Combinatorial Chemistry Approach Research Articles

Exploring the Polyamine Regulatory Site of the NMDA Receptor: a Parallel Synthesis Approach

The elongated structures of polyamine inverse agonists such as 1,12-diaminododecane (N12N) and 5-(4-aminobutyl)-2-thiopheneoctanamine (N4T8N) lend themselves to a combinatorial chemistry approach to explore a potential polyamine pharmacophore at the NMDA receptor. Herein we describe more than 100 new analogues of N4T8N obtained by breaking up the long octanamine arm into a dipeptide chain of equivalent length. Solid-phase parallel synthesis based on cross-linked polystyrene and a Wang anchor allowed the low-scale preparation of four small libraries based on the combination of two amino acid residues (out of Gly, Leu, Phe, Lys, phenylglycine, Tyr, Trp, His, and Arg). The obtained compounds were tested as modulators of [(3) H]MK-801 binding to rat brain membranes and of NMDA-induced currents in cultured rat hippocampal neurons. Compounds with two aromatic residues acted as binding inhibitors (inverse agonists). Compounds with two Lys residues acted as binding stimulators (agonists) and had stimulatory and inhibitory effects on NMDA-induced currents, depending on the holding potential. High sensitivity of binding inhibition to spermine was conferred by a Tyr residue, whereas a His residue favored high potency at acidic pH.

A Combinatorial Library of Bi-functional Polymeric Vectors for siRNA Delivery In Vitro

To apply a combinatorial chemistry approach toward the design of polymeric vectors, and to evaluate their effectiveness as siRNA delivery systems in vitro. Poly(acrylic acid) (pAA) was synthesized via RAFT polymerization with well-controlled molecular weights (M (n): 3kDa, 5kDa, 10kDa and 21kDa). A polymer library was generated from the pAA precursors by conjugating two distinct moieties, agmatine (Agm) and D-(+)-galactosamine (Gal), at various ratios. Biophysical and cellular characterization was evaluated in vitro for these polymeric vectors using MDA-MB-231-luc+ cells. A critical balance between Agm/Gal content and polymer molecular weight must be attained to achieve favorable transfection efficacies. From the library of 22 polymers, only a few had knockdown efficiencies commensurate with effective siRNA delivery, particularly those with polymer precursor M (n) of 5kDa and 10kDa. Highest protein knockdown of 84% was achieved by a polymer conjugate with a 5kDa pAA backbone with a side chain composition of 55% Agm and 17% Gal. Effective delivery of siRNA was found to be highly dependent on the molecular structure of the polymeric vector. The combinatorial approach employed provided the tools to identify optimal structural properties leading to efficient siRNA delivery for this class of vector.

Abstract 378: Multifunctional bladder cancer targeting micelle nanocarriers with both imaging and therapeutic potentials

