63 research outputs found
Biological characterization of the lectins DC-SIGN and FimH : putative targets for novel anti-infectives
Urinary tract infection (UTI) is one of the most common infections, with millions of people affected every year. Besides women, who bear a risk of 40 - 50% to experience at least one symptomatic UTI episode during a life-time, patients with diabetes, spinal cord injuries, and suppressed immune system are particularly at risk. Without treatment UTI may lead to bladder infection (cystitis) and, in a later infection state, to kidney infection (pyelonephritis). The initial and most fundamental step in the pathogenesis of UTIs is the type 1 pili-dependent adhesion of uropathogenic Escherichia coli (UPEC) to ?-mannoside-containing glycoprotein receptors on the surface of uroepithelial cells, such as uroplakin Ia (UPIa). The bacterial adhesion is mediated by the lectin FimH, localized at the tip of type 1 pili, which recognizes mono- and oligomannosides. The adhesion triggers the bacterial cell invasion, resulting in the development of an infection. FimH antagonists such as alpha-D-mannopyranosides have been shown to interfere with the attachment of UPEC to their host cells, thus providing a novel therapeutic opportunity for the treatment and prevention of UTIs as an alternative to antibiotic treatment.
A potent FimH antagonist has to fulfill several requirements to also achieve a high in vivo efficacy. Besides a high affinity for FimH (KD), slow off-rates and irreversibility of the antagonist-FimH interaction are beneficial for the in vivo efficacy, as prolonged occupancy of the target by the drug results in an extended duration of the pharmacological effect. Furthermore, target selectivity of FimH antagonists is a pivotal concern, since the reported FimH antagonists are alpha-D-mannopyranosides and therefore are potential ligands for mannose receptors of the human host system. Non-selective interactions of FimH antagonists with the various mannose receptors would have a profound impact on physiological processes and could cause severe side effects.
This thesis adresses some major issues in the development and biological evaluation of FimH antagonists:
-Development of a cell-based competition assay for the determination of IC50 values of FimH antagonists using flow cytometry.
-Determination of kinetic properties and KD values of FimH antagonists by surface plasmon resonance.
- Investigation of the selectivity of FimH antagonists towards human mannose binding receptors.
Dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is a pathogen recognition receptor (PRR) and abundantly expressed on immature dendritic cells (DCs). The binding of pathogens via PRRs mediates phagocytosis, DC maturation and migration from peripheral tissues to draining lymph nodes. After lysosomal degradation, the processed antigen particles are presented to naïve T-cells, resulting in the stimulation of adaptive immune responses. However, a variety of pathogens including HIV-1 use the interaction with DC-SIGN on DCs as initial entry port to their host. These pathogens are able to circumvent the intracellular degradation process and impair DC maturation. DC-SIGN recognizes mannose-containing glycoconjugates and fucose-containing blood-group antigens, such as Lewisx (Lex), Lewisa (Lea), and Lewisy (Ley), in the envelope of viruses and the membranes of parasites. DC-SIGN is therefore considered as a potential drug target for the treatment and prevention of a number of infectious diseases. Consequently, considerable efforts are made to develop DC-SIGN antagonists. These new anti-infectives would inhibit DC-SIGN-pathogen interaction and block the initial step of an infection, as well as the pathogen dissemination.
