Three-dimensional models for integral membrane proteins: Possibilities and pitfalls

Summary Some three-dimensional model-building techniques applicable to membrane proteins are presented. Requirements for a successful modeling project are outlined, and methodological limitations are discussed. One example of a modeling exercise for the ₂ adrenergic receptor is reviewed briefly, and the utility of such studies is explored. The explosion of data for integral membrane protein sequences and properties make 3D modeling studies increasingly feasible and necessary. Model-building exercises should enable us to take better advantage of new protein sequence data, and studies of this type are likely to proliferate in the future.     

Challenges and opportunities for new protein crystallization strategies in structure-based drug design

Structure-based drug design (SBDD) has emerged as a valuable pharmaceutical lead discovery tool, showing potential for accelerating the discovery process, while reducing developmental costs and boosting potencies of the drug that is ultimately selected. SBDD is an iterative, rational, lead compound sculpting process that involves both the synthesis of new derivatives and the evaluation of their binding to the target structure either through computational docking or elucidation of the target structure as a complex with the lead compound. This method heavily relies on the production of high-resolution (< 2 Å) 3D structures of the drug target, obtained through X-ray crystallographic analysis, in the presence or absence of the drug candidate. The lack of generalized methods for high quality crystal production is still a major bottleneck in the process of macromolecular crystallization. This review provides a brief introduction to SBDD and describes several macromolecular crystallization strategies, with an emphasis on advances and challenges facing researchers in the field today. Recent trends in the development of more universal macromolecular crystallization techniques, particularly nucleation-based techniques that are applicable to both soluble and integral membrane proteins, are also discussed.     

A Novel Pharmacophore Model for the Design of Anthrax Lethal Factor Inhibitors

This study aims at the identification of novel structural features on the surface of the Zn-dependant metalloprotease lethal factor (LF) from anthrax onto which to design novel and selective inhibitors. We report that by targeting an unexplored region of LF that exhibits ligand-induced conformational changes, we could obtain inhibitors with at least 30-fold LF selectivity compared to two other most related human metalloproteases, MMP-2 and MMP-9. Based on these results, we propose a novel pharmacophore model that, together with the preliminarily identified compounds, should help the design of more potent and selective inhibitors against anthrax.     

From Zn to Mn: the study of novel manganese-binding groups in the search for new drugs against tuberculosis

In most eubacteria, apicomplexans, and most plants, including the causal agents for diseases such as malaria, leprosy, and tuberculosis, the methylerythritol phosphate pathway is the route for the biosynthesis of the C(5) precursors to the essential isoprenoid class of compounds. Owing to their absence in humans, the enzymes of the methylerythritol phosphate pathway have become attractive targets for drug discovery. This work investigates a new class of inhibitors against the second enzyme of the pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Inhibition of this enzyme may involve the chelation of a crucial active site Mn ion, and the metal-chelating moieties studied here have previously been shown to be successful in application to the zinc-dependent metalloproteinases. Quantum mechanics and docking calculations presented in this work suggest the transferability of these metal-chelating compounds to Mn-containing 1-deoxy-D-xylulose 5-phosphate reductoisomerase enzyme, as a promising starting point to the development of potent inhibitors.     

Molecular dynamics in drug design: new generations of compstatin analogs

We report the computational and rational design of new generations of potential peptide-based inhibitors of the complement protein C3 from the compstatin family. The binding efficacy of the peptides is tested by extensive molecular dynamics-based structural and physicochemical analysis, using 32 atomic detail trajectories in explicit water for 22 peptides bound to human, rat or mouse target protein C3, with a total of 257 ns. The criteria for the new design are: (i) optimization for C3 affinity and for the balance between hydrophobicity and polarity to improve solubility compared to known compstatin analogs; and (ii) development of dual specificity, human-rat/mouse C3 inhibitors, which could be used in animal disease models. Three of the new analogs are analyzed in more detail as they possess strong and novel binding characteristics and are promising candidates for further optimization. This work paves the way for the development of an improved therapeutic for age-related macular degeneration, and other complement system-mediated diseases, compared to known compstatin variants.     

Rational Design of Multitargeted Tyrosine Kinase Inhibitors: A Novel Approach

The non‐receptor Src tyrosine kinase is known to cooperate with the epidermal growth factor receptor in a mechanism leading to invasion and metastasis of solid tumours. With the purpose of developing agents targeted to both epidermal growth factor receptor and Src or related kinases, we embarked on the design of chimeric molecules termed combi‐molecules capable of blocking both Src and epidermal growth factor receptor. To this end, we have chosen to design molecules containing a quinazoline moiety (directed at epidermal growth factor receptor) and a 7‐phenyl‐pyrazolopyrimidine (directed at Src). Molecular modelling showed that the optimal position to attach the linker was the 6‐position of the quinazoline and the 9‐position of the pyrazolopyrimidine. This has led to the synthesis of SB162, SB166 and SB163. SB163 containing the longest linker was the only molecule capable of inducing a dose‐dependent inhibition of both Src and epidermal growth factor receptor. SB163 also induced a dose inhibition of Abl and PDGFR.     

