The integration of genomic and structural information in the development of high affinity plasmepsin inhibitors

The plasmepsins are key enzymes in the life cycle of the Plasmodium parasites responsible for malaria. Since plasmepsin inhibition leads to parasite death, these enzymes have been acknowledged to be important targets for the development of new antimalarial drugs. The development of effective plasmepsin inhibitors, however, is compounded by their genomic diversity which gives rise not to a unique target for drug development but to a family of closely related targets. Successful drugs will have to inhibit not one but several related enzymes with high affinity. Structure-based drug design against heterogeneous targets requires a departure from the classic 'lock-and-key' paradigm that leads to the development of conformationally constrained molecules aimed at a single target. Drug molecules designed along those principles are usually rigid and unable to adapt to target variations arising from naturally occurring genetic polymorphisms or drug-induced resistant mutations. Heterogeneous targets need adaptive drug molecules, characterised by the presence of flexible elements at specific locations that sustain a viable binding affinity against existing or expected polymorphisms. Adaptive ligands have characteristic thermodynamic signatures that distinguish them from their rigid counterparts. This realisation has led to the development of rigorous thermodynamic design guidelines that take advantage of correlations between the structure of lead compounds and the enthalpic and entropic components of the binding affinity. In this paper, we discuss the application of the thermodynamic approach to the development of high affinity (K(i) - pM) plasmepsin inhibitors. In particular, a family of allophenylnorstatine-based compounds is evaluated for their potential to inhibit a wide spectrum of plasmepsins.     

Developing hybrid molecule therapeutics for diverse enzyme inhibitory action: Active role of coumarin-based structural leads in drug discovery

Hybrid drugs featuring two or more potentially bioactive pharmacophores have been recognized as advanced and superior chemical entities to simultaneously modulate multiple drug targets of multifactorial diseases, thus overcoming the severe side effects associated with a single drug molecule. The selection of these chemical moieties to produce hybrid structures with druggable properties is generally facilitated by the observed and/or anticipated synergistic pharmacological activities of the individual molecules. In this perspective, coumarin template has extensively been studied in pursuit of structurally diverse leads for drug development due to high affinity and specificity to different molecular targets. This review highlights the most commonly exploited approaches conceptualizing the design and construction of hybrid molecules by coupling two or more individual fragments with or without an appropriate linker. In addition to the design strategies, this review also summarizes and reflects on the therapeutic potential of these hybrid molecules for diverse enzyme inhibitory action as well as their observed structure-activity relationship (SAR). Several key features of the synthesized hybrid structures that assert a profound impact on the inhibitory function have also been discussed alongside computational investigations, inhibitor molecular diversity and selectivity toward multiple drug targets. Finally, these drug discovery and development efforts should serve as a handy reference aiming to provide a useful platform for the exploration of new coumarin-based compounds with enhanced enzyme inhibitory profile.     

High affinity targets of protein kinase inhibitors have similar residues at the positions energetically important for binding

Inhibition of protein kinase activity is a focus of intense drug discovery efforts in several therapeutic areas. Major challenges facing the field include understanding of the factors determining the selectivity of kinase inhibitors and the development of compounds with the desired selectivity profile. Here, we report the analysis of sequence variability among high and low affinity targets of eight different small molecule kinase inhibitors (BIRB796, Tarceva, NU6102, Gleevec, SB203580, balanol, H89, PP1). It is observed that all high affinity targets of each inhibitor are found among a relatively small number of kinases, which have similar residues at the specific positions important for binding. The findings are highly statistically significant, and allow one to exclude the majority of kinases in a genome from a list of likely targets for an inhibitor. The findings have implications for the design of novel inhibitors with a desired selectivity profile (e.g. targeted at multiple kinases), the discovery of new targets for kinase inhibitor drugs, comparative analysis of different in vivo models, and the design of "a-la-carte" chemical libraries tailored for individual kinases.     

Biosensor-based screening and characterization of HIV-1 inhibitor interactions with Sap 1, Sap 2, and Sap 3 from Candida albicans

