C3 activation is inhibited by analogs of compstatin but not by serine protease inhibitors or peptidyl alpha-ketoheterocycles

C3 convertase is a key enzyme in the complement cascade and is an attractive therapeutic target for drug design. Recent studies have demonstrated that this enzyme is inhibited by compstatin (Morikis, D. , Assa-Munt, N., Sahu, A., Lambris, J.D., 1998. Solution structure of Compstatin, a potent complement inhibitor. Protein Sci. (7) 619-627; Sahu, A., Kay, B.K., Lambris, J.D., 1996. Inhibition of human complement by a C3-binding peptide isolated from a phage-displayed random peptide library. J. Immunol. (157) 884-891), a 13 amino acid cyclic peptide that binds to C3. Since the enzyme exhibits some homology to serine proteases, substrate-based design could be another avenue for drug design. In this study, we confirm the activity of compstatin using different sources of enzyme and different assay systems. We also tested the activity of substituted compstatin analogs and compared the selectivity and toxicity of these compounds to peptidyl alpha-ketoheterocyclic compounds. Our work confirms the activity of compstatin in both alternative and classical complement pathways, describes 11 new active analogs of this cyclic peptide, and provides evidence for key segments of the peptide for activity. Compstatin and related active analogs showed little or no inhibition of clotting or key enzymes in the clotting cascade nor did they appear to have significant cytotoxicity. The characteristics of compstatin suggest that this peptide and its analogs could be attractive candidates for further clinical development. By contrast, known serine protease inhibitors, including peptidyl alpha-ketoheterocycles, did not inhibit C3 convertase illustrating the atypical nature of this enzyme.     

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.     

Synthetic small-molecule complement inhibitors

During the past few years, several large molecular-weight compounds with complement-inhibitory activities have entered clinical trials for a wide variety of acute and chronic inflammatory conditions. Various small synthetic compounds that offer several advantages over the larger complement inhibitors are also being discovered at a rapid pace. In this review, the focus will be on three of these small molecules, a C3-binding peptide, compstatin; a synthetic peptidic antagonist of the C5a anaphylatoxin receptor, 3D53; and a non-peptidergic antagonist of the C3a anaphylatoxin receptor, SB-290157. In recent years, compstatin has undergone a series of optimizations that have led to more active and stable analogs, while 3D53 and SB-290157 have been more extensively tested in animal models of various human inflammatory diseases. These compounds have been shown to be effective and display little or no toxicity, and as such may be promising new candidates for further therapeutic development.     

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.     

Design and NMR characterization of active analogues of compstatin containing non-natural amino acids

We present new findings in our drug discovery effort to develop an anticomplement therapeutic. We have designed several active analogues of compstatin by altering its amino acid composition at positions 4 and 9. The most effective analogues have tryptophan or fused-ring non-natural amino acids at position 4 and alanine or an unbranched single-methyl amino acid at position 9. Twenty-one of these analogues have 2-99-fold higher activities compared to the parent peptide compstatin. The analogue Ac-V4(2Nal)/H9A-NH(2) has the highest inhibitory activity with IC(50) 500 nM. NMR data, through NOE and chemical shift analysis, suggest the presence of interconverting conformers spanning the extended and helical regions of the Ramachandran plot, and they detect a predominant averaged conformer with coil structure and at least one flexible beta-turn, of type I. The fused-ring non-natural amino acids at position 4 contribute to the formation of the hydrophobic cluster of compstatin, which has been previously proposed, together with the beta-turn and a disulfide bridge, to be essential for binding to the target of compstatin, complement component C3. We propose that additional mechanisms may contribute to the structural stability of the analogues and to binding to C3, involving intra- and intermolecular electrostatic interactions of the pi-electron system of side chain aromatic rings. The presence of pi-pi interactions for Trp4-Trp7 was confirmed with a molecular dynamics simulation for the most active analogue with natural amino acids, Ac-V4W/H9A-NH(2). Alanine or aminobutyric acid at position 9 contribute to the weak propensity for helical structure of the residue segment 4-10 of the analogues, which may also play a role in increased activity.     