Salinomycin: a new cancer drug candidate

Very recently, it has been shown that it is possible to selectively kill breast cancer stem cells using the ionophore antibiotic, salinomycin. Its ability to kill cancer stem cells and apoptosis-resistant cancer cells may define salinomycin as a novel anticancer drug.     

Virtual design of chemical penetration enhancers for transdermal drug delivery

Traditional drug design is a laborious and expensive process that often challenges the pharmaceutical industries. As a result, researchers have turned to computational methods for computer-assisted molecular design. Recently, genetic and evolutionary algorithms have emerged as efficient methods in solving combinatorial problems associated with computer-aided molecular design. Further, combining genetic algorithms with quantitative structure-property relationship analyses has proved effective in drug design. In this work, we have integrated a new genetic algorithm and nonlinear quantitative structure-property relationship models to develop a reliable virtual screening algorithm for the generation of potential chemical penetration enhancers. The genetic algorithms-quantitative structure-property relationship algorithm has been implemented successfully to identify potential chemical penetration enhancers for transdermal drug delivery of insulin. Validation of the newly identified chemical penetration enhancer molecular structures was conducted through carefully designed experiments, which elucidated the cytotoxicity and permeability of the chemical penetration enhancers.     

Bacterial toxins: an outlook

An overview of some current issues in bacterial toxinology is presented.     

Multitarget ligands in antibacterial research: progress and opportunities

Introduction: Resistance to current antibacterial therapies is an inevitability that represents a significant global health concern. Bacteria have the capacity to render all current drug treatments ineffective, which places a demand on the drug discovery community to constantly develop new antibacterial agents. Compounds that inhibit multiple biological targets, often referred to as multitarget ligands, are an inviting prospect in antibacterial research because, although they will not solve the issue of resistance, they might help to delay the onset.

Areas covered: This review covers some of the recent progress in identifying new ligands that deliberately interact with more than one essential biological target in bacteria. The two principal areas covered are inhibitors of DNA replication and cell wall biosynthesis.

Expert opinion: Antibacterial programs for the design of multitarget ligands present an important opportunity for production of antibacterial agents. Their longevity, due to slow development of resistance, is comparable to that seen with other successful agents – but is much improved over single-targeted agents for which resistance can appear in vitro overnight. The preclinical development of these agents will have to overcome the standard problems of antibacterial discovery. Such problems include optimization of characteristics favoring cell entry and particularly the demonstration of selectivity of inhibition of the desired multiple targets without inhibition of other bacterial or any mammalian functions.     

Kinetic parameters for the reaction of 1,2,4‐trioxolanes and artemisinin with iron(II): New evidence for the source of antimalarial activity

The Fenton‐like reductive cleavage of antimalarial peroxides like artemisinin by iron(II) species is a chemical reaction whose mechanistic pathway has not been yet fully understood; it is, however, known that there is considerable production of radical species centered at both the oxygen and carbon, which are important to the therapeutical effects of those compounds. This article reports kinetic data for the reaction of artemisinin and two model 1,2,4‐trioxolanes with iron(II) species and also a mechanistic interpretation of this reductive cleavage from transition state thermodynamics. The suggestion of the presence of an enhancing specific factor inside the plasmodium is made.     

Discovery of TDI-10229: A Potent and Orally Bioavailable Inhibitor of Soluble Adenylyl Cyclase (sAC,ADCY10)

Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 (12), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound.     

A Protein Relational Database and Protein Family Knowledge Bases to Facilitate Structure-Based Design Analyses

The Protein Data Bank is the most comprehensive source of experimental macromolecular structures. It can, however, be difficult at times to locate relevant structures with the Protein Data Bank search interface. This is particularly true when searching for complexes containing specific interactions between protein and ligand atoms. Moreover, searching within a family of proteins can be tedious. For example, one cannot search for some conserved residue as residue numbers vary across structures. We describe herein three databases, Protein Relational Database, Kinase Knowledge Base, and Matrix Metalloproteinase Knowledge Base, containing protein structures from the Protein Data Bank. In Protein Relational Database, atom–atom distances between protein and ligand have been precalculated allowing for millisecond retrieval based on atom identity and distance constraints. Ring centroids, centroid–centroid and centroid–atom distances and angles have also been included permitting queries for π-stacking interactions and other structural motifs involving rings. Other geometric features can be searched through the inclusion of residue pair and triplet distances. In Kinase Knowledge Base and Matrix Metalloproteinase Knowledge Base, the catalytic domains have been aligned into common residue numbering schemes. Thus, by searching across Protein Relational Database and Kinase Knowledge Base, one can easily retrieve structures wherein, for example, a ligand of interest is making contact with the gatekeeper residue.     

