Since FimH emerged as the most appropriate target for the development of anti-adhesive therapeutic strategies, several studies began, decades ago, to analyze the effects of FimH antagonists. Duguid and Gillis were the first authors to report mannose as an anti-adhesive substance in
E. coli bacteria in 1957
[24][40]. Then, in 1977, the anti-adhesive activity of mannose was described in detail by Ofek, Mirelman and Sharon for
E. coli [24][41], and later on for other uropathogens
[42][43][44]. Up until now, different categories of soluble mono- and polyvalent FimH inhibitors (α-
d-mannosides, and their chemically modified derivatives and glycodendrimers, respectively) have been selected, synthesized and analyzed
[17][26]. Due to the huge number of available molecules, researchers in this field established standardized protocols and techniques to test FimH inhibitory activity. The relative inhibitory potency (RIP) index describes the extent of the affinity of the various FimH antagonists, compared to known synthetic mannoside derivatives such as the Methyl α-
d-mannoside (MeMan), which is considered as a high-affinity molecule with regards to FimH
[19][45]. The application of this method ensures the comparability of the results obtained by applying different experimental procedures. High RIP values result in the strong affinity of the analyzed molecules. For example, it has been shown that a monovalent antagonist with a high RIP value possessed the same affinity toward a variety of UPEC strains, while a polyvalent antagonist showing high RIP displayed a strain-dependent affinity. This result points out that more studies should be performed in assessing the therapeutic efficacy of polyvalent molecules
[46]. However, despite their high and broad activity, monovalent glycosides, such as natural
d-mannose, have no stable structures in vivo, and are rapidly hydrolyzed within the mouth, gastrointestinal tract and other organs and tissues, or they are quickly excreted from the body
[47][48]. So, the application of antagonist therapeutic strategies, such as glycosidic drugs, represents a big challenge. The advances in understanding carbohydrate–protein interactions led to the development of a new class of small-molecule drugs for the treatment of several human diseases, known as glycomimetics. These molecules mimic the bioactive function of pure carbohydrates without presenting their drawbacks, such as low activity and stability
[49]. Thus, specific chemical modifications represented a good approach to enhancing the bioavailability and metabolic stability of glycoside molecules
[47]. Today, several glycomimetics, based on α-
d-mannose derivatives, have been selected that show a strong affinity with FimH
[34]. Glycosides are chemically linked to the aglycone (non-carbohydrate) portion in order to improve the glycomimetics’ affinity to FimH, as well as their stability and bioavailability. Alkyl-, aryl- biaryl-, biphenyl-, butyl- dioxocyclobutenylaminophenyl-, indolylphenyl-, methyl-, phenyl-, triazolyl-, thiazolylamine- and umbelliferyl- are the most common aglycone groups combined with monovalent α-
d-mannosides
[24][26][34][36][50][51]. Vice versa, the polyvalent
d-mannosides include cyclodextrin-based heptyl mannosides (CD-based HMs), divalent mannosides, glycoclusters, glycodendrimers, neoglycoproteins and trivalent mannosides, which contain more than one mannose subunit
[24][34][36][52]. Indeed, lectins may carry several carbohydrate-binding sites (CBS), and this property enables them to significantly increase their affinity towards sugar residues. Exploiting this feature, multivalent glycomimetics are bound by FimH with high affinity, and this multivalent effect is known as molecule avidity
[53][54]. These types of modifications result in an inhibition effect on FimH a million times greater than that exerted by the
d-mannose sugar
[33][55][56].
4. FimH Antagonists, Biochemical Characteristics and Bioavailability
According to the type of interaction with the
d-mannosylated molecules, FimH adhesin changes its conformational structure, leading to different binding affinities. As outlined above, the low affinity (T-state) conformational structure of FimH, in which the LD and PD domains are in strict contact, occurs in the absence of shear stress. Vice versa, the high affinity conformation (R-state), in which FimH
LD and Fim
PD are separated, represents the shear stress-induced allosteric regulation of its mannose-binding affinity, resulting in the strong attachment of FimH
LD to the host urothelial cell receptors
[26]. Thus, the balance between R- and T-states regulates the capability of the bacteria to colonize the urothelial niche or to spread the infection. At the molecular level, it is known that the interactions between α-
d-mannose molecules (and related derivatives) and MBP in FimH
LD occur in the presence of water, because water molecules support the hydrogen bonds between the hydroxyl groups of α-
d-mannose molecules and the amino acid residues within MBP. Moreover, the presence of water drives the proper binding of α-anomer molecules to the MBP of FimH, increasing the affinity of the α-anomeric configuration of mannose and its derivatives with MBP
[24][35][57][58]. Biochemical analyses of the interaction between FimH
LD and α-
d-mannose revealed that mannosides with an apolar (hydrophobic) substituent are able to mimic the interactions of high-mannose glycans with the MBD of FimH
[56]. For this reason, n-hexyl- and n-heptyl-modified mannosides (i.e., MeMan) have a significant high affinity towards FimH
[24][59]. This hydrophobic portion of aglycone interacts with the tyrosine gate through aromatic stacking (non-covalent interaction between aromatic rings) and Van der Waals bonds
[51][59][60][61]. Moreover, it was shown that glycomimetics with inhibition constants in the range of 1–20 nM can be obtained by combining the α-anomeric configurations of
d-mannose
[58][62]. Hence, Wellens et al. generated a set of α-
d-mannosides carrying alkyl and aryl hydrophobic moieties. The determination of the crystal structure of FimH
LD with the eight synthesized inhibitors, together with the analyses of their thermodynamic parameters, demonstrated that the presence of alkyl and aryl groups in the aglycone can induce the increased dynamics in the tyrosine gate responsible for the proper orientation of the interacting mannosides. This dynamic behavior of the tyrosine gate could contribute to FimH’s ability to deal with less compatible high-mannose structures, while still making bacterial adhesion plausible
[37]. Moreover, aromatic aglycone compounds mediate several interactions within the tyrosine gate in its hydrophobic space, increasing the affinity of the antagonist to the MBP of FimH
LD [54][58]. An increase in the length of alkyl chains results in the higher affinity of the molecule with the FimH
LD and, in particular, with the tyrosine gate area, showing that the affinity of the alkyl group with FimH adhesin is 100-fold greater than that exhibited by mannose
[24][63][64].
