Tagged fragment method for evolutionary structure-based de novo lead generation and optimization

Here we describe a computer-assisted de novo drug design method, EAISFD, which combines the de novo design engine EA-Inventor with a scoring function featuring the molecular docking program Surflex-Dock. This method employs tagged fragments, which are preserved substructures in EA-Inventor used for base fragment matching in Surflex-Dock, for constructing ligand structures under specific binding motifs. In addition, a target score mechanism is adopted that allows EAISFD to deliver a diverse set of desired structures. This method can be used to design novel ligand scaffolds (lead generation) or to optimize attachments on a fixed scaffold (lead optimization). EAISFD has successfully suggested many known inhibitor scaffolds as well as a number of new scaffold types when applied to p38 MAP kinase.     

Predicting synthetic accessibility: application in drug discovery and development

The estimation and use of synthetic accessibility in the drug discovery process is discussed. A distinction is drawn between synthetic feasibility and accessibility and the practical uses of an accessibility score are examined. Various techniques used in the estimation of accessibility are described and their utility and potential accuracy compared.     

Computational combinatorial chemistry for de novo ligand design: Review and assessment

Summary Computational combinatorial chemistry divides the ligand design problem into three parts: the search for optimal positions and orientations of functional groups in the binding site, the connection of such optimally placed fragments to form candidate ligands, and the estimation of their binding constants. In this review, approaches to each of these problems are described. The present limitations of methodologies are indicated and efforts to improve them are outlined. Applications to HIV-1 aspartic proteinase, which is a target for the development of AIDS therapeutic agents, and human thrombin, a multifunctional enzyme that has a central role in both haemostasis and thrombosis, are presented. The relation between combinatorial methods for drug discovery on the computer and in the laboratory is addressed.Inquiries concerning the CHARMM, MCSS, and HOOK programs should be addressed to M. Karplus (e-mail: marci@tammy.harvard.edu). Inquiries concerning the program CONNECT should be addressed to A. Caflisch at Department of Biochemistry, University of Zürich, CH-8057 Zürich, Switzerland.     

Scaffold hopping in de novo design. Ligand generation in the absence of receptor information

We report here the de novo generation of chemotypes and scaffolds for the estrogen receptor, without use of the receptor structure in the assembly phase. Through use of ligand superpositions or a single bound conformation of a known active, a pseudoreceptor can be generated as a design envelope, within which novel structures are readily assembled. Many of these structures have high similarity to known chemotypes. Scaffold hopping is readily achieved within this pseudoreceptor, indicating the advantages of such an approach in discovery research.     

SkelGen: a general tool for structure-based de novo ligand design

The recent lapse in productivity in the pharmaceutical industry has facilitated the emergence of experimental and in silico structure-based design methodologies, based on identification of biologically active low molecular weight fragments that can be exploited to produce potential drug candidates with diverse chemistries. SkelGen, an in silico example of this methodology, is reviewed. The ability of this algorithm to identify chemically diverse low molecular weight fragments that would potentially bind to DNA gyrase is recounted, as is the first purely de novo structure-based design of five compounds that show at least micromolar activity against the estrogen receptor. The ability of the algorithm to incorporate partial protein flexibility during its design of compounds to the estrogen receptor is discussed, and an opinion as to the near and long-term futures for de novo design algorithms is expressed.     

Structure-based generation of a new class of potent Cdk4 inhibitors: new de novo design strategy and library design.

As a first step in structure-based design of highly selective and potent Cdk4 inhibitors, we performed structure-based generation of a novel series of Cdk4 inhibitors. A Cdk4 homology model was constructed according to X-ray analysis of an activated form of Cdk2. Using this model, we applied a new de novo design strategy which combined the de novo design program LEGEND with our in-house structure selection supporting system SEEDS to generate new scaffold candidates. In this way, four classes of scaffold candidates including diarylurea were identified. By constructing diarylurea informer libraries based on the structural requirements of Cdk inhibitors in the ATP binding pocket of the Cdk4 model, we were able to identify a potent Cdk4 inhibitor N-(9-oxo-9H-fluoren-4-yl)-N'-pyridin-2-ylurea 15 (IC(50) = 0.10 microM), together with preliminary SAR. We performed a docking study between 15 and the Cdk4 model and selected a reasonable binding mode which is consistent with the SAR. Further modification based on the proposed binding mode provided a more potent compound, N-[(9bR)-5-oxo-2,3,5,9b-tetrahydro-1H-pyrrolo[2,1-a]isoindol-9-yl]-N'-pyridin-2-ylurea 26a (IC(50) = 0.042 microM), X-ray analysis of which was accomplished by the soaking method. The predicted binding mode of 15 in Cdk4 was validated by X-ray analysis of the Cdk2-26a complex.     