New leads for DPP IV inhibition: structure-based pharmacophore mapping and virtual screening study

Dipeptidyl peptidase IV (DPP IV) is an attractive target for the development of new antidiabetic drugs. DPP IV inhibitors improve glycemic control by preventing the rapid inactivation of the incretin hormones; glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide. In the current study, virtual screening, using 2D and 3D filters implemented in a hierarchical cascade, was employed to identify new DPP IV inhibitors. Co-crystallized ligands, with potent DPP IV-inhibitory activities, were utilized to generate structure-based pharmacophore models using DS Visualizer software. The derived pharmacophore maps were validated using in-house built database containing active and inactive DPP IV inhibitors. Subsequently, the optimum validated pharmacophore model was used as a search query against two 3D-databases (NCI and in-house built drug databases). Further hit filtration was carried out employing 2D virtual filters based on Lipinski’s rule of 5; number of rotatable bonds and other physicochemical filters. 3D filter using high-throughput molecular docking was also applied. As a result, 5 novel DPP IV inhibitors were discovered as potential lead compounds and later confirmed via in vitro bioassay.     

Hydrophobic effect and hydrogen bonds account for the improved activity of a complement inhibitor, compstatin

Tryptophans at positions 4 and 7 of compstatin, a peptide complement inhibitor, are crucial for its interaction with C3. However, the nature of their involvement has not been studied to date. Here we investigate the molecular forces involved in the C3-compstatin interactions, mediated by aromatic residues, by incorporating in these two positions various tryptophan analogues (5-methyltryptophan, 5-fluorotryptophan, 1-methyltryptophan, and 2-naphthylalanine) and assessing the resulting peptides for activity by enzyme-linked immunosorbent assay (ELISA) and binding by isothermal titration calorimetry (ITC). Of all the compstatin analogues, peptides containing 1-methyltryptophan at position 4 exhibited the highest binding affinity (Kd = 15 nM) and activity (IC50 = 0.205 microM), followed by a peptide containing 5-fluorotryptophan at position 7. Our observations suggest that hydrophobic interactions involving residues at position 4 and the hydrogen bond initiated by the indole nitrogen are primarily responsible and crucial for the increase in activity. These findings have important implications for the design of clinically useful complement inhibitors.     

Discovery and synthesis of namalide reveals a new anabaenopeptin scaffold and peptidase inhibitor

The discovery, structure elucidation, and solid-phase synthesis of namalide, a marine natural product, are described. Namalide is a cyclic tetrapeptide; its macrocycle is formed by only three amino acids, with an exocyclic ureido phenylalanine moiety at its C-terminus. The absolute configuration of namalide was established, and analogs were generated through Fmoc-based solid phase peptide synthesis. We found that only natural namalide and not its analogs containing l-Lys or l-allo-Ile inhibited carboxypeptidase A at submicromolar concentrations. In parallel, an inverse virtual screening approach aimed at identifying protein targets of namalide selected carboxypeptidase A as the third highest scoring hit. Namalide represents a new anabaenopeptin-type scaffold, and its protease inhibitory activity demonstrates that the 13-membered macrolactam can exhibit similar activity as the more common hexapeptides.     

Evolution of compstatin family as therapeutic complement inhibitors

Introduction: Therapeutic modulation of complement activation is considered as a promising approach for the treatment of host tissue damage in several inflammatory and autoimmune diseases. Complement component protein C3 is a particularly attractive drug target for complement inhibitors, due to its central role in three pathways of complement activation cascade.

Areas covered: The author provides a comprehensive review on compstatin family peptides which have been discovered and optimized as potent and selective C3 inhibitors via a combination of chemical, biophysical and computational approaches. New generations of the compstatin family with improved potency and therapeutic properties have been developed in recent years. Over two decades, compstatin demonstrated therapeutic potential as a first-of-its-kind complement inhibitor in a series of disease models, with encouraging efforts in clinical trials.

Expert opinion: Compstatin holds promise for new therapeutic implications in blocking the effect of the complement cascade in a variety of disease conditions. The development of cost-effective treatment options with suitable dosing route and schedule will be critical for patients with complement mediated chronic diseases.     