Abstract Background: Human and dog invasive bladder cancers share similar attributes, such as pathologic changes, response to chemotherapy, and extremely poor prognosis. Via combinatorial chemistry approach, we previously identified a novel bladder cancer-specific ligand, PLZ4, that can specifically bind to both human and dog bladder cancer cells in vitro and in vivo. We also developed a micelle nanocarrier drug delivery system with multifunctional loading capacity. Here, we assessed whether the targeting micelles impregnated with PLZ4 on the surface could promote targeting efficacy against both human and dog bladder cancers for diagnostic imaging and therapeutic purposes. Materials and Methods: Micelle-building monomers (i.e., telodendrimers) were synthesized through conjugation of polyethylene glycol with cholic acid cluster at one end and PLZ4 at the other end, which then self-assembled in aqueous solution to form micelles. Near-infrared dye DiD and chemotherapeutic drugs (paclitaxel or daunorubicin) were co-loaded for the experiments. Cellular uptake and distribution of nanomicelles were evaluated by fluorescence intensity and high resolution topography, while drug delivery efficacy was monitored by cytotoxicity assay. Using orthotopic/xenograft mouse model generated from human clinical bladder cancer specimen or dog bladder cancer line, in vivo and ex vivo fluorescence imaging as well as efficacy study were performed. Results: Compared to non-targeting micelles, targeting PLZ4-decorated-micelles loaded with an imaging agent DiD and chemotherapeutic drugs were more efficient in dye delivery, and caused a comparable to higher degree of cytotoxicity with free drugs in bladder cancer cell lines. According to topography, targeting micelles tended to localize at the membrane, perinuclear, and nucleus. In vivo and ex vivo orthotopic xenograft studies showed that both targeting (14.3X of free dye, p<0.01) and non-targeting micelles (9.6X, p<0.01) rapidly accumulated at the tumor sites, while targeting micelles exhibited significantly higher efficiency in homing property than non-targeting micelles (p<0.05). Considerably higher DiD signal was microscopically observed in targeting group than non-targeting group as well as free dye group, but all exhibited similar blood vessel density. Our preliminary efficacy study also supported a better drug delivery efficiency using targeting micelle formulation than non-targeting micelles/free drug in mouse xenografts formed from human clinical bladder tumors. Conclusions: Targeting micelles impregnated with PLZ4 can selectively and efficiently target both human and dog bladder cancer cells and can be potentially developed as imaging and therapeutic agents in both human and veterinary medicine. Preclinical studies of targeting micelles can be performed in dogs with spontaneous bladder cancer before proceeding to human patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 378. doi:1538-7445.AM2012-378

Combinatorially-generated library of 6-fluoroquinolone analogs as potential novel antitubercular agents: a chemometric and molecular modeling assessment

The virtual combinatorial chemistry approach as a methodology for generating chemical libraries of structurally-similar analogs in a virtual environment was employed for building a general mixed virtual combinatorial library with a total of 53.871 6-FQ structural analogs, introducing the real synthetic pathways of three well known 6-FQ inhibitors. The druggability properties of the generated combinatorial 6-FQs were assessed using an in-house developed drug-likeness filter integrating the Lipinski/Veber rule-sets. The compounds recognized as drug-like were used as an external set for prediction of the biological activity values using a neural-networks (NN) model based on an experimentally-determined set of active 6-FQs. Furthermore, a subset of compounds was extracted from the pool of drug-like 6-FQs, with predicted biological activity, and subsequently used in virtual screening (VS) campaign combining pharmacophore modeling and molecular docking studies. This complex scheme, a powerful combination of chemometric and molecular modeling approaches provided novel QSAR guidelines that could aid in the further lead development of 6-FQs agents.

Targeting Oncogenic Protein-Protein Interactions by Diversity Oriented Synthesis and Combinatorial Chemistry Approaches

We are currently witnessing a decline in the development of efficient new anticancer drugs, despite the salient efforts made on all fronts of cancer drug discovery. This trend presumably relates to the substantial heterogeneity and the inherent biological complexity of cancer, which hinder drug development success. Protein-protein interactions (PPIs) are key players in numerous cellular processes and aberrant interruption of this complex network provides a basis for various disease states, including cancer. Thus, it is now believed that cancer drug discovery, in addition to the design of single-targeted bioactive compounds, should also incorporate diversity-oriented synthesis (DOS) and other combinatorial strategies in order to exploit the ability of multi-functional scaffolds to modulate multiple protein-protein interactions (biological hubs). Throughout the review, we highlight the chemistry driven approaches to access diversity space for the discovery of small molecules that disrupt oncogenic PPIs, namely the p53-Mdm2, Bcl-2/Bcl-xL-BH3, Myc-Max, and p53-Mdmx/Mdm2 interactions.

