBD, NI, HBA, HBA) based on inhibition (IC50) of proliferation of isogenic Chinese hamster ovary (CHO) cells engineered to express only human PCFT or only RFC. Our results revealed substantial differences in structural features required for transport of novel molecules by these transporters which can be utilized for developing transporter-selective antifolates.

The Influenza Virus Polymerase Complex: An Update on Its Structure, Functions, and Significance for Antiviral Drug Design.

Influenza viruses cause seasonal epidemics and pandemic outbreaks associated with significant morbidity and mortality, and a huge cost. Since resistance to the existing anti‐influenza drugs is rising, innovative inhibitors with a different mode of action are urgently needed. The influenza polymerase complex is widely recognized as a key drug target, given its critical role in virus replication and high degree of conservation among influenza A (of human or zoonotic origin) and B viruses. We here review the major progress that has been made in recent years in unravelling the structure and functions of this protein complex, enabling structure‐aided drug design toward the core regions of the PA endonuclease, PB1 polymerase, or cap‐binding PB2 subunit. Alternatively, inhibitors may target a protein–protein interaction site, a cellular factor involved in viral RNA synthesis, the viral RNA itself, or the nucleoprotein component of the viral ribonucleoprotein. The latest advances made for these diverse pharmacological targets have yielded agents in advanced (i.e., favipiravir and VX‐787) or early clinical testing, besides several experimental inhibitors in various stages of development, which are all covered here.

Human sterol 14α-demethylase as a target for anticancer chemotherapy: towards structure-aided drug design

Rapidly multiplying cancer cells synthesize greater amounts of cholesterol to build their membranes. Cholesterol-lowering drugs (statins) are currently in clinical trials for anticancer chemotherapy. However, given at higher doses, statins cause serious side effects by inhibiting the formation of other biologically important molecules derived from mevalonate. Sterol 14α-demethylase (CYP51), which acts 10 steps downstream, is potentially a more specific drug target because this portion of the pathway is fully committed to cholesterol production. However, screening a variety of commercial and experimental inhibitors of microbial CYP51 orthologs revealed that most of them (including all clinical antifungals) weakly inhibit human CYP51 activity, even if they display high apparent spectral binding affinity. Only one relatively potent compound, (R)-N-(1-(3,4'-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VFV), was identified. VFV has been further tested in cellular experiments and found to decrease proliferation of different cancer cell types. The crystal structures of human CYP51-VFV complexes (2.0 and 2.5 Å) both display a 2:1 inhibitor/enzyme stoichiometry, provide molecular insights regarding a broader substrate profile, faster catalysis, and weaker susceptibility of human CYP51 to inhibition, and outline directions for the development of more potent inhibitors.

Virtual Screening and Experimental Validation Identify Novel Inhibitors of the Plasmodium falciparum Atg8-Atg3 Protein-Protein Interaction.

New therapies are needed against malaria, a parasitic infection caused by Plasmodium falciparum, as drug resistance emerges against the current treatment, artemisinin. We previously characterized the Atg8-Atg3 protein-protein interaction (PPI), which is essential for autophagy and parasite survival. Herein we illustrate the use of virtual library screening to selectively block the PPI in the parasite without inhibiting the homologous interaction in humans by targeting the A-loop of PfAtg8. This A-loop is important for Atg3 binding in Plasmodium, but is absent from the human Atg8 homologues. In this proof-of-concept study, we demonstrate a shift in lipidation state of PfAtg8 and inhibition of P. falciparum growth in both blood- and liver-stage cultures upon drug treatment. Our results illustrate how in silico screening and structure-aided drug design against a PPI can be used to identify new hits for drug development. Additionally, as we targeted a region of Atg8 that is conserved within apicomplexans, we predict that our small molecule will have cross-reactivity against other disease-causing apicomplexans, such as Toxoplasma, Cryptosporidium, Theileria, Neospora, Eimeria, and Babesia.

Creating novel activated factor XI inhibitors through fragment based lead generation and structure aided drug design.

Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC50 of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds.

Investigation of the essentiality of glutamate racemase in Mycobacterium smegmatis.

The mycobacterial cell wall frequently has been used as a target for drug development, and d-glutamate, synthesized by glutamate racemase (MurI), is an important component of peptidoglycan. While the essentiality of the murI gene has been shown in several bacterial species, including Escherichia coli, Bacillus anthracis, and Streptococcus pneumoniae, studies in mycobacteria have not yet provided definitive results. This study aimed to determine whether murI is indeed essential and can serve as a possible target for structure-aided drug design. We have achieved this goal by creating a ΔmurI strain of Mycobacterium smegmatis, a close relative of Mycobacterium tuberculosis. The deletion of the murI gene in M. smegmatis could be achieved only in minimal medium supplemented with D-glutamate, demonstrating that MurI is essential for growth and that glutamate racemase is the only source of D-glutamate for peptidoglycan synthesis in M. smegmatis.