Activation of de novo synthetic pathway of ceramides is responsible for the initiation of hydrogen peroxide-induced apoptosis in HL-60 cells

Sphingolipid metabolites in HL-60 cells were analyzed to gain an understanding of their roles in early events underlying hydrogen peroxide (H2O2)-induced apoptosis. Incubation of cells with H2O2 increased the intracellular levels of ceramides and sphinganine, but decreased those of ceramide 1-phosphates (ceramide 1-P) and sphingosine. The levels of sphingomyelins and sphingomyelinase (SMase) activities were not affected by H2O2 treatment. These results were similar to the profiles induced by daunorubicin, an activator of serine palmitoyl CoA transferase (SPT), suggesting that H2O2 stimulated the de novo synthetic pathway of ceramides. L-cycloserine and fumonisin B1 (FB1), specific inhibitors of de novo ceramide biosynthesis, suppressed the elevation of ceramides and sphinganine induced by H2O2, which consequently reduced apoptotic cell death. Collectively, these results demonstrated that H2O2 increased the intracellular concentrations of ceramides via activation of a de novo biosynthetic pathway, and the enhanced ceramides might initiate apoptosis in HL-60 cells.     

Carbopeptides: carbohydrates as potential templates for de novo design of protein models

Abstract: De novo design of proteins has evolved into a powerful approach for studying the factors governing protein folding and stability. Among the families of structures frequently studied is the ‘four‐helix bundle’ in which four α‐helical peptide strands, linked by loops, form a hydrophobic core. Assembly of protein models on a template has been suggested as a way to reduce the entropy of folding. Here we describe the potential use of a carbohydrate as such a template. The monosaccharide d ‐galactose was per‐O‐acylated with (N^β‐Fmoc‐βAla)₂O to give a penta‐substituted derivative, which was converted to the corresponding glycosyl bromide and used for the glycosylation of 4‐hydroxymethylbenzoic acid pentafluorophenyl ester (HMBA‐OPfp). The β‐glycosidic carbohydrate template (N^β‐Fmoc‐βAla)₄‐β‐d ‐Galp‐(1‐O)‐MBA‐OPfp thus obtained was coupled to a PAL‐PEG‐PS resin and simultaneously extended at the four arms to yield, after cleavage from the solid support, a carbopeptide with four identical peptide strands. Extension of this concept to, for example, synthesis of novel multiple antigenic peptides (MAPs) and synthesis of carbohydrate clusters can be easily envisioned. The ability to efficiently synthesize such structures sets the stage for further studies to test whether the carbohydrate templates do indeed nucleate folding.     

Sphingoid bases and de novo ceramide synthesis: enzymes involved, pharmacology and mechanisms of action.

The sphingoid base backbones of sphingolipids (sphingosines, sphinganines, 4-hydroxysphinganines and others) are highly bioactive species directly and-in most cases-as their metabolites, the N-acyl-sphingoid bases (ceramides) and sphingoid base 1-phosphates. The complexity of these compounds affords many opportunities to prepare synthetic analogs for studies of sphingolipid metabolism and the functions of the sphingoid bases and metabolites. Described in this review are methods for the preparation of libraries of sphingoid bases, including a series of 1-deoxy-analogs, as well as information about their metabolism and biological activities. Findings with these compounds have uncovered some of the complications of working with compounds that mimic a naturally occurring biomodulator-such as that they are sometimes metabolized by enzymes that handle the endogenous compounds and the products may have potent (and unexpected) biological activities. Through studying such compounds, there is now a greater understanding of the metabolism and mechanism(s) of action of naturally occurring sphingoid bases as well as of these analogs.     