CDK-Taverna 2.0: migration and enhancements of an open-source pipelining solution

Pipelining or workflow tools allow for the LegoTM-like, graphical assembly of I/O modules and algorithms into a complex workflow which can be easily deployed, modified and tested without the hassle of implementing it into a monolithic application. The CDK-Taverna project aims at building an opensource pipelining solution through combination of different open-source projects such as Taverna [1], the Chemistry Development Kit (CDK) [2,3] or Bioclipse [4]. A first integrated version of CDK-Taverna was recently released to the public [5]. Current developments in CDK-Taverna refactor all workers as well as the complete setup on the basis of Taverna 2.2 and CDK 1.3.5 which themselves introduce major improvements to the whole platform. In addition the CDK is enhanced with specific functions and options for reaction enumeration based on a reaction template and corresponding reactant libraries. Reaction enumeration supports combinatorial chemistry approaches in the drug discovery process of the pharmaceutical industry. The CDK enhancements are applied and illustrated by corresponding CDK-Tavernaworkflows.

Abstract 366: Novel self-folding tricyclic cholic acid-peptide hybrids as nanotherapeutic ligands targeting prostate and breast cancers and human embryonic stem cells

Abstract Prostate cancer is the second leading cause of death in American men accounting for 10% of all cancer-related mortalities. According to NCI statistics from 2005-2007, 1 in 6 men will be diagnosed with prostate cancer during their lifetime. Despite the use of chemotherapy for the treatment of advanced or metastatic disease, cellular resistance to anticancer drugs and adverse side effects to healthy organs limit therapeutic efficacy. A targeted pro-apoptotic drug delivery system has been developed for the selective destruction of malignant tumors and tumorigenic stem cells. A novel self-folding tricyclic branched library was synthesized. Fixed hydrophobic amino acids near the N-terminus (position 5) of twin branched peptide arms fold back onto the hydrophobic face of the planar cholic acid molecule to form a compact tricyclic molecule. The novel bracelet library was screened using a One Bead Two Compounds (OB2C) combinatorial chemistry approach. Receptors expressed on cancerous cells serve as the targets for the tricyclic molecules to induce apoptotic signaling. Receptors for LDO 18, found on prostate cancer and stem cells, serve as the carrier vector, enabling the targeted delivery of the anticancer drug to the tumor mass. This approach spares normal tissues from toxic side effects of the peptidomimetic compounds. Preliminary studies suggest that tumors of ovarian and breast origin can also be targeted using LDO18. Lead therapeutic compounds will be conjugated to LDO18 nanocarriers along with LHRH analogues to evaluate the efficacy and combined potency to prostate, ovarian, and breast cancer xenografts in nude mice and orthotropic models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 366. doi:10.1158/1538-7445.AM2011-366

Large-area SnO 2: F thin films by offline APCVD

In this paper, we reported the successful preparation of fluorine-doped tin oxide (FTO) thin films on large-area glass substrates (1245 mm × 635 mm × 3 mm) by self-designed offline atmospheric pressure chemical vapor deposition (APCVD) process. The FTO thin films were achieved through a combinatorial chemistry approach using tin tetrachloride, water and oxygen as precursors and Freon (F-152, C2H4F2) as dopant. The deposited films were characterized for crystallinity, morphology (roughness) and sheet resistance to aid optimization of materials suitable for solar cells. We got the FTO thin films with sheet resistance 8–11 Ω/□ and direct transmittance more than 83%. X-ray diffraction (XRD) characterization suggested that the as-prepared FTO films were composed of multicrystal, with the average crystal size 200–300 nm and good crystallinity. Further more, the field emission scanning electron microscope (FESEM) images showed that the films were produced with good surface morphology (haze). Selected samples were used for manufacturing tandem amorphous silicon (a-Si:H) thin film solar cells and modules by plasma enhanced chemical vapor deposition (PECVD). Compared with commercially available FTO thin films coated by online chemical vapor deposition, our FTO coatings show excellent performance resulting in a high quantum efficiency yield for a-Si:H solar cells and ideal open voltage and short circuit current for a-Si:H solar modules.