This thesis addresses the issue of improving the recombinant expression of the carbohydrate recognition domain of DC-SIGN by investigating the effect of different signal peptides on the expression of recombinant protein in CHO-K1 cells. The usage of the luciferase signal peptide of the copepod Gaussia princeps led to a drastic improvement of the protein yield compared to the standard interleukin-2 signal peptide. The recombinant protein was used for the evaluation of binding potentials of novel FimH antagonists. A target-based binding assay was developed and a series of antagonists were measured, with the focus on Lewis type structures. Combination of binding assays, mutational analysis, STD NMR studies, and computational modeling revealed a new binding mode with improved affinity for phenyl group-substituted Lea compounds. Therefore these compounds display a novel class of potential anti-infectives
Target Selectivity of FimH Antagonists
Mannose-based FimH antagonists are considered new therapeutics
for the treatment of urinary tract infections (UTIs). They prevent
the adhesion of uropathogenic <i>Escherichia coli</i> (UPEC)
to urothelial cell surfaces triggered by the lectin FimH, which is
located at the tip of bacterial type 1 pili. Because all reported
FimH antagonists are α-d-mannosides, they are also
potential ligands of mannose receptors of the human host system. We
therefore investigated the selectivity range of five FimH antagonists
belonging to different compound families by comparing their affinities
for FimH and eight human mannose receptors. On the basis of the detected
selectivity range of approximately 5 orders of magnitude, no adverse
side effects resulting from nonselective binding to the human receptors
have to be expected. FimH antagonists can therefore be further considered
as potential therapeutics for the treatment of UTI
Anti-adhesion therapy for urinary tract infections - a balanced PK/PD-profile proved to be key for success
FimH Antagonists for the Oral Treatment of Urinary Tract Infections : From Design and Synthesis to In Vitro and In Vivo Evaluation
Blockade of activin type II receptors with a dual anti-ActRIIA/IIB antibody is critical to promote maximal skeletal muscle hypertrophy
The TGF-β family ligands myostatin, GDF11, and activins are negative regulators of skeletal muscle mass, which have been reported to primarily signal via the ActRIIB receptor on skeletal muscle and thereby induce muscle wasting described as cachexia. Use of a soluble ActRIIB-Fc “trap,” to block myostatin pathway signaling in normal or cachectic mice leads to hypertrophy or prevention of muscle loss, perhaps suggesting that the ActRIIB receptor is primarily responsible for muscle growth regulation. Genetic evidence demonstrates however that both ActRIIB- and ActRIIA-deficient mice display a hypertrophic phenotype. Here, we describe the mode of action of bimagrumab (BYM338), as a human dual-specific anti-ActRIIA/ActRIIB antibody, at the molecular and cellular levels. As shown by X-ray analysis, bimagrumab binds to both ActRIIA and ActRIIB ligand binding domains in a competitive manner at the critical myostatin/activin binding site, hence preventing signal transduction through either ActRII. Myostatin and the activins are capable of binding to both ActRIIA and ActRIIB, with different affinities. However, blockade of either single receptor through the use of specific anti-ActRIIA or anti-ActRIIB antibodies achieves only a partial signaling blockade upon myostatin or activin A stimulation, and this leads to only a small increase in muscle mass. Complete neutralization and maximal anabolic response are achieved only by simultaneous blockade of both receptors. These findings demonstrate the importance of ActRIIA in addition to ActRIIB in mediating myostatin and activin signaling and highlight the need for blocking both receptors to achieve a strong functional benefit
FimH Antagonists: Bioisosteres To Improve the in Vitro and in Vivo PK/PD Profile
Urinary tract infections (UTIs), predominantly caused by uropathogenic Escherichia coli (UPEC), belong to the most prevalent infectious diseases worldwide. The attachment of UPEC to host cells is mediated by FimH, a mannose-binding adhesin at the tip of bacterial type 1 pili. To date, UTIs are mainly treated with antibiotics, leading to the ubiquitous problem of increasing resistance against most of the currently available antimicrobials. Therefore, new treatment strategies are urgently needed. Here, we describe the development of an orally available FimH antagonist. Starting from the carboxylate substituted biphenyl α-d-mannoside 9, affinity and the relevant pharmacokinetic parameters (solubility, permeability, renal excretion) were substantially improved by a bioisosteric approach. With 3'-chloro-4'-(α-d-mannopyranosyloxy)biphenyl-4-carbonitrile (10j) a FimH antagonist with an optimal in vitro PK/PD profile was identified. Orally applied, 10j was effective in a mouse model of UTI by reducing the bacterial load in the bladder by about 1000-fold
Exploring host-pathogen interactions through genome wide protein microarray analysis
During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ∼2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis.</p
- …