A surface plasmon resonance (SPR) biosensor-based strategy for identification and characterization of compounds has been devised as a tool for the discovery of specific drugs for treatment of Candida albicans infections. Three secreted aspartic proteases (Saps 1-3) from C. albicans were used as parallel targets. The stepwise procedure involved screening of 104 HIV-1 pro-tease inhibitors at a single concentration for binding to the targets. Twenty-four compounds that appeared to interact with the targets were identified in the screen. False positives and compounds with low affinities or very fast dissociation rates could be removed after a series of additional measurements of these compounds at 3 different concentrations. Kinetic characterization was performed with 13 compounds, giving information about the interaction mechanism and interaction kinetic parameters (k(on), k(off), and K(D)). The pH dependence of the interaction and the inhibitory effect of a final small set of compounds were also evaluated. The strategy resulted in the identification of ritonavir as the compound generally exhibiting the highest affinity for the Candida enzymes. It had similar interaction kinetic characteristics for Sap 1 and Sap 2 but a lower affinity for Sap 3 due to a slower association rate. Several additional compounds with high affinity and/or slow dissociation rates for the targets were identified, revealing 2 other structural scaffolds for Sap inhibitors. In addition, important differences in the specificity for these types of compounds by the Saps were identified. The stepwise biosensor-based strategy was consequently efficient for identification and characterization of new lead compounds for 3 important drug targets.     

Celastrol analogues as inducers of the heat shock response. Design and synthesis of affinity probes for the identification of protein targets

The natural product celastrol (1) possesses numerous beneficial therapeutic properties and affects numerous cellular pathways. The mechanism of action and cellular target(s) of celastrol, however, remain unresolved. While a number of studies have proposed that the activity of celastrol is mediated through reaction with cysteine residues, these observations have been based on studies with specific proteins or by in vitro analysis of a small fraction of the proteome. In this study, we have investigated the spatial and structural requirements of celastrol for the design of suitable affinity probes to identify cellular binding partners of celastrol. Although celastrol has several potential sites for modification, some of these were not synthetically amenable or yielded unstable analogues. Conversion of the carboxylic acid functionality to amides and long-chain analogues, however, yielded bioactive compounds that induced the heat shock response (HSR) and antioxidant response and inhibited Hsp90 activity. This led to the synthesis of biotinylated celastrols (23 and 24) that were used as affinity reagents in extracts of human Panc-1 cells to identify Annexin II, eEF1A, and β-tubulin as potential targets of celastrol.     

Nuclear magnetic resonance spectroscopy reveals the functional state of the signalling protein CheY in vivo in Escherichia coli

Two-component signal transduction (TCST) pathways are regulatory systems that are highly homologous throughout the bacterial kingdom. Their established role in virulence and absence in vertebrates has made TCST an attractive target for therapeutic intervention. However, such systems have yet to yield success in the development of novel antibiotics. CheY serves as a prototype for the analysis of response regulator function. The protein structure exhibits several conformations by both X-ray and nuclear magnetic resonance (NMR) analyses. Knowledge of which structures are relevant in vivo would be valuable in a rational drug design project. Our aim was to probe the in vivo conformation and ligand binding of CheY in Escherichia coli under resting conditions by in-cell NMR methods. CheY was selectively labelled with 15N by the control of growth and expression conditions. NMR spectra obtained in vivo demonstrated that the Mg2+ complex was the predominant form even though cells were resuspended in metal-free buffers and the intracellular free Mg2+ was low. In-cell NMR also confirmed the uptake and in vivo binding mode to CheY of small-molecular-weight compounds identified in vitro. This paper reports the first observation of the structure and interactions with a potential drug of a regulator protein in its native host in vivo using NMR spectroscopy.     

Synthesis of methyl 5-S-alkyl-5-thio-D-arabinofuranosides and evaluation of their antimycobacterial activity

The emergence of drug resistant tuberculosis necessitates a search for new antimycobacterial compounds. The antigen 85 (ag85) complex is a family of mycolyl transferases involved in the synthesis of trehalose-6,6'-dimycolate and the mycolated hexasaccharide motif found at the terminus of the arabinogalactan in mycobacterium. Enzymes involved in the synthesis of cell wall structures like these are potential targets for the development of new antiinfectives. To potentially inhibit the ag85 complex, methyl 5-S-alkyl-5-thio-arabinofuranoside analogues were designed based on docking studies with ag85C derived from Mycobacterium tuberculosis. The target arabinofuranosides were then synthesized and the antibacterial activity evaluated against Mycobacterium smegmatis ATCC 14468. Two of the compounds, 5-S-octyl-5-thio-alpha-d-arabinofuranoside (8) and 5-S-octyl-5-thio-beta-d-arabinofuranoside (11), showed MICs of 256 and 512microg/mL, respectively. Attempts to directly evaluate acyltransferase inhibitory activity of the arabinofuranosides against ag85C are also described. In conclusion, a new class of antimycobacterial arabinofuranosides has been discovered.     