Search for new phosphors in Eu 2+ doped MgAl 2O 4–SrAl 2O 4–BaAl 2O 4 ternary system by combinatorial approach

Eu 2+ doped MgAl 2O 4–SrAl 2O 4–BaAl 2O 4 ternary phosphors were systematically studied by a sol–gel based combinatorial chemistry approach. The photoluminescence properties were optimized by chemically tuning of the compositions and a green phosphor and a blue one, with their compositions of Mg 0.350Sr 0.500Ba 0.125Al 2O 4:Eu 2+ 0.025 and Mg 0.855Sr 0.100Ba 0.025Al 2O 4:Eu 2+ 0.020 each were identified. The luminescent intensity of the green phosphor Mg 0.350Sr 0.500Ba 0.125Al 2O 4:Eu 2+ 0.025 was about 2.5 times to that of SrAl 2O 4:Eu 2+ 0.015. For the blue one, Mg 0.855Sr 0.100Ba 0.025Al 2O 4:Eu 2+ 0.020, the luminescent intensity was about 19 times to that of MgAl 2O 4:Eu 2+ 0.015. The quenching temperatures of Mg 0.350Sr 0.500Ba 0.125Al 2O 4:Eu 2+ 0.025 (green) and Mg 0.855Sr 0.100Ba 0.025Al 2O 4:Eu 2+ 0.020 (blue) were high enough for application in LEDs. In addition, they had very good excitation characteristics in the UV region as confirmed by the excitation spectra. We propose these two materials could be used as potential green and blue phosphors. More importantly, our sol–gel based combinatorial chemistry approach could be further applied to optimize the other phosphors systems.

Combinatorial chemistry approach to searching phosphors for white light-emitting diodes in (Gd-Y-Bi-Eu)VO4 quaternary system

A red phosphor is desired to improve the color rendering index (Ra) of white light-emitting diodes (LEDs). Instead of developing a novel red phosphor individually, this work exploits a special yellow phosphor that contains red and green components to be matched with blue phosphor to produce white light. The desired emission color of yellow phosphor is tuned up by tailoring the green emission of Bi3+ and the red emission of Eu3+, and the luminescence efficiency and the thermal stability of luminescence are maximized by optimizing the composition of the host in the GdVO4-YVO4 system. A dynamic and virtual variation of samples emission color and intensity with Bi3+, Eu3+, Y3+ and Gd3+ concentrations and excitation wavelengths is clearly displayed in the luminescence images of the combinatorial libraries. The production of white light with high color rendering is demonstrated by preparing a practical white LED with a Ra of up to 90 from commercially available blue phosphor and optimized yellow phosphor.

Identification of a bladder cancer-specific ligand using a combinatorial chemistry approach

ObjectivesTo develop bladder cancer-specific ligands using a combinatorial chemistry approach. Materials and methodsWe performed a high-throughput one-bead one-compound combinatorial chemistry approach to identify ligands that bound to bladder transitional cell carcinoma cells. The whole-cell binding assay allowed successful identification of a few peptides that bound selectively to bladder cancer cells. Single cell suspensions derived from clinical bladder cancer specimens and cell lines were used to determine the binding specificity. Studies with mouse xenografts were performed to determine the in vivo binding and targeting efficiency, specificity, and biodistribution of one of the ligands. ResultsOne cyclic peptide named PLZ4 (amino acid sequence: cQDGRMGFc) was identified that could selectively bind to bladder cancer cell lines and all of the 5 primary bladder cancer cells from human patients, but not to normal urothelial cells, cell mixtures from normal bladder specimens, fibroblasts, and blood cells. Comparison of PLZ4 binding to cell lines of different cancer origins showed that it was bladder cancer-specific (P < 0.05). PLZ4 could bind to tumor cells treated with urine at pH 6.0, but not to noncancerous cells collected from the urine of 4 patients actively being treated with intravesical Bacillus Calmette-Guerin therapy. In vivo and ex vivo imaging studies showed that PLZ4 linked to Cy5.5 fluorescent dye administered via tail vein injection was specifically taken up in mouse xenografts developed from excised fresh human bladder cancer specimens. Several ligands contain the same DGR motif, but only PLZ4 was bladder cancer-specific. We performed alanine walk and rainbow bead coding experiments, and found that the C-terminal GF residues were also important for cell binding and modulated the binding specificity. ConclusionsPLZ4 has the potential to be used for targeted therapy and imaging detection during diagnosis and follow-up/surveillance of noninvasive and advanced bladder cancer.