Cyclic GMP and protein kinase-G in myocardial ischaemia-reperfusion: opportunities and obstacles for survival signaling

It is clear that multiple signalling pathways regulate the critical balance between cell death and survival in myocardial ischaemia-reperfusion. Recent attention has focused on the activation of survival or salvage kinases, particularly during reperfusion, as a common mechanism of many cardioprotective interventions. The phosphatidyl inositol 3'-hydroxy kinase/Akt complex (PI3K/Akt) and p42/p44 mitogen-activated protein kinase cascades have been widely promoted in this respect but the cyclic guanosine 3',5'-monophosphate/cGMP-dependent protein kinase (cGMP/PKG) signal transduction cassette has been less systematically investigated as a survival cascade. We propose that activation of the cGMP/PKG signalling pathway, following activation of soluble or particulate guanylate cyclases, may play a pivotal role in survival signalling in ischaemia-reperfusion, especially in the classical preconditioning, delayed preconditioning and postconditioning paradigms. The resurgence of interest in reperfusion injury, largely as a result of postconditioning-related research, has confirmed that the cGMP/PKG pathway is a pivotal salvage mechanism in reperfusion. Numerous studies suggest that the infarct-limiting effects of preconditioning and postconditioning, exogenously donated nitric oxide (NO), natriuretic peptides, phosphodiesterase inhibitors, and other diverse drugs and mediators such as HMG co-A reductase inhibitors (statins), Rho-kinase inhibitors and adrenomedullin, whether given before and during ischaemia, or specifically at the onset of reperfusion, may be mediated by activation or enhancement of the cGMP pathway, either directly or indirectly via endogenous NO generation downstream of PI3K/Akt. Putative mechanisms of protection include PKG regulation of Ca(2+) homeostasis through the modification of sarcoplasmic reticulum Ca(2+) uptake mechanisms, and PKG-induced opening of ATP-sensitive K(+) channels during ischaemia and/or reperfusion. At present, significant technical obstacles in defining the precise roles played by cGMP/PKG signalling include the heavy reliance on pharmacological PKG inhibitors of uncertain selectivity, difficulties in determining PKG activity in intact tissue, and the growing recognition that intracellular compartmentalisation of the cGMP pool may contribute markedly to the nucleotide's biological actions and biochemical determination. Overall, the body of experimental evidence suggests that cGMP/PKG survival signalling ameliorates irreversible injury associated with ischaemia-reperfusion and may be a tractable therapeutic target.     

Combinatorial design of biomaterials for drug delivery: opportunities and challenges

Background: The rational design of biodegradable polymeric devices for controlled drug delivery and tissue engineering is an important area of research for advancing new therapies for cancer, diabetes and immune-related disorders. In an era of escalating costs for discovery-based research, there is an urgent need to develop new and rapid methods to design drug delivery systems. Objective/methods: By merging this field of study with rapid and high throughput methods of design, optimization and development, researchers have been able to accelerate the discovery and design processes for these devices. Combinatorial research enables the rapid identification of key regions of interest. Conclusion: This review focuses on the opportunities and challenges in the area of combinatorial biomaterials design for drug delivery, as there has been a great deal of significant progress over the past decade to propel this approach for the rational design of biomaterials.     

Adaptive clinical trials for new drug applications in Japan

Adaptive design is regarded as an efficient method for clinical trials in order to increase the success rate of a new drug in development, and recently has been actively discussed among regulatory agencies, industry and academia. Since adaptive design involves interim analyses and is more complex than traditional fixed design, some points such as possibility of introducing statistical and operational bias should be considered when planning and implementing such trials. In this article, we share our perspectives in the consideration of adaptive design clinical trials based on our experiences discussing adaptive design in clinical trial consultation meetings in Japan.     

A new generation of potent complement inhibitors of the Compstatin family

Compstatin family peptides are potent inhibitors of the complement system and promising drug candidates against diseases involving under-regulated complement activation. Compstatin is a 13-residue cyclized peptide that inhibits cleavage of complement protein C3, preventing downstream complement activation. We present three new compstatin variants, characterized by tryptophan replacement at positions 1 and/or 13. Peptide design was based on physicochemical reasoning and was inspired by earlier work, which identified tryptophan substitutions at positions 1 and 13 in peptides with predicted C3c binding abilities [Bellows M.L., Fung H.K., Taylor M.S., Floudas C.A., López de Victoria A., Morikis D. (2010) Biophys J; 98: 2337-2346]. The new variants preserve distinct polar and nonpolar surfaces of compstatin, but have altered local interaction capabilities with C3. All three peptides exhibited potent C3 binding by surface plasmon resonance and potent complement inhibition by enzyme-linked immunosorbent assay. We also present enzyme-linked immunosorbent assay data and detailed surface plasmon resonance kinetic data of three peptides from previous computational design.