Investigating Apoptozole as a Chemical Probe for HSP70 Inhibition

The use of chemical tools to validate clinical targets has gained in popularity over recent years and the importance of understanding the activity, selectivity and mechanism of action of these compounds is well recognized. Dysregulation of the HSP70 protein family has been linked to multiple cancer types and drug resistance, highlighting their importance as popular targets for anti-cancer drug development. Apoptozole is a recently identified small molecule, which has been reported to possess strong affinity for the HSP70 isoforms HSP72 and HSC70. We investigated apoptozole as a potential chemical tool for HSP70 inhibition. Unfortunately, using both biochemical and biophysical techniques, we were unable to find any experimental evidence that apoptozole binds to HSP70 in a specific and developable way. Instead, we provide experimental evidence that apoptozole forms aggregates under aqueous conditions that could interact with HSP70 proteins in a non-specific manner.     

A call for using natural compounds in the development of new antimalarial treatments - an introduction

Natural compounds, mostly from plants, have been the mainstay of traditional medicine for thousands of years. They have also been the source of lead compounds for modern medicine, but the extent of mining of natural compounds for such leads decreased during the second half of the 20th century. The advantage of natural compounds for the development of drugs derives from their innate affinity for biological receptors. Natural compounds have provided the best anti-malarials known to date. Recent surveys have identified many extracts of various organisms (mostly plants) as having antiplasmodial activity. Huge libraries of fractionated natural compounds have been screened with impressive hit rates. Importantly, many cases are known where the crude biological extract is more efficient pharmacologically than the most active purified compound from this extract. This could be due to synergism with other compounds present in the extract, that as such have no pharmacological activity. Indeed, such compounds are best screened by cell-based assay where all potential targets in the cell are probed and possible synergies identified. Traditional medicine uses crude extracts. These have often been shown to provide many concoctions that deal better with the overall disease condition than with the causative agent itself. Traditional medicines are used by ~80 % of Africans as a first response to ailment. Many of the traditional medicines have demonstrable anti-plasmodial activities. It is suggested that rigorous evaluation of traditional medicines involving controlled clinical trials in parallel with agronomical development for more reproducible levels of active compounds could improve the availability of drugs at an acceptable cost and a source of income in malaria endemic countries.     

Selective ligand purification using high-performance affinity beads

Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.     

Design, synthesis, and biological evaluation of alcyopterosin A and illudalane derivatives as anticancer agents

The synthesis of alcyopterosin A and a series of new derivatives possessing an illudalane skeleton is described. The DNA binding properties of these compounds have been examined and compared to those of reference drugs using a UV spectroscopy technique. The antitumor activity of selected compounds against a panel of 60 human tumor cell lines was tested in the in vitro anticancer screening of the National Cancer Institute. Redox properties were also evaluated. Tested compounds showed significant DNA affinity, derivatives 6 and 15 exhibited remarkable antiproliferative activity and have been identified as new leads in the antitumor strategies.     

Inhibition of the function of class IIa HDACs by blocking their interaction with MEF2

Enzymes that modify the epigenetic status of cells provide attractive targets for therapy in various diseases. The therapeutic development of epigenetic modulators, however, has been largely limited to direct targeting of catalytic active site conserved across multiple members of an enzyme family, which complicates mechanistic studies and drug development. Class IIa histone deacetylases (HDACs) are a group of epigenetic enzymes that depends on interaction with Myocyte Enhancer Factor-2 (MEF2) for their recruitment to specific genomic loci. Targeting this interaction presents an alternative approach to inhibiting this class of HDACs. We have used structural and functional approaches to identify and characterize a group of small molecules that indirectly target class IIa HDACs by blocking their interaction with MEF2 on DNA.Weused X-ray crystallography and (19)F NMRto show that these compounds directly bind to MEF2. We have also shown that the small molecules blocked the recruitment of class IIa HDACs to MEF2-targeted genes to enhance the expression of those targets. These compounds can be used as tools to study MEF2 and class IIa HDACs in vivo and as leads for drug development.     

A critique of the molecular target-based drug discovery paradigm based on principles of metabolic control: advantages of pathway-based discovery

Contemporary drug discovery and development (DDD) is dominated by a molecular target-based paradigm. Molecular targets that are potentially important in disease are physically characterized; chemical entities that interact with these targets are identified by ex vivo high-throughput screening assays, and optimized lead compounds enter testing as drugs. Contrary to highly publicized claims, the ascendance of this approach has in fact resulted in the lowest rate of new drug approvals in a generation. The primary explanation for low rates of new drugs is attrition, or the failure of candidates identified by molecular target-based methods to advance successfully through the DDD process. In this essay, I advance the thesis that this failure was predictable, based on modern principles of metabolic control that have emerged and been applied most forcefully in the field of metabolic engineering. These principles, such as the robustness of flux distributions, address connectivity relationships in complex metabolic networks and make it unlikely a priori that modulating most molecular targets will have predictable, beneficial functional outcomes. These same principles also suggest, however, that unexpected therapeutic actions will be common for agents that have any effect (i.e., that complexity can be exploited therapeutically). A potential operational solution (pathway-based DDD), based on observability rather than predictability, is described, focusing on emergent properties of key metabolic pathways in vivo. Recent examples of pathway-based DDD are described. In summary, the molecular target-based DDD paradigm is built on a na?ve and misleading model of biologic control and is not heuristically adequate for advancing the mission of modern therapeutics. New approaches that take account of and are built on principles described by metabolic engineers are needed for the next generation of DDD.     