Substrate Channel Flexibility in Pseudomonas aeruginosa MurB Accommodates Two Distinct Substrates

Biosynthesis of UDP-N-acetylmuramic acid in bacteria is a committed step towards peptidoglycan production. In an NADPH- and FAD-dependent reaction, the UDP-N-acetylglucosamine-enolpyruvate reductase (MurB) reduces UDP-N-acetylglucosamine-enolpyruvate to UDP-N-acetylmuramic acid. We determined the three-dimensional structures of the ternary complex of Pseudomonas aeruginosa MurB with FAD and NADP+ in two crystal forms to resolutions of 2.2 and 2.1 Å, respectively, to investigate the structural basis of the first half-reaction, hydride transfer from NADPH to FAD. The nicotinamide ring of NADP+ stacks against the si face of the isoalloxazine ring of FAD, suggesting an unusual mode of hydride transfer to flavin. Comparison with the structure of the Escherichia coli MurB complex with UDP-N-acetylglucosamine-enolpyruvate shows that both substrates share the binding site located between two lobes of the substrate-binding domain III, consistent with a ping pong mechanism with sequential substrate binding. The nicotinamide and the enolpyruvyl moieties are strikingly well-aligned upon superimposition, both positioned for hydride transfer to and from FAD. However, flexibility of the substrate channel allows the non-reactive parts of the two substrates to bind in different conformations. A potassium ion in the active site may assist in substrate orientation and binding. These structural models should help in structure-aided drug design against MurB, which is essential for cell wall biogenesis and hence bacterial survival.

Transcription and replication complex of influenza virus, a new antiviral drugs target.

Transcription and replication by influenza virus are carried out by protein-RNA complexes named RNPs. There are eight of these complexes, each containing one of the eight segments of viral RNA, multiple copies of the viral nucleoprotein and each complex carries a copy of the viral RNA-dependent RNA polymerase. The polymerase itself is a complex of three subunits: PB1, PB2 and PA. Through an effort by laboratories from all over the world, atomic structures have been determined of nucleoproteins of several viral strains and of protein domains of PA and PB2. For PB1, only the structures of the small interfaces with PA and PB2 have been determined. Even though a full understanding of the fundamental processes in the viral life cycle is still lacking, the structures have revealed how nucleoprotein can oligomerize and binds to RNA, how PB1 binds to PA and how the polymerase binds to capped cellular pre-messenger RNA (mRNA) and cleaves this RNA in order to make a capped primer for its own mRNAs (cap-snatching mechanism). The structures also stimulated structure-aided drug design efforts and first generation inhibitors against nucleoprotein oligomerization, binding of PB1 to PA and the cap-snatching activity have been published. Such inhibitors may be developed into new anti-influenza drugs.

Foreword for EDICT Special Edition, volume 2

A central aim of the EDICT consortium was to obtain high resolution structures of a range of integral membrane proteins as a route to structure-aided drug design. The first issue of our special EDICT edition described efforts to express and isolate a wide range of membrane proteins in a number of different expression systems together with selected structureactivity studies. In this second issue we present further structure-activity studies, especially those detailing how structural information can be translated into platforms for the discovery of novel drugs. When no experimental structures are available, researchers depend upon homology models for their studies. Vaziri et al describe the validation of one such model of a major facilitator superfamily (MFS) transporter, using cysteine mutagenesis. In contrast, Patching et al utilized solid state NMR and sample deuteration to explore the low affinity binding site of the E. coli MFS transporter, GalP, demonstrating the usefulness of this technique for investigation of binding site dynamics. Gul et al explored the osmoregulatory properties of the ProUABC transporter, comprised of ProX, ProY and ProW, from E. coli following isolation and reconstitution into proteoliposomes. The findings reveal novel insights into the regulation of this interesting protein complex. Monne et al review the mutagenic data currently available as a means of shedding light on substrate specificity of mitochondrial carriers. This is complementedby the functional characterization of the human mitochondrial ADP/ATP carrier AAC1 described by Mifsud et al. Wohri et al have used isothermal calorimetry to investigate the thermodynamic changes in the pentameric glycine receptor upon ligand binding. In the latter part of the project, members of the EDICT consortium made significant progress in the identification and characterization of novel drug molecules, for a number of different membrane protein targets. Simmons et al used a structurebased virtual high-throughput screening (vHTS) technique initially developed for soluble proteins to identify inhibitors of LeuT and achieved a very impressive hit rate of 45%. Paulsen et al built on earlier extensive structural analysis to confirm the importance of the two C-terminal residues of the Na, K-ATPase in sodium binding. They also describe a combined computer docking and experimental approach to the preliminary characterization of dipeptides with potential to act as novel Na, K-ATPase inhibitors. Jurkowski et al describe a computational approach to the identification of key regions of the galanin receptors with roles in ligand interaction. Finally, Infed et al describe an approach involving modification of a known small molecule to generate novel inhibitors of multi-drug resistance transporters. Taken together, 19 manuscripts published in two special issues of Molecular Membrane Biology provide an overview of almost all of the research expertise required to take a protein from gene to early stage drug discovery. The results of the EDICT consortium have significantly moved the field of membrane protein drug discovery forward and have provided the basis for a range of different studies on these vitally important molecules.