Beta-diketo acid pharmacophore hypothesis. 1. Discovery of a novel class of HIV-1 integrase inhibitors

HIV-1 Integrase (IN) is an essential enzyme for viral replication. The discovery of beta-diketo acids was crucial in the validation of IN as a legitimate target in drug discovery against HIV infection. In this study, we discovered a novel class of IN inhibitors using a 3D pharmacophore guided database search. We used S-1360 (1), the first IN inhibitor to undergo clinical trials, and three other analogues to develop a common feature pharmacophore hypothesis. Testing this four-featured pharmacophore against a multiconformational database of 150,000 structurally diverse small molecules yielded 1,700 compounds that satisfied the 3D query. Subsequently, all 1,700 compounds were docked into the active site of IN. On the basis of docking scores, Lipinski's rule-of-five, and structural novelty, 110 compounds were selected for biological screening. We found that compounds that contain both salicylic acid and a 2-thioxo-4-thiazolidinone (rhodanine) group (e.g. 5-13) showed significant inhibitory potency against IN, while the presence of either salicylic acid or a rhodanine group alone did not. Although some of the compounds containing only a salicylic acid showed inhibitory potency against IN, none of the compounds containing only rhodanine exhibited considerable potency. Of the 52 compounds reported in this study, 11 compounds (5, 6, 8, 10-13, 32-33, 51, and 53) inhibited 3'-processing or strand transfer activities of IN with IC(50) < or = 25 microM. This is the first reported use of S-1360 and its analogues as leads in developing a pharmacophore hypothesis for IN inhibition and for identification of new compounds with potent inhibition of this enzyme.     

Pyridazin-3(2H)-ones: the versatile pharmacophore of medicinal significance

Pyridazin-3(2H)-one derivatives have attracted the attention of medicinal chemists during the last decade due to their diverse pharmacological activities. Easy functionalization of various ring positions of pyridazinones makes them an attractive synthetic building block for designing and synthesis of new drugs. The incorporation of this versatile biologically accepted pharmacophore in established medicinally active molecules results in wide range of pharmacological effects. Pyridazinones constitute an interesting group of compounds, many of which possess wide spread pharmacological properties such as antihypertensive, platelet aggregation inhibitory, cardiotonic activities and some are also well known for their pronounced analgesic, anti-inflammatory, antinociceptive, and antiulcer activities. Recently pyridazinones have also been reported as antidiabetic, anticonvulsant, antiasthmatic, and antimicrobial agents. These encouraging reports suggest that this privileged skeleton should be extensively studied for the therapeutic benefits. In view of this, a detailed and updated account of the pharmacological properties of pyridazinones is described in this review. The wide range of synthesized pyridazinone analogs along with their medicinal significance is also presented.     

Analogs of methyllycaconitine as novel noncompetitive inhibitors of nicotinic receptors: pharmacological characterization, computational modeling, and pharmacophore development

As a novel approach to drug discovery involving neuronal nicotinic acetylcholine receptors (nAChRs), our laboratory targeted nonagonist binding sites (i.e., noncompetitive binding sites, negative allosteric binding sites) located on nAChRs. Cultured bovine adrenal cells were used as neuronal models to investigate interactions of 67 analogs of methyllycaconitine (MLA) on native alpha3beta4* nAChRs. The availability of large numbers of structurally related molecules presents a unique opportunity for the development of pharmacophore models for noncompetitive binding sites. Our MLA analogs inhibited nicotine-mediated functional activation of both native and recombinant alpha3beta4* nAChRs with a wide range of IC(50) values (0.9-115 microM). These analogs had little or no inhibitory effects on agonist binding to native or recombinant nAChRs, supporting noncompetitive inhibitory activity. Based on these data, two highly predictive 3D quantitative structure-activity relationship (comparative molecular field analysis and comparative molecular similarity index analysis) models were generated. These computational models were successfully validated and provided insights into the molecular interactions of MLA analogs with nAChRs. In addition, a pharmacophore model was constructed to analyze and visualize the binding requirements to the analog binding site. The pharmacophore model was subsequently applied to search structurally diverse molecular databases to prospectively identify novel inhibitors. The rapid identification of eight molecules from database mining and our successful demonstration of in vitro inhibitory activity support the utility of these computational models as novel tools for the efficient retrieval of inhibitors. These results demonstrate the effectiveness of computational modeling and pharmacophore development, which may lead to the identification of new therapeutic drugs that target novel sites on nAChRs.     

