Synthesis of potent inhibitors of anthrax toxin based on poly-L-glutamic acid

We report the synthesis of biodegradable polyvalent inhibitors of anthrax toxin based on poly-L-glutamic acid (PLGA). These biocompatible polyvalent inhibitors are at least 4 orders of magnitude more potent than the corresponding monovalent peptides in vitro and are comparable in potency to polyacrylamide-based inhibitors of anthrax toxin assembly. We have elucidated the influence of peptide density on inhibitory potency and demonstrated that these inhibitory potencies are limited by kinetics, with even higher activities seen when the inhibitors are preincubated with the heptameric receptor-binding subunit of anthrax toxin prior to exposure to cells. These polyvalent inhibitors are also effective at neutralizing anthrax toxin in vivo and represent attractive leads for designing biocompatible anthrax therapeutics.     

Bivalent inhibitors of the tyrosine kinases ABL and SRC: determinants of potency and selectivity

We recently reported a chemical genetic method for generating bivalent inhibitors of protein kinases. This method relies on the use of the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (AGT) to display an ATP-competitive inhibitor and a ligand that targets a secondary binding domain. With this method potent and selective inhibitors of the tyrosine kinases SRC and ABL were identified. Here, we dissect the molecular determinants of the potency and selectivity of these bivalent ligands. Systematic analysis of ATP-competitive inhibitors with varying linker lengths revealed that SRC and ABL have differential sensitivities to ligand presentation. Generation of bivalent constructs that contain ligands with differential affinities for the ATP-binding sites and SH3 domains of SRC and ABL demonstrated the modular nature of inhibitors based on the AGT scaffold. Furthermore, these studies revealed that the interaction between the SH3 domain ligand and the kinase SH3 domain is the major selectivity determinant amongst closely-related tyrosine kinases. Finally, the potency of bivalent inhibitors against distinct phospho-isoforms of SRC was determined. Overall, these results provide insight into how individual ligands can be modified to provide more potent and selective bivalent inhibitors of protein kinases.     

Statistical pattern matching facilitates the design of polyvalent inhibitors of anthrax and cholera toxins

Numerous biological processes involve the recognition of a specific pattern of binding sites on a target protein or surface. Although ligands displayed by disordered scaffolds form stochastic rather than specific patterns, theoretical models predict that recognition will occur between patterns that are characterized by similar or "matched" statistics. Endowing synthetic biomimetic structures with statistical pattern matching capabilities may improve the specificity of sensors and resolution of separation processes. We demonstrate that statistical pattern matching enhances the potency of polyvalent therapeutics. We functionalized liposomes with an inhibitory peptide at different densities and observed a transition in potency at an interpeptide separation that matches the distance between ligand-binding sites on the heptameric component of anthrax toxin. Pattern-matched polyvalent liposomes inhibited anthrax toxin in vitro at concentrations four orders of magnitude lower than the corresponding monovalent peptide, and neutralized this toxin in vivo. Statistical pattern matching also enhanced the potency of polyvalent inhibitors of cholera toxin. This facile strategy should be broadly applicable to the detection and neutralization of toxins and pathogens.     

Biofunctionalized surfactant mesophases as polyvalent inhibitors of cholera toxin

The cubic lyotropic mesophase composed of the ganglioside G(M1) and the synthetic surfactant phytantriol has been employed as a scaffold to prepare a polyvalent inhibitor of cholera toxin (CT). Surfactant mixtures containing up to 20% (w/w) G(M1)/phytantriol afforded a hydrated insoluble gel-like material, which retained an inverse cubic phase (Q) structure in excess water and at 22 degrees C, as confirmed using small-angle X-ray scattering. The G(M1)-functionalized mesophases bind up to 98.8% of CT from solution containing 100 ng/mL of CT with a dissociation constant (K(d)) of 67-73 nM. The estimated IC50 values for the mesophase systems were 0.1-0.27 microM. The inhibitory effect of the mesophases may be enhanced through the high internal surface area of the inverse cubic phase in addition to the optimal self-orientation of G(M1) ligand to match the distribution of binding sites on the toxin surface. As a result, polyvalent inhibitor materials manufactured using these mesophase structures do not require precise control of ligand distribution, which offers advantages with respect to complexity of synthesis and formulation when compared to more rigid and conformationally restricted materials. This approach provides a route to a unique class of polyvalent inhibitors, which should be broadly applicable to a range of bacterial and plant toxins.     