Towards a Structural Comprehension of Bacterial Type VI Secretion Systems: Characterization of the TssJ-TssM Complex of an Escherichia coli Pathovar

Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded β-sheets protein that exhibits a transthyretin fold with an additional α-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membrane-anchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS.

Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist

The α(7) acetylcholine receptor (AChR) mediates pre- and postsynaptic neurotransmission in the central nervous system and is a potential therapeutic target in neurodegenerative, neuropsychiatric and inflammatory disorders. We determined the crystal structure of the extracellular domain of a receptor chimera constructed from the human α(7) AChR and Lymnaea stagnalis acetylcholine binding protein (AChBP), which shares 64% sequence identity and 71% similarity with native α(7). We also determined the structure with bound epibatidine, a potent AChR agonist. Comparison of the structures revealed molecular rearrangements and interactions that mediate agonist recognition and early steps in signal transduction in α(7) AChRs. The structures further revealed a ring of negative charge within the central vestibule, poised to contribute to cation selectivity. Structure-guided mutational studies disclosed distinctive contributions to agonist recognition and signal transduction in α(7) AChRs. The structures provide a realistic template for structure-aided drug design and for defining structure-function relationships of α(7) AChRs.

Structural basis of catalysis by monometalated methionine aminopeptidase.

Methionine aminopeptidase (MetAP) removes the amino-terminal methionine residue from newly synthesized proteins, and it is a target for the development of antibacterial and anticancer agents. Available x-ray structures of MetAP, as well as other metalloaminopeptidases, show an active site containing two adjacent divalent metal ions bridged by a water molecule or hydroxide ion. The predominance of dimetalated structures leads naturally to proposed mechanisms of catalysis involving both metal ions. However, kinetic studies indicate that in many cases, only a single metal ion is required for full activity. By limiting the amount of metal ion present during crystal growth, we have now obtained a crystal structure for a complex of Escherichia coli MetAP with norleucine phosphonate, a transition-state analog, and only a single Mn(II) ion bound at the active site in the position designated M1, and three related structures of the same complex that show the transition from the mono-Mn(II) form to the di-Mn(II) form. An unliganded structure was also solved. In view of the full kinetic competence of the monometalated MetAP, the much weaker binding constant for occupancy of the M2 site compared with the M1 site, and the newly determined structures, we propose a revised mechanism of peptide bond hydrolysis by E. coli MetAP. We also suggest that the crystallization of dimetalated forms of metallohydrolases may, in some cases, be a misleading experimental artifact, and caution must be taken when structures are generated to aid in elucidation of reaction mechanisms or to support structure-aided drug design efforts.

Crystallization to obtain protein–ligand complexes for structure-aided drug design

The use of X-ray crystallography to derive three-dimensional structures for structure-aided drug design (SADD) is a common activity in drug discovery today. In this process, the structures of inhibitors or other ligands of interest complexed with their macromolecular target are solved and the structural information is used iteratively to design new molecules. The ability to form cocrystal complexes between a target protein and a ligand is essential to this process and therefore is of considerable interest to anyone practicing in this field. In the course of obtaining the necessary ligand-protein crystals, even with crystallization conditions well established for a protein of interest, obtaining co-structures with inhibitors either through cocrystallization or soaking is too often not successful. There are numerous potential reasons for this lack of success and this article outlines a number of possible factors that may be involved and discusses considerations that should be taken into account when designing successful experiments to obtain iterative costructures.