Ligand-Based Peptide Design and Combinatorial Peptide Libraries to Target G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are considered to represent the most promising drug targets; it has been repeatedly said that a large fraction of the currently marketed drugs elicit their actions by binding to GPCRs (with cited numbers varying from 30-50%). Closer scrutiny, however, shows that only a modest fraction of (≈60) GPCRs are, in fact, exploited as drug targets, only ≈20 of which are peptide-binding receptors. The vast majority of receptors in the humane genome have not yet been explored as sites of action for drugs. Given the drugability of this receptor class, it appears that opportunities for drug discovery abound. In addition, GPCRs provide for binding sites other than the ligand binding sites (referred to as the "orthosteric site"). These additional sites include (i) binding sites for ligands (referred to as "allosteric ligands") that modulate the affinity and efficacy of orthosteric ligands, (ii) the interaction surface that recruits G proteins and arrestins, (iii) the interaction sites of additional proteins (GIPs, GPCR interacting proteins that regulate G protein signaling or give rise to G protein-independent signals). These sites can also be targeted by peptides. Combinatorial and natural peptide libraries are therefore likely to play a major role in identifying new GPCR ligands at each of these sites. In particular the diverse natural peptide libraries such as the venom peptides from marine cone-snails and plant cyclotides have been established as a rich source of drug leads. High-throughput screening and combinatorial chemistry approaches allow for progressing from these starting points to potential drug candidates. This will be illustrated by focusing on the ligand-based drug design of oxytocin (OT) and vasopressin (AVP) receptor ligands using natural peptide leads as starting points.

Combinatorial Approach to the Development of a Single Mass YVO4:Bi3+,Eu3+ Phosphor with Red and Green Dual Colors for High Color Rendering White Light-Emitting Diodes

Instead of developing a novel red phosphor individually, this work proposes the production of white light by combining a near-ultraviolet/ultraviolet diode chip with blue and special yellow phosphors: the yellow phosphor includes the red and green components with high color saturation. The availability of this scheme is demonstrated by preparing a white light-emitting diode (WLED) with color rendering index (Ra) up to 90.3. The desired single-mass yellow phosphor is successfully screened out from the YVO(4):Bi(3+),Eu(3+) system by using a combinatorial chemistry approach. When the emission color and luminous efficiency are both considered, the best composition for producing white light is (Y(1-s-t)Bi(s)Eu(t))VO(4) with 0.040 < or = s < or = 0.050 and 0 < t < or = 0.015. The red component that is required for a high-Ra WLED is obtained through sensitizing luminescence of Eu(3+) by Bi(3+) in a YVO(4) host; meanwhile, both Bi(3+) and Eu(3+) emission are improved by keeping the Bi(3+) and Eu(3+) contents close to the critical concentration.

The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region

Previously selected by the combinatorial chemistry approach, potent fluorogenic substrate of proteinase 3 was used as the starting structure to design new substrates. The general formula of the synthesized peptides is as follows: ABZ-Tyr-Tyr-Abu-ANB-X-NH 2, where ANB (5-amino-2-nitrobenzoic acid) served as a chromophore and an acceptor of fluorescence, ABZ (aminobenzoic acid) is a donor of fluorescence in these fluorescence resonance energy transfer (FRET) peptides, and X is a proteinogenic amino acid (except Cys). The introduced modifications influenced substrate activity of the synthesized peptides. The highest value of specificity constant for proteinase 3 was obtained for the single peptide with Gln in the discussed position ( k cat/ K M = 275,000 M −1 s −1), which was nearly twice as active as the reference compound (lacking a substituent in the X position). In addition, more efficient energy transfer was observed, due mainly to the bathochromic effect for the introduced modification. This approach opens a new possibility to design potent and highly specific substrates of proteinase 3 and other proteinases optimized in the prime site region.