Pharmacophoric features of Pseudomonas aeruginosa deacetylase LpxC inhibitors: an electronic and structural analysis

Various electronic properties of structurally diverse synthetic LpxC inhibitors containing oxazoline, aroylserine and thiazoline rings were calculated and correlated with biological activity. These electronic features include the magnitude and locations of 3-dimensional molecular electrostatic potentials, hydrogen bond acceptor/donor density, lowest unoccupied molecular orbital, and highest occupied molecular orbital. Strong correlation of these stereo-electronic properties with LpxC inhibitory potency reveals the potential pharmacophoric features of specific LpxC inhibitors. Thus, these pharmacophoric features of LpxC inhibitors based on electronic and surface analysis could be successfully exploited for designing more potent LpxC inhibitors.     

Synthesis of copolymers containing an active ester of methacrylic acid by RAFT: controlled molecular weight scaffolds for biofunctionalization

We report the controlled radical copolymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with a monomer containing an active ester, N-methacryloyloxysuccinimide (NMS), by reversible addition fragmentation chain transfer (RAFT). The large difference in the reactivity ratios of HPMA and NMS resulted in significant variations in copolymer composition with increasing conversion during batch copolymerization. The use of a semi-batch copolymerization method, involving the gradual addition of the more reactive NMS, allowed uniformity of copolymer composition to be maintained during the polymerization. We synthesized polymers in a wide range of molecular weights (M(n) = 3000-50,000 Da) with low polydispersities (1.1-1.3). The effect of the ratio of monomer to chain transfer agent (CTA) on the molecular weight of the polymer was investigated. Given the numerous applications of poly(HPMA)-based conjugates in designing polymeric therapeutics, these controlled molecular weight activated polymers represent attractive scaffolds for biofunctionalization. As a demonstration, we attached a peptide to the activated polymer backbone to synthesize a potent controlled molecular weight polyvalent inhibitor of anthrax toxin.     

Novel bicyclic lactam inhibitors of thrombin: highly potent and selective inhibitors

The potency and selectivity of a previous series of low molecular weight thrombin inhibitors were improved through modifications of the P1 and P3 residues. Introduction of diphenyl substituted sulfonamides in the P3 moiety led to highly efficacious compounds. By correctly selecting the combination of P1 and P3 residues, high levels of potency, selectivity and in vivo efficacy were obtained.     

Polyvalency: a promising strategy for drug design

The simultaneous binding of multiple ligands on one entity to multiple receptors on another can result in an affinity that is significantly greater than that for the binding of a single ligand to a single receptor. This concept of "polyvalency" can be used to design molecules that are potent inhibitors of toxins and pathogens. We describe the design of potent polyvalent inhibitors that neutralize anthrax toxin in vivo as well as our attempts to elucidate the relationship between inhibitor structure and activity. We also highlight promising future avenues for research in polyvalent drug design.     

Structure-based design of novel groups for use in the P1 position of thrombin inhibitor scaffolds. Part 2: N-acetamidoimidazoles

Guided by X-ray crystallography of thrombin-inhibitor complexes and molecular modeling, alkylation of the N1 nitrogen of the imidazole P1 ligand of the pyridinoneacetamide thrombin inhibitor 1 with various acetamide moieties furnished inhibitors with significantly improved thrombin potency, trypsin selectivity, functional in vitro anticoagulant potency and in vivo antithrombotic efficacy. In the pyrazinoneacetamide series, oral bioavailability was also improved.     

