Several examples of glycomimetics have illustrated that a reduction in ligand polar surface area can enhance binding affinities by both generating new hydrophobic contacts with the protein, as well as reducing the enthalpic cost of ligand desolvation. Therefore, the removal of polar functional groups uninvolved in protein binding, most commonly the hydroxy moieties, has been well demonstrated to enhance binding affinities.
The thermodynamics of ligand-protein binding can be quantified by calculating the Gibbs free energy of an interaction, ΔG (Equation (1)), from its individual enthalpic (ΔH) and entropic (TΔS) terms, where a negative free energy is essential for productive binding events:
To better understand the high enthalpic cost of desolvation, one can compare the thermodynamic quantities calculated by Cabani et al. [19
]. The enthalpic penalty of desolvating a single hydroxy group, ΔH = 35 kJ/mol, is only partially offset by the favorable entropy term that results from the release of structured water molecules into bulk solvent, ΔS = 10 kJ/mol. This results in a net free energy of +25 kJ/mol, which cannot be compensated for by the energy gain afforded by a single H-bond (approx. ΔG = −18 kJ/mol). Although vicinal hydroxy groups experience a somewhat reduced desolvation penalty in comparison to individual hydroxy moieties (approx. ΔG = 34 kJ/mol for two vicinal hydroxy groups), the high enthalpic penalty of desolvation is still unfavorable for a binding event. This suggests that a minimum of two H-bonds should form between a ligand hydroxy group and the protein in order for the free energy of binding to be considered favorable. As exemplified in the literature, the removal of hydroxy groups forming only a single H-bond with the protein binding site typically enhances binding affinity. In order to optimize affinities in the design of glycomimetic ligands, the aim should be to form a larger number of high-quality H-bonds between each of the ligand’s polar groups and the protein surface.
Although the desolvation penalty is often very high for carbohydrate-binding proteins, resulting in part from shallow and solvent-exposed binding sites, proteins with deeper binding pockets are inherently more hydrophobic, less solvated, and therefore often display enhanced affinities. In these hydrophobic binding cavities, the H-bonds between ligand and protein are considerably stronger (approx. 10-fold), experience less competition from bulk solvent, and also have improved residence times (reduced koff
]. In these particular cases, where a lower desolvation penalty exists in combination with a higher enthalpic gain per H-bond, the requirement for generating such extensive H-bonding networks is reduced.
Alternative strategies have been used to reduce ligand solvation and thereby minimize the desolvation penalty: although not exemplified with a glycomimetic, Gao et al. nicely illustrated that the addition of a hydrophobic group in a non-binding, non-relevant position of a ligand was successful in disrupting water structure around the ligand and, therefore, reduced the enthalpic cost of desolvation [43
]. Even though this portion of the molecule displayed no interactions with the protein surface, it was successful in enhancing the free energy of binding.
Deoxygenation can also provide other beneficial effects. By reducing overall polarity of the molecule, this can increase the electron density on the pyranose ring and thereby enhance nucleophilicity of its remaining hydroxy groups. This enhanced nucleophilicity can strengthen interactions involving complexation of metal ions or salt bridges. Alternatively, deoxygenation of 6-OH groups removes a rotational degree of freedom yet still leaves the C-6 methyl group intact for influencing 4C1/1C4 pyranose conformational preference, which can further reduce the entropic costs associated with ligand binding.
Replacement of a hydroxy group with a fluorine atom has been used to experimentally probe the necessity of individual hydroxy moieties for H-bonding. This same strategy of OH → F substitution can also be applied in computational modelling. The fluorine atom is useful as a bioisosteric mimic of the hydroxy group, yet is also more hydrophobic and therefore, can retain important characteristics of the hydroxy moiety yet reduce polar surface area of the ligand [44
]. Several studies have revealed that upon fluorine substitution, recognition of the mimetic by its native receptor is still possible; for example, in studying the transport of d
-glucose across the endothelial membrane of red blood cells, 2-deoxy-2-fluoroglucose and 3-deoxy-3-fluoroglucose afforded very similar transport rates as compared to the native ligand [45
]. In another study, fluorinated mimetics of MUC1-based glycopeptides were observed to be cross-reactive with serum antibodies from mice that had been vaccinated with native antigen (compound 7
; Figure 3
]. This widespread recognition of fluorinated glyco-analogues has been a contributing factor to the success of 18
) as a radiotracer for diagnosing neoplasia through positron emission tomography (PET scans) [47
2.2. Biomimetic Replacement of Functional Groups
Biomimetic functional groups, i.e., those with comparable electronic and steric properties, can sometimes be used to replace existing functional groups to improve properties of a drug candidate. Bioisosteric replacement is a common practice in medicinal chemistry, with many families of bioisosteres having been reported and evaluated (Table 1
). Given the specific requirements for a functional group in a particular binding event (e.g., steric restrictions, H-bond donor/acceptor properties), different bioisosteres can be considered as suitable replacements in different situations.