Studies on bioorganic chemistry of peptides active against insects and plants

Peptides are key molecules in various biological events, and certain types of animals, such as scorpions, have venom, which includes a variety of peptide toxins. Since venoms contain peptides with various structures and activities, they are recognized as “natural libraries” for pesticide and drug discovery. On the other hand, “synthetic libraries” can be readily prepared by utilizing combinatorial chemistry approaches. Millions of peptide sequences generated by this method can be tested for the biological activity of interest using a high-throughput screening system. Here we introduce the isolation and characterization of novel insecticidal peptides from scorpion venom, and plant immunity-activating peptides from synthetic random libraries.

Exploring novel target space: a need to partner high throughput docking and ligand-based similarity searches?

Recent advances in combinatorial chemistry (CC) and High throughput screening (HTS) approaches for use in drug discovery have made it possible to synthesize and/or screen large repositories of chemically diverse scaffolds in search of small molecules that disrupt or regulate macromolecular function. Although successful in the discovery of novel therapeutics this approach is both costly and time consuming. In silico computer aided drug discovery (CADD) approaches including; structure based virtual screening (SBVS) or high throughput docking (HTD) and/or ligand based virtual screening (LBVS) are areas experiencing renewed interest both in the pharmaceutical industry and academia. The emerging success of these approaches alone or partnered with HTS platforms in search of, and/or optimization of, novel therapeutic compounds represents a potential approach for the identification of therapies that target novel space. Here we will discuss how LBVS has been and continues to be partnered with HTS in early stage compound identification and/or triage. We will also provide a significant overview of how SBVS when partnered with LBVS can overcome the limitations inherent to each approach when used alone. We will discuss this partnered approach in the context of both traditional drug discovery targets and provide thoughts on its applicability to study novel chemical space including protein-protein and/or other historical intractable interfaces.

Selective regulation of aromatase expression for drug discovery

Aromatase is a particularly attractive drug target in the treatment of hormone-responsive breast cancer, and aromatase activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Complex regulation of aromatase expression in human tissues involves alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase ( CYP19) gene expression and the expression of cyclooxygenase ( COX) genes. Additionally, COX selective inhibitors can suppress CYP19 gene expression and decrease aromatase activity. Our current hypothesis is that pharmacological regulation of aromatase can act locally to decrease the biosynthesis of estrogen and may provide additional therapy options for patients with hormone-dependent breast cancer. Two pharmacological approaches are being developed, one approach utilizing small molecule drug design and the second approach involving mRNA silencing technology. The small molecule drug design approach focuses on the synthesis and biological evaluation of a novel series of sulfonanilide analogs derived from COX-2 selective inhibitors. Combinatorial chemistry approaches were used to generate diversely substituted novel sulfonanilides. The compounds suppress aromatase enzyme activity in SK-BR-3 breast cancer cells in a dose and time dependent manner, and structure activity analysis does not find a correlation between aromatase suppression and COX inhibition. Real-time PCR analysis demonstrates that the sulfonanilide analogs decrease aromatase gene transcription in breast cells. Furthermore, the sulfonanilide compounds selectively decrease aromatase gene expression in several breast cancer cells, without exhibiting cytotoxic or apoptotic effects at low micromole concentrations. A ligand-based pharmacophore model for selective aromatase modulation (SAM) by the novel sulfonanilides identified an aromatic ring, two hydrogen bond acceptors, and a hydrophobic function as four key chemical features. In the second approach, short interfering RNAs (siRNA) were designed targeting human aromatase mRNA. Treatment of breast cancer cells with siRNAs targeting aromatase (siAROMs) completely masked the aromatase enzyme activity and resulted in suppression of CYP19 mRNA. Thus, these results suggest that the novel sulfonanilides and the siRNAs targeting aromatase expression may be valuable tools for selective regulation of aromatase in breast cancer.