Multiscale computational study of ligand binding pathways: Case of p38 MAP kinase and its inhibitors

Protein kinases are one of the most important drug targets in the past 10 years. Understanding the inhibitor association processes will profoundly impact new binder designs with preferred binding kinetics. However, after more than a decade of effort, a complete atomistic-level study of kinase inhibitor binding pathways is still lacking. As all kinases share a similar scaffold, we used p38 kinase as a model system to investigate the conformational dynamics and free energy transition of inhibitor binding toward kinases. Two major kinase conformations, Asp-Phe-Gly (DFG)-in and DFG-out, and three types of inhibitors, type I, II, and III, were thoroughly investigated in this work. We performed Brownian dynamics simulations and up to 340 μs Gaussian-accelerated molecular dynamics simulations to capture the inhibitor binding paths and a series of conformational transitions of the p38 kinase from its apo to inhibitor-bound form. Eighteen successful binding trajectories, including all types of inhibitors, are reported herein. Our simulations suggest a mechanism of inhibitor recruitment, a faster ligand association step to a pre-existing DFG-in/DFG-out p38 protein, followed by a slower molecular rearrangement step to adjust the protein-ligand conformation followed by a shift in the energy landscape to reach the final bound state. The ligand association processes also reflect the energetic favor of type I and type II/III inhibitor binding through ATP and allosteric channels, respectively. These different binding routes are directly responsible for the fast (type I binders) and slow (type II/III binders) kinetics of different types of p38 inhibitors. Our findings also echo the recent study of p38 inhibitor dissociation, implying that ligand unbinding could undergo a reverse path of binding, and both processes share similar metastates. This study deepens the understanding of molecular and energetic features of kinase inhibitor-binding processes and will inspire future drug development from a kinetic point of view.     

Structure-based design of novel human Pin1 inhibitors (II)

Following the discovery of a novel series of phosphate-containing small molecular Pin1 inhibitors, the drug design strategy shifted to replacement of the phosphate group with an isostere with potential better pharmaceutical properties. The initial loss in potency of carboxylate analogs was likely due to weaker charge–charge interactions in the putative phosphate binding pocket and was subsequently recovered by structure-based optimization of ligand–protein interactions in the proline binding site, leading to the discovery of a sub-micromolar non-phosphate small molecular Pin1 inhibitor.     

Species specificity of amidine-based urokinase inhibitors

Inhibition of the proteolytic activity of urokinase has been shown to inhibit the progression of tumors in rodent models and is being investigated for use in human disease. Understanding the rodent/human species-specificity of urokinase inhibitors is therefore critical for interpretation of rodent cancer progression models that use these inhibitors. We report here studies with a panel of 11 diverse urokinase inhibitors in both human and mouse enzymatic assays. Inhibitors such as amiloride, B428, and naphthamidine, that occupy only the S1 subsite pocket were found to be nearly equipotent between the human and the murine enzymes. Inhibitors that access additional, more distal, pockets were significantly more potent against the human enzyme but there was no corresponding potency increase against the murine enzyme. X-ray crystallographic structures of these compounds bound to the serine protease domain of human urokinase were solved and examined in order to explain the human/mouse potency differences. The differences in inhibitor potency could be attributed to four amino acid residues that differ between murine and human urokinases: 60, 99, 146, and 192. These residues are Asp, His, Ser, and Gln in human and Gln, Tyr, Glu, and Lys in mouse, respectively. Compounds bearing a cationic group that interacts with residue 60 will preferentially bind to the human enzyme because of favorable electrostatic interactions. The hydrogen bonding to residue 192 and steric considerations with residues 99 and 146 also contribute to the species specificity. The nonparallel human/mouse enzyme inhibition observations were extended to a cell-culture assay of urokinase-activated plasminogen-mediated fibronectin degradation with analogous results. These studies will aid the interpretation of in vivo evaluation of urokinase inhibitors.     

Macrophage growth inhibitors derived from the murine peritoneal cavity

Summary The murine peritoneal cavity contains factors that inhibit the in vitro growth and colony formation of macrophages. The inhibition of macrophage growth is not due to cell death. In the presence of inhibitors, the growth of colony-forming macrophages is suppressed, and small clusters are formed as a result of limited proliferation. The more mature mono-nuclear phagocytes (blood monocytes and peritoneal exudate macrophages) are more sensitive to the overall inhibitory effect of the peritoneal inhibitors than the less mature bone marrow mononuclear phagocytes. Furthermore, using dialysis and Amicon ultrafiltration, at least two inhibitors with differential inhibitory effects can be demonstrated. The colony formation of bone marrow mononuclear phagocytes is suppressed mainly by a protease-resistant, small molecular weight (<1,000) dialyzable inhibitor. In contrast, peritoneal exudate macrophages are sensitive to both the small molecular weight inhibitor and a protease-sensitive, large molecular weight (>12,000), nondialyzable inhibitor. The data suggest a possible existence of a dual inhibitor control on the proliferation of mononuclear phagocytes in vivo. In addition, the in vitro cultured peritoneal exudate cells are capable of producing inhibitors that mimic the activity of the in vivo inhibitors. This investigation was supported by Grants CA 09 11(SY) and AI15563(CCS) from the National Institutes of Health, Bethesda, MD     