It has been shown that O- and C-linked α-
d-mannosides with hydrophobic and aryl substituents are potent
E. coli FimH antagonists, having an affinity in the same range as that of nanomolar
[65]. Indeed, the conformation and lipophilicity of aglycone moieties, their position with respect to the core sugar structure and the type of chemical group determine the RIP of antagonist molecules
[34][54]. Para-substituted biphenyl derivatives were shown to be particularly appealing, owing to their numerous favorable binding interactions within the tyrosine gate. Thus, the structural and functional analyses of a series of O-, C-, and S-linked mannoside derivatives, incorporating the 1,1′-biphenyl pharmacophore and diverse aglycone atoms, demonstrated the suitability of these antagonists, establishing the possibility of further exploring these chemically modified mannosides
[65][66]. Furthermore, it was shown that the biphenyl group linked to mannosides can be efficiently absorbed if orally administered
[26][67]. Indeed, these mannosides show increased metabolic stability, bioavailability and intestinal permeability in in vivo pharmacokinetic studies, thereby recommending them for preclinical evaluation
[68]. In addition, the reabsorption of biphenyl groups by renal tubuli results in stable and regular excretion into urine, leading to their high availability in the site of infection
[26][67][69]. It was also demonstrated that 3′-chloro-4′-(α-
d-mannopyranosyloxy) biphenyl-4-carbonitriler (
Figure 4), synthesized using the bioisostere approach, is a highly effective FimH antagonist, also presenting optimal pharmacokinetic characteristics, such as proper solubility, low toxicity, intestinal permeability and renal excretion in mouse models
[18]. Moreover, its oral application reduced the bacterial load in the bladder by almost 1000-fold 3 h after infection, highlighting its therapeutic potential
[18].
Figure 4. The successful linkage between Tyrosine Gate (Fim
LD MBP) and the bioisostere of 3′-chloro-4′-(α-
d-mannopyranosyloxy)-biphenyl-4-carbonitrile (4CST PDB file)
[18].
The polyvalent adhesin inhibitors (carbohydrate dendrimers) were designed to better mimic the interaction of FimH with high-mannose eukaryotic receptors
[34]. The affinity, avidity and selectivity of mannosylated glycodendrimers are strengthened throughout by the presence of several mannose residues in the molecule; the so-called “cluster effect”
[54][59][70]. Despite their higher affinity with the MBP of FimH, mannosylated glycodendrimers are large-size polar molecules, and these chemical properties reduce their absorption in the gastrointestinal tract, affecting their oral usage
[34].
Apart from chemically synthesized mannose-based molecules, natural compounds, including cranberry and its derivatives, such as myricetin, cranberry extract standardized in proanthocyanidins (PACs) and PAC-derived polyphenol metabolites, have anti-adhesive effects on UPEC
[25]. The mechanism by which these compounds exert their anti-adhesive activity is not totally understood yet. The complex molecular composition of these natural extracts can influence the establishment of the infection at different levels, acting on both bacteria and human physiology. Several investigations showed that PACs efficiently block the P fimbriae
[71][72][73]. Vice versa, it was indicated that PAC-metabolites could be responsible for anti-adhesive effects on FimH
[25]. Moreover, it was also suggested that cranberry induces the expression/secretion of the Tamm–Horsfall proteins by the kidney, thereby leading to its accumulation in the bladder. Thus, the interaction between UPEC FimH and the mannosylated Tamm–Horsfall glycoproteins causes bacterial release within the urine flux
[74]. As such, cranberry-based supplements represent a source of natural compounds that are biochemically active against UPEC, which deserves further investigation.