Optimization of Solar Cell Performance using Atmospheric Pressure Chemical Vapour Deposition deposited TCOs

Photovoltaic cell performance can be greatly improved by optimising the transparent conducting oxide used as the front contact. We have developed an advanced atmospheric pressure chemical vapour deposition (APCVD) process, by applying fast experimentation and using a combinatorial chemistry approach to aid the studies. By use of this process, F-doped SnO2 has been deposited using either monobutyl tin trichloride or tin tetrachloride with aqueous HF or trifluoro-acetic acid as the dopant source. The deposited films were characterised for crystallinity, morphology, resistivity and growth rate to aid optimisation of material suitable for solar cells. The results from use of the different tin precursors and dopants were compared. The most striking changes were related to resistivity and surface morphology. Compared with commercially available TCO CVD coated glasses, our coatings show excellent performance resulting in a high quantum efficiency yield for a-Si:H solar cells.

Ligand-Induced Changes in the Structure and Dynamics of Escherichia coli Peptide Deformylase

Peptide deformylase (PDF) is an enzyme that is responsible for removing the formyl group from nascently synthesized polypeptides in bacteria, attracting much attention as a potential target for novel antibacterial agents. Efforts to develop potent inhibitors of the enzyme have progressed on the basis of classical medicinal chemistry, combinatorial chemistry, and structural approaches, yet the validity of PDF as an antibacterial target hangs, in part, on the ability of inhibitors to selectively target this enzyme in favor of structurally related metallohydrolases. We have used (15)N NMR spectroscopy and isothermal titration calorimetry to investigate the high-affinity interaction of EcPDF with actinonin, a naturally occurring potent EcPDF inhibitor. Backbone amide chemical shifts, residual dipolar couplings, hydrogen-deuterium exchange, and (15)N relaxation reveal structural and dynamic effects of ligand binding in the immediate vicinity of the ligand-binding site as well as at remote sites. A comparison of the crystal structures of free and actinonin-bound EcPDF with the solution data suggests that most of the consequences of the ligand binding to the protein are lost or obscured during crystallization. The results of these studies improve our understanding of the thermodynamic global minimum and have important implications for structure-based drug design.

Selection of peptomeric inhibitors of bovine α-chymotrypsin and cathepsin G based on trypsin inhibitor SFTI-1 using a combinatorial chemistry approach

A peptomeric library consisting of 360 monocyclic analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds was designed and synthesized by a solid-phase approach in order to select chymotrypsin and cathepsin G inhibitors. All peptomers contained a proteinogenic-Phe-mimicking N-benzylglycine (Nphe) at positions 5 and 12. Into the synthesized library, different peptoid monomers were introduced in the 7-10 segment. Deconvolution of the library against both proteinases through an iterative method in solution revealed that the strongest chymotrypsin inhibitory activity was displayed by two analogues, [Nphe(5,12)]SFTI-1 (1) and [Nphe(5,12), Naem(8)]SFTI-1 (2), where Naem stands for N-(2-morpholinoethyl)glycine. After deconvolution against a cathepsin G analogue, [Nphe(5,12), Npip(8,9), Nnle(10)] SFTI-1 (3) (Npip = N-(3,4-methylenedioxybenzyl)glycine) appeared to be the most potent inhibitor with a high serum stability. It is worth noting that the analogues obtained by a combinatorial approach display high specificity towards one of the experimental enzymes. Another interesting feature is the lack of Pro8 in analogues 2 and 3, the amino acid residue absolutely conserved in the family of Bownan-Birk inhibitors.