Molecular docking studies on quinazoline antifolate derivatives as human thymidylate synthase inhibitors

We have performed molecular docking on quinazoline antifolates complexed with human thymidylate synthase to gain insight into the structural preferences of these inhibitors. The study was conducted on a selected set of one hundred six compounds with variation in structure and activity. The structural analyses indicate that the coordinate bond interactions, the hydrogen bond interactions, the van der Waals interactions as well as the hydrophobic interactions between ligand and receptor are responsible simultaneously for the preference of inhibition and potency. In this study, fast flexible docking simulations were performed on quinazoline antifolates derivatives as human thymidylate synthase inhibitors. The results indicated that the quinazoline ring of the inhibitors forms hydrophobic contacts with Leu192, Leu221 and Tyr258 and stacking interaction is conserved in complex with the inhibitor and cofactor.     

Enhanced potency of bivalent small molecule gp41 inhibitors

Low molecular weight peptidomimetic inhibitors with hydrophobic pocket binding properties and moderate fusion inhibitory activity against HIV-1 gp41-mediated cell fusion were elaborated by increasing the available surface area for interacting with the heptad repeat-1 (HR1) coiled coil on gp41. Two types of modifications were tested: 1) increasing the overall hydrophobicity of the molecules with an extension that could interact in the HR1 groove, and 2) forming symmetrical dimers with two peptidomimetic motifs that could potentially interact simultaneously in two hydrophobic pockets on the HR1 trimer. The latter approach was more successful, yielding 40–60 times improved potency against HIV fusion over the monomers. Biophysical characterization, including equilibrium binding studies by fluorescence and kinetic analysis by Surface Plasmon Resonance, revealed that inhibitor potency was better correlated to off-rates than to binding affinity. Binding and kinetic data could be fit to a model of bidentate interaction of dimers with the HR1 trimer as an explanation for the slow off-rate, albeit with minimal cooperativity due to the highly flexible ligand structures. The strong cooperativity observed in fusion inhibitory activity of the dimers implied accentuated potency due to the transient nature of the targeted intermediate. Optimization of monomer, dimer or higher order structures has the potential to lead to highly potent non-peptide fusion inhibitors by targeting multiple hydrophobic pockets.     

Semiquantitative determination of low molecular weight proteinase inhibitors in the presence of high molecular weight inhibitors by a modified radial diffusion assay

The radial diffusion assay is very suitable for the determination of proteinase inhibitors in biological fluids. By combining radial diffusion and ultrafiltration, it has become possible to directly determine low molecular weight proteinase inhibitors in mixtures with inhibitors of higher molecular weight. By this modification the inhibitor solutions to be investigated are not pipetted into wells as usually, but are applied on small pieces of dialysis membranes lying on the gel. The exclusion limit of the membrane must be of a magnitude that the inhibitors of higher molecular weight are retained, whereas the inhibitors of lower molecular weight can diffuse into the gel. The modified method can be used for the direct determination of e.g. aprotinin (Mr 6500) in the presence of alpha 1-proteinase inhibitor (Mr 54,000), ovoinhibitor (Mr 50,000) and ovomucoid (Mr 27,000), respectively. The modified method is suitable for the direct determination of low molecular weight inhibitors of trypsin and papain in serum, synovial fluid and saliva. Tissue extracts containing 4 M guanidine hydrochloride or 6 M urea can be investigated directly, too.     