In silico studies on recreational drugs: 3D quantitative structure activity relationship prediction of classified and de novo designer benzodiazepines

Currently, increasing availability and popularity of designer benzodiazepines (DBZDs) constitutes a primary threat to public health. To assess this threat, the biological activity/potency of DBZDs was investigated using in silico studies. Specific Quantitative Structure Activity Relationship (QSAR) models were developed in Forge™ for the prediction of biological activity (IC₅₀) on the γ-aminobutyric acid A receptor (GABA-AR) of previously identified classified and unclassified DBDZs. A set of new potential ligands resulting from scaffold hopping studies conducted with MOE^® was also evaluated. Two generated QSAR models (i.e. 3D-field QSAR and RVM) returned very good performance statistics (r² = 0.98 [both] and q² = 0.75 and 0.72, respectively). The DBZDs predicted to be the most active were flubrotizolam, clonazolam, pynazolam and flucotizolam, consistently with what reported in literature and/or drug discussion fora. The scaffold hopping studies strongly suggest that replacement of the pendant phenyl moiety with a five-membered ring could increase biological activity and highlight the existence of a still unexplored chemical space for DBZDs. QSAR could be of use as a preliminary risk assessment model for (newly) identified DBZDs, as well as scaffold hopping for the creation of computational libraries that could be used by regulatory bodies as support tools for scheduling procedures.     

Structure-based de novo design,synthesis, and biological evaluation of non-azole inhibitors specific for lanosterol 14alpha-demethylase of fungi

The active site of lanosterol 14alpha-demethylase (CYP51) was investigated via MCSS functional group mapping and LUDI calculations. Several non-azole lead molecules were obtained by coupling structure-based de novo design with chemical synthesis and biological evaluation. All of the lead molecules exhibited a strong inhibitory effect on CYP51 of Candida albicans. They occupy the substrate-binding site and interfere with the binding of azole antifungal agents in a competitive manner. The mode of action of the lead molecules was validated by spectrophotomeric analysis and SAR studies. This is the first successful example reported for the inhibitor design of the cytochrome P450 superfamily using the de novo design strategy. Because the affinity of the lead molecules for CYP51 was mainly attributed to their nonbonding interaction with the apoprotein, the studies presented here afford the opportunity to develop novel antifungal agents that specifically interact with the residues in the active site and avoid the serious toxicity arising from coordination binding with the heme of mammalian P450s.     

FTY720, a novel immunomodulator in de novo kidney transplant patients: pharmacokinetics and exposure-response relationship

The pharmacokinetics, safety, and preliminary efficacy of FTY720, a novel immunomodulator, were examined in de novo renal transplant patients. Both noncompartmental and population methods were used to estimate pharmacokinetic estimates in the patients. The steady-state plasma concentrations of FTY720 increased in accordance with maintenance dose level, indicating linearity in clearance and volume of distribution over the 0.25- to 2.5-mg dose range. The pharmacokinetics of FTY720 in de novo renal transplant patients were characterized by the long terminal phase half-life of approximately 200 hours across doses, high volume of distribution (>3000 L), and low clearance (10.8 L/h). The intersubject variation of clearance was 55%, and the intrasubject variation of FTY720 concentrations was 28%. The population analysis revealed significant positive relationships between baseline alkaline phosphatase and clearance, as well as between baseline body weight on apparent volume of distribution. There was no relationship between FTY720 concentrations within a given FTY720 dose cohort and the rate of allograft rejection.     

Myoglobin structure and regulation of solvent accessibility of heme pocket

The effects of heme removal on the molecular structure of tuna and sperm whale myoglobin have been investigated by comparing the solvent accessibility to the heme pocket of the two proteins with that of the corresponding apoproteins. Although the heme microenvironment of tuna myoglobin is more polar than that of sperm whale myoglobin, the accessibility of solvent to heme is identical in the two proteins as revealed by thermal perturbation of Soret absorption. The removal of heme produces loss of helical folding and increase of solvent accessibility but the effects are rather different for the two proteins. More precisely, the loss of helical structure upon heme removal is 50% for tuna myoglobin and 15% for sperm whale myoglobin; moreover, the solvent accessibility of the heme pocket of tuna apomyoglobin is 2–3-fold greater than that of sperm whale apomyoglobin. These results have been explained in terms of the lack of helical folding in segment D, the structural organization of which may have a relevant effect in regulating the accessibility of ligands to the heme. The effects produced by charged quenchers reveal that the ligand path from the surface of the molecule to the ion atom of the heme involves a positively charged residue which may reasonably be identified as Arg-45 (sperm whale myoglobin) or Lys-41 (tuna myoglobin) on the basis of recent X-ray crystallographic information.     