Mammalian spermidine synthase--identification of cysteine residues and investigation of the putrescine binding site--

Homology modeling and inhibitory studies using substrate analogs were undertaken to construct a possible three-dimensional structure, including the putrescine-binding site, of rat spermidine synthase based on its primary sequence. Of the ten cysteine residues of the enzyme, six residues were chemically determined as sulfhydryl; similarly, one residue (C25) was determined as the disulfide. Using the model obtained from the Swiss-Model protein-modeling server, and based on the crystal structure of the Thermotoga maritima enzyme, the three remaining residues were assigned as sulfhydryl. Discussions are presented on the counterpart of the C25 residue, based on the apparent role of the bacterial N-terminal peptide region in reinforcing the binding between protomers in a functional oligomeric form. The active sites of the bacterial and mammalian versions of the enzyme were very similar. The putrescine-binding site of the rat enzyme was investigated using IC(50) values of the analogs of two known potent inhibitors, n-butylamine and trans-4-methylcyclohexylamine (4MCHA). Our results indicated that 5-amino-1-pentene and 4MCHA possess comparable inhibitory activities towards the enzyme.     

Optimization of a polyglutamine aggregation inhibitor peptide (QBP1) using a thioflavin T fluorescence assay

Polyglutamine protein aggregates are a hallmark of several neurodegenerative diseases, including Huntington's disease, and increasing evidence suggests that reducing or inhibiting aggregation produces a therapeutic benefit in animal models of disease. Part of the challenge in designing compounds that interfere with protein aggregation is having a sensitive and consistent in vitro assay that allows for efficient screening and lead optimization. Here we describe a simplified polyglutamine assay that uses a soluble, pathological-length polyglutamine construct (62 glutamines [Q62]) fused to glutathione-S-transferase (GST) and measure aggregate formation with fluorescence generated by thioflavin T binding. Controlled release of Q62 from GST using proteolytic cleavage resulted in time-dependent aggregate formation that was not observed for a non-pathological-length GST-Q19 construct. Cleavage of the polyglutamine domain from GST increased the rate of Q62 aggregation from days to hours, significantly decreasing the time for compound analysis. Controlled aggregate formation combined with the fluorescence sensitivity of the dye thioflavin T allowed us to screen a series of peptide analogs for lead optimization of a previously identified peptide aggregation inhibitor, QBP1. QBP1 analogs showed the greatest inhibitory potency when added prior to Q62 aggregate initiation, suggesting that the mechanism of inhibition was interference with early formed aggregates that were not detectable by ultraviolet or dye binding. The assay detected activities that differed by three orders of magnitudes with Z' = 0.56, which is suitable for high-throughput screening and allowed us to do lead optimization of QBP1 analogs for pharmacophore model building.     

Simplified receptor based pharmacophore approach to retrieve potent PTP-LAR inhibitors using apoenzyme

The design of biological active compounds from the apoenzyme is still a challenging task. Herein a simple yet efficient technique is reported to generate a receptor based pharmacophore solely using a ligand-free protein crystal structure. Human leukocyte antigen-related phosphatase (PTP-LAR) is an apoenzyme and a receptor like transmembrane phosphatase that has emerged as a drug target for diabetes, obesity and cancer. The prior knowledge of the active residues responsible for the mechanism of action of the protein was used to generate the LUDI interaction map. Then, the complement negative image of the binding site was used to generate the pharmacophore features. A unique strategy was followed to design a pharmacophore query maintaining crucial interactions with all the active residues, essential for the enzyme inhibition. The same query was used to screen several databases consisting of the Specs, IBS, MiniMaybridge, NCI and an in-house PTP inhibitor databases. In order to overcome the common bioavailability problem associated with phosphatases, the hits obtained were filtered by Lipinski's Rule of Five, SADMET properties and validated by docking studies in Glide and GOLD. These docking studies not only suggest the essential ligand binding interactions but also the binding patterns necessary for the LAR inhibition. The ligand pharmacophore mapping studies further validated the screened protocol and supported that the final screened molecules, presumably, showed potent inhibitory activity. Subsequently, these molecules were subjected to Derek toxicity predictions and nine new molecules with different scaffold were obtained as non-toxic PTP-LAR inhibitors. The present prospective strategy is a powerful technique to identify potent inhibitors using the protein 3D structure alone and is a valid alternative to other structure-based and random docking approaches.     