Designing a polyvalent inhibitor of anthrax toxin

Screening peptide libraries is a proven strategy for identifying inhibitors of protein-ligand interactions. Compounds identified in these screens often bind to their targets with low affinities. When the target protein is present at a high density on the surface of cells or other biological surfaces, it is sometimes possible to increase the biological activity of a weakly binding ligand by presenting multiple copies of it on the same molecule. We isolated a peptide from a phage display library that binds weakly to the heptameric cell-binding subunit of anthrax toxin and prevents the interaction between cell-binding and enzymatic moieties. A molecule consisting of multiple copies of this nonnatural peptide, covalently linked to a flexible backbone, prevented assembly of the toxin complex in vitro and blocked toxin action in an animal model. This result demonstrates that protein-protein interactions can be inhibited by a synthetic, polymeric, polyvalent inhibitor in vivo.     

Recent advances in the development of steroid sulphatase inhibitors

Inhibition of steroid sulphatase is now an important target for the development of new drugs for the treatment of women with endocrine-dependent breast tumours. The first potent sulphatase inhibitor identified, oestrone-3-O-sulphamate (EMATE) proved, unexpectedly, to be oestrogenic. A number of strategies have therefore been adopted to design and synthesize a non-oestrogenic inhibitor. For this, a number of modifications have been made to the A and D rings of the oestrone nucleus. 2 Methoxyoestrone-3-O-sulphamate, while having similar in vitro and in vivo sulphatase inhibitory potency to that of EMATE, was devoid of oestrogenic activity when tested at 2 mg/kg in an ovariectomised rat uterine weight gain assay. 17-Deoxyoestrone-3-O-sulphamate was also a potent steroid sulphatase inhibitor and while it was devoid of oestrogenic activity when tested at 0.1 mg/kg, did stimulate uterine growth at 1.0 mg/kg. As an alternative approach to the use of steroid-based inhibitors a number of single ring, bicyclic non-fused ring, and two fused ring sulphamate analogues were designed, synthesized and tested for their ability to inhibit steroid sulphatase activity. In general, although the single ring and bicyclic non-fused ring sulphamate analogues could inhibit sulphatase activity, they were considerably less potent than EMATE. The mono- and bis-sulphamate derivatives of 5,7-dihydroxyisoflavone were relatively potent, inhibiting in vivo steroid sulphatase activity by 62 and 81% respectively at a single oral dose of 10 mg/kg. A study of the structure-activity relationship of a series of coumarin-based sulphamates has led to the development of a number of potent non-steroidal inhibitors, one of which has a similar potency to that of EMATE. The identification of potent steroid- and non-steroid-based sulphatase inhibitors will enable the therapeutic value of this therapy to be examined in the near future.     

Beta-amino acid substitutions and structure-based CoMFA modeling of hepatitis C virus NS3 protease inhibitors

In an effort to develop a new type of HCV NS3 peptidomimetic inhibitor, a series of tripeptide inhibitors incorporating a mix of alpha- and beta-amino acids has been synthesized. To understand the structural implications of beta-amino acid substitution, the P(1), P(2), and P(3) positions of a potent tripeptide scaffold were scanned and combined with carboxylic acid and acyl sulfonamide C-terminal groups. Inhibition was evaluated and revealed that the structural changes resulted in a loss in potency compared with the alpha-peptide analogues. However, several compounds exhibited muM potency. Inhibition data were compared with modeled ligand-protein binding poses to understand how changes in ligand structure affected inhibition potency. The P(3) position seemed to be the least sensitive position for beta-amino acid substitution. Moreover, the importance of a proper oxyanion hole interaction for good potency was suggested by both inhibition data and molecular modeling. To gain further insight into the structural requirements for potent inhibitors, a three-dimensional quantitative structure-activity relationship (3D-QSAR) model has been constructed using comparative molecular field analysis (CoMFA). The most predictive CoMFA model has q(2)=0.48 and r(pred)(2)=0.68.     

Synthesis and anti-influenza evaluation of polyvalent sialidase inhibitors bearing 4-guanidino-Neu5Ac2en derivatives

We synthesized polyvalent sialidase inhibitors bearing 4-guanidino-Neu5Ac2en analogues on the polyglutamic acid back bone, via a spacer of alkyl ether at the C-7 position. These multivalent conjugates 9 and 10 showed enhancement of antiviral activity against infuenza A virus and more potent efficacy in vivo relative to a monomeric sialidase inhibitor.