Tricyclic GyrB/ParE (TriBE) Inhibitors: A New Class of Broad-Spectrum Dual-Targeting Antibacterial Agents

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.     

Arginine-aminoglycoside conjugates that bind to HIV transactivation responsive element RNA in vitro

HIV gene expression is crucially dependent on binding of the viral Tat protein to the transactivation RNA response element. A number of synthetic Tat-transactivation responsive element interaction inhibitors of peptide/peptoid nature were described as potential antiviral drug prototypes. We present a new class of peptidomimetic inhibitors, conjugates of L-arginine with aminoglycosides. Using a gel-shift assay and affinity chromatography on an L-arginine column we found that these compounds bind specifically to the transactivation responsive element RNA in vitro with Kd values in the range of 20-400 nM, which is comparable to the Kd of native Tat bound to the transactivation responsive element (10-12 nM). Confocal microscopy studies demonstrated that fluorescein-labelled conjugate penetrates into live cells. High affinity to the transactivation responsive element, low toxicity, and relative simplicity of synthesis make these compounds attractive candidates for antiviral drug design.     

NMR fragment-based screening for development of the CD44-binding small molecules

The cell-surface protein CD44, a primary receptor for hyaluronic acid (HA), is one of the most promising targets for cancer therapies. It is prominently involved in the process of tumor growth and metastasis. The possibility of modulating the CD44-HA interaction with a pharmacological inhibitor is therefore of great importance, yet until now there are only few small molecules reported to bind to CD44. Here, we describe the results of the NMR fragment-based screening conducted against CD44 by which we found eight new hit compounds that bind to the receptor with the affinity in milimolar range. The NMR-based characterization revealed that there are two possible binding modes for these compounds, and for some of them the binding is no longer possible in the presence of hyaluronic acid. This could provide an interesting starting point for the development of new high-affinity ligands targeting the CD44-HA axis.     

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.     

Accessing target information by virtual parallel screening—The impact on natural product research

Secondary metabolites exhibit an astonishing multitude of functionalities and enormous chemical diversity and these qualities are responsible for their favoured selection as drug leads. The complex process of finding natural products’ bioactivities is largely based on trial and error, and is therefore risky, time- and cost-intensive. In recent decades, computer-assisted techniques have emerged as promising tools to manage the huge amount of available structural data of macromolecular targets and compounds annotated to specific functions, and to extract knowledge from these data for the prediction of new events. The novel concept of virtual parallel screening aims to access a pharmacological profile for each compound screened using an array of macromolecular targets. Providing putative ligand–target interactions, this in silico multitarget application meets the requirements for natural product research in a complementary way. It enables (i) a fast identification of potential targets (target fishing), (ii) insight into a putative molecular mechanism, and (iii) an estimation of the bioactivity profile which allows for prioritizing experimental investigations. The first application examples in natural product research are described.     

Biomolecular NMR: a chaperone to drug discovery

Biomolecular NMR now contributes routinely to every step in the development of new chemical entities ahead of clinical trials. The versatility of NMR--from detection of ligand binding over a wide range of affinities and a wide range of drug targets with its wealth of molecular information, to metabolomic profiling, both ex vivo and in vivo--has paved the way for broadly distributed applications in academia and the pharmaceutical industry. Proteomics and initial target selection both benefit from NMR: screenings by NMR identify lead compounds capable of inhibiting protein-protein interactions, still one of the most difficult development tasks in drug discovery. NMR hardware improvements have given access to the microgram domain of phytochemistry, which should lead to the discovery of novel bioactive natural compounds. Steering medicinal chemists through the lead optimisation process by providing detailed information about protein-ligand interactions has led to impressive success in the development of novel drugs. The study of biofluid composition--metabonomics--provides information about pharmacokinetics and helps toxicological safety assessment in animal model systems. In vivo, magnetic resonance spectroscopy interrogates metabolite distributions in living cells and tissues with increasing precision, which significantly impacts the development of anticancer or neurological disorder therapeutics. An overview of different steps in recent drug discovery is presented to illuminate the links with the most recent advances in NMR methodology.