3-硝基-2H-色烯衍生物的合成及抗菌活性研究

目的设计并合成一系列3-硝基-2H-色烯衍生物,研究其抗菌活性与构效关系。方法以水杨醛和2-硝基乙醇为原料合成目标化合物,采用肉汤稀释法测定抗菌活性。结果目标化合物结构均经过1HNMR和HRMS确证,3-硝基-2H-色烯衍生物对金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌具有很好的抑制活性(MIC=4μg·mL-1)。结论 3-硝基-2H-色烯衍生物具有进一步深入研究的价值。     

Biological activities of retro and diastereo analogs of a 13‐residue peptide with antimicrobial and hemolytic activities

Abstract: The biological activities of synthetic retro and diastereo analogs of PKLLKTFLSKWIG (SPFK), a 13‐residue peptide with antimicrobial and hemolytic activities, have been investigated. Retro peptides with C‐terminal acid and amide exhibited antibacterial activities comparable with those of SPFK. Their hemolytic activities were, however, only marginally lower. The diastereo analog with C‐terminal acid was not antibacterial and was weakly hemolytic. Amidation of this analog could restore antibacterial activity. Both retro analogs were unordered in aqueous medium but had a propensity for a helical structure in trifluoroethanol. However, diastereo analogs were unordered in both aqueous medium and trifluoroethanol. Thus, reversing the sequence in a short amphiphilic peptide may not always result in the selective loss of biological activity such as hemolytic activity. Also, introduction of enantiomeric amino acids in a short peptide to generate a diastereomer may result in loss of structure as well as antimicrobial and hemolytic activities, unless compensated by an increase in positive charges.     

Developing a dynamic pharmacophore model for HIV-1 integrase

We present the first receptor-based pharmacophore model for HIV-1 integrase. The development of "dynamic" pharmacophore models is a new method that accounts for the inherent flexibility of the active site and aims to reduce the entropic penalties associated with binding a ligand. Furthermore, this new drug discovery method overcomes the limitation of an incomplete crystal structure of the target protein. A molecular dynamics (MD) simulation describes the flexibility of the uncomplexed protein. Many conformational models of the protein are saved from the MD simulations and used in a series of multi-unit search for interacting conformers (MUSIC) simulations. MUSIC is a multiple-copy minimization method, available in the BOSS program; it is used to determine binding regions for probe molecules containing functional groups that complement the active site. All protein conformations from the MD are overlaid, and conserved binding regions for the probe molecules are identified. Those conserved binding regions define the dynamic pharmacophore model. Here, the dynamic model is compared to known inhibitors of the integrase as well as a three-point, ligand-based pharmacophore model from the literature. Also, a "static" pharmacophore model was determined in the standard fashion, using a single crystal structure. Inhibitors thought to bind in the active site of HIV-1 integrase fit the dynamic model but not the static model. Finally, we have identified a set of compounds from the Available Chemicals Directory that fit the dynamic pharmacophore model, and experimental testing of the compounds has confirmed several new inhibitors.     

Generation of predictive pharmacophore models for CCR5 antagonists: study with piperidine- and piperazine-based compounds as a new class of HIV-1 entry inhibitors

Predictive pharmacophore models were developed for a large series of piperidine- and piperazine-based CCR5 antagonists as anti-HIV-1 agents reported by Schering-Plough Research Institute in recent years. The pharmacophore models were generated using a training set consisting of 25 carefully selected antagonists based on well documented criteria. The activity spread, expressed in K(i), of training set molecules was from 0.1 to 1300 nM. The most predictive pharmacophore model (hypothesis 1), consisting of five features, namely, two hydrogen bond acceptors and three hydrophobic, had a correlation (r) of 0.920 and a root mean square of 0.879, and the cost difference between null cost and fixed cost was 44.46 bits. The model was cross-validated by randomizing the data using the CatScramble technique. The results confirmed that the pharmacophore models generated from the test set were not due to chance correlation. The best model (hypothesis 1) was validated using test set molecules (total of 78) and performed well in classifying active and inactive molecules correctly. The model was further validated by mapping onto it a diverse set of six CCR5 antagonists identified by five different pharmaceutical companies. The best model correctly predicted these compounds as being highly active. These multiple validation approaches provide confidence in the utility of the predictive pharmacophore model developed in this study as a 3D query tool in virtual screening to retrieve new chemical entities as potent CCR5 antagonists. The model can also be used in predicting biological activities of compounds prior to undertaking their costly synthesis.