The quest for novel chemical matter and the contribution of computer-aided de novo design

Identifying novel chemical matter is the focus of many drug discovery efforts. Through these efforts, computer-based de novo design of drug-like molecules, which aim to build an entire molecule 'from scratch', has emerged as a valuable approach to identify novel chemical matter. In this paper, the author discusses the recent research efforts that aim to build, in silico, more chemically accessible molecules, sample more efficiently the chemical space and rank the proposed molecules. The author reviews de novo design algorithms developed between 2008 and 2010 and the issue of validation, and highlights some recent successful applications of de novo design to drug discovery projects. Although research has addressed the lack of synthetic accessibility of the molecules proposed by the first generation of de novo design tools, the lack of accurate scoring function remains a major limitation of structure-based de novo design. However, de novo design is a valuable approach to generate either chemical starting points or ideas.     

De novo generation of histamine in sputum and the effect of antibiotics

We have performed experiments to test the hypothesis that bacteria may contribute to the presence of histamine in sputum. Sputum samples obtained from 7 patients with exacerbations of chronic bronchitis and 7 patients with cystic fibrosis were incubated at 37 degrees C for 72 hours. Serial sputum histamine estimations, performed by a recently-developed HPLC technique, showed large, progressive increases in both groups of samples. Both the pre-heating of samples at 100 degrees C prior to incubation and the addition of antibiotics to the incubates substantially reduced these increases. These findings strongly suggest that bacteria may contribute to sputum histamine in infective lung disease.     

Improving antivenom availability and accessibility: Science, technology, and beyond

Snakebite envenomings constitute a serious and neglected public health problem. Despite the fact that effective treatment exists, i.e. administration of animal-derived antivenoms, the availability and accessibility of these life-saving immunobiologicals is deficitary in various parts of the world, particularly in sub-Saharan Africa and some regions of Asia. This article discusses some of the problems that need to be circumvented in order to improve the availability and accessibility of antivenoms. The conglomerate of antivenom manufacturers is highly heterogeneous in terms of technological base, qualification of staff, implementation of Good Manufacturing Practices (GMPs), and volume of production. Therefore, improvements in antivenom quality and availability should be based on strategies tailored to the situation of each region or country; in this context, three different scenarios are discussed. Accessibility of antivenoms demands concerted efforts at multiple levels, including raising the awareness of public health authorities on the relevance of the problem, implementing innovative antivenom purchasing schemes, strengthening national distribution channels on the basis of robust epidemiological information, improving the cold chain and the provision of health services in remote rural settings, supporting the correct use of antivenoms, and promoting the involvement of local community organizations in various aspects of prevention and management. These tasks should be envisaged in terms of synergistic, interprogrammatic and intersectorial interventions, with the participation of many players.     

合成多肽药物结构确证和质量研究

文中主要是结合药品注册技术审评过程中遇到的常见问题对《合成多肽药物药学研究技术指导原则》中结构确证、质量研究部分的一些关键问题进行归纳和分析,对指导原则中的一些技术要求,如有关物质研究方法的要求等进行深入和细化的阐述,同时对指导原则起草过程中存在争议的一些问题进行了说明。     

合成多肽药物结构确证和质量研究

文中主要是结合药品注册技术审评过程中遇到的常见问题对《合成多肽药物药学研究技术指导原则》中结构确证、质量研究部分的一些关键问题进行归纳和分析，对指导原则中的一些技术要求，如有关物质研究方法的要求等进行深入和细化的阐述，同时对指导原则起草过程中存在争议的一些问题进行了说明。