Reviews in Angewandte Chemie: Influence, Language, Competition

Reviews in <i>Angewandte Chemie</i>: Influence, Language, Competition

The 1.9 Å Crystal Structure of Alanine Racemase from Mycobacterium tuberculosis Contains a Conserved Entryway into the Active Site,

We report the crystal structure of alanine racemase from Mycobacterium tuberculosis (Alr(Mtb)) at 1.9 A resolution. In our structure, Alr(Mtb) is found to be a dimer formed by two crystallographically different monomers, each comprising 384 residues. The domain makeup of each monomer is similar to that of Bacillus and Pseudomonas alanine racemases and includes both an alpha/beta-barrel at the N-terminus and a C-terminus primarily made of beta-strands. The hinge angle between these two domains is unique for Alr(Mtb), but the active site geometry is conserved. In Alr(Mtb), the PLP cofactor is covalently bound to the protein via an internal aldimine bond with Lys42. No guest substrate is noted in its active site, although some residual electron density is observed in the enzyme's active site pocket. Analysis of the active site pocket, in the context of other known alanine racemases, allows us to propose the inclusion of conserved residues found at the entrance to the binding pocket as additional targets in ongoing structure-aided drug design efforts. Also, as observed in other alanine racemase structures, PLP adopts a conformation that significantly distorts the planarity of the extended conjugated system between the PLP ring and the internal aldimine bond.

Structure-aided drug design's next generation.

Has structural bioinformatics advanced enough to form the core of a drug discovery program? A new generation of companies exploiting structure-focused technologies is counting on it.

PROTEASES OF INFECTIOUS AGENTS

J. Erickson and M. Eissenstat, HIV Protease as a Target for the Design of Antiviral Agents for AIDS. C. Steinkuhler, A. Urbani, and R. De Francesco, Proteases of the Hepatitis C Virus. S. Abdel-Meguid and X. Qiu, Human Herpesvirus Proteases. C. Abad-Zapatero, R. Goldman, and K. Stewart, The Secreted Proteinases from Candida: Challenges for Structure-Aided Drug Design. M.N.G. James and E. Bergman, Proteolytic Enzymes of the Viruses of the Family Picornaviridae. C. Berry, Proteases as Drug Targets in the Treatment of Malaria. J. Cazzulo, Chagas Disease. H. Kido, Y. Chen, M. Murakami, Y. Beppu, and T. Towatari, Cellular Proteinases and Viral Infection: Influenza Virus, Sendai Virus, and HIV-1. M. Lively, Bacterial Type 1 Signal Peptidases. J.A. Garcia, M.R. Fernandez-Fernandez, and J.J. Lopez-Moya, Proteinases Involved in Plant Virus Genome Expression. Subject Index.

Structure of extracellular tissue factor complexed with factor VIIa inhibited with a BPTI mutant

The event that initiates the extrinsic pathway of blood coagulation is the association of coagulation factor VIIa (VIIa) with its cell-bound receptor, tissue factor (TF), exposed to blood circulation following tissue injury and/or vascular damage. The natural inhibitor of the TF·VIIa complex is the first Kunitz domain of tissue factor pathway inhibitor (TFPI-K1). The structure of TF·VIIa reversibly inhibited with a potent (Ki = 0.4 nM) bovine pancreatic trypsin inhibitor (BPTI) mutant (5L15), a homolog of TFPI-K1, has been determined at 2.1 Å resolution. When bound to TF, the four domain VIIa molecule assumes an extended conformation with its light chain wrapping around the framework of the two domain TF cofactor. The 5L15 inhibitor associates with the active site of VIIa similar to trypsin-bound BPTI, but makes several unique interactions near the perimeter of the site that are not observed in the latter. Most of the interactions are polar and involve mutated positions of 5L15. Of the eight rationally engineered mutations distinguishing 5L15 from BPTI, seven are involved in productive interactions stabilizing the enzyme-inhibitor association with four contributing contacts unique to the VIIa·5L15 complex. Two additional unique interactions are due to distinguishing residues in the VIIa sequence: a salt bridge between Arg20 of 5L15 and Asp60 of an insertion loop of VIIa, and a hydrogen bond between Tyr34O of the inhibitor and Lys192NZ of the enzyme. These interactions were used further to model binding of TFPI-K1 to VIIa and TFPI-K2 to factor Xa, the principal activation product of TF·VIIa. The structure of the ternary protein complex identifies the determinants important for binding within and near the active site of VIIa, and provides cogent information for addressing the manner in which substrates of VIIa are bound and hydrolyzed in blood coagulation. It should also provide guidance in structure-aided drug design for the discovery of potent and selective small molecule VIIa inhibitors.