Bioisosteric replacement can afford enhanced affinities in a number of ways; for example, in the previously described OH → F substitutions [45
], the fluorine atom can still facilitate polar interactions with the protein surface yet reduces overall hydrophilicity of the ligand. The fluorine atom can also be used as a suitable replacement for hydrogen, owing to its small size and relative hydrophobicity; replacement of the axial C-3 proton of sialic acid (Neu5Ac), to afford the glycomimetic 10
was successful in generating an inhibitor of sialyltransferase (Figure 4
]. Substitution with the fluorine atom afforded a ligand which was sterically compatible with the binding site, yet the unique electronic properties of fluorine generated a much more electrophilic anomeric carbon (C-2), improving antagonist ability. To overcome the negligible oral availability associated with such a polar substrate, the drug candidate was peracetylated; treatment in mice successfully impaired the progression of murine melanoma by inhibiting the attachment of metastatic cancer cells to the extracellular matrix and was also observed to slow down tumor growth in vivo.
In alternative biomimetic approaches, hydroxy groups binding to an active-site metal ion can be replaced with improved metal ligands, assuming that this modification is well tolerated by the binding site. Non-covalent interactions between sulfur and π-systems are typically stronger than those with oxygen atoms, suggesting a suitable route for further enhancing binding enthalpies. Aside from enhancing the enthalpic and entropic contributions of binding, bioisosteric replacement can also be useful for the removal of groups prone to metabolic degradation, or those that facilitate rapid excretion; these effects on the pharmacokinetic properties of a drug candidate will be discussed in more detail later.
2.3. Targeting Neighboring Regions of the Binding Site
For lectins with a well-structured binding pocket (which facilitates reduced entropic penalties upon generating additional interactions), it can be beneficial to look for new, enthalpically-favorable binding opportunities. The most promising approaches have targeted nearby aromatic or aliphatic residues and hydrophobic pockets, since ligand modification with hydrophobic groups has the added advantage of reducing the overall polar surface area of the ligand. Although, in general, hydrophobicity is preferred, additional interactions with neighboring ionic groups can also be realized, either through salt bridges or cation-π interactions. The overall approach for developing high-affinity glycomimetics is to optimize the individual entropic and enthalpic binding contributions; the majority of efforts in developing carbohydrate derivatives have focused on targeting surrounding protein sites that can both positively enhance binding contributions and also improve ligand selectivity against a particular target, with some examples highlighted below.
A large body of work has been focused on developing FimH antagonists, as an anti-adhesive approach to treating urinary tract infections (UTIs). UTIs are one of the most common causes of infection in developed countries, typically caused by uropathogenic Escherichia coli
]. Antibiotic resistance has been of increasing concern for treating these infections, and therefore, the possibility of anti-adhesive treatment offers a promising alternative. Type 1-fimbriae on E. coli
facilitate bacterial adherence to the bladder epithelium and enable the pathogen to avoid clearance during micturition; the FimH protein is located at the tip of the fimbriae, and binds to the highly mannosylated glycoprotein uroplakin 1a present at the epithelial surface [54
]. Examination of the FimH crystal structure was very beneficial for glycomimetic development, as it provided pertinent information on the ligand binding mode and also suggested further modifications to improve ligand affinity [55
]. It was observed that the 2-, 3-, 4-, and 6-OH groups of the d
-mannose residue form an important H-bond network in the buried ligand cavity with amino acid side chains Asp54, Gln133, Asn135, and Asp140, and backbone atoms from Phe1 and Asp47. Not unexpectedly, attempts to modify these positions have generally proven unsuccessful. Alternatively, the region surrounding the binding site entrance contains two tyrosine residues and one isoleucine residue (Tyr48, Ile52, and Tyr137), often referred to as the ‘tyrosine gate’, which can form hydrophobic contacts with glycomimetics and have been a major target for improving the affinities of FimH antagonists. First developed were aryl and n
-alkyl mannosides, which displayed increased affinities due to interactions with a hydrophobic rim surrounding the deep binding pocket and the aforementioned tyrosine gate. The groups of Janetka and Hultgren improved affinities by using 4′-biaryl mannosides with a meta substituent that could act as an H-bond acceptor (11
), in which the aromatic extension formed an optimal π-π interaction with Tyr48 and a new H-bonding electrostatic interaction with Arg98/Glu50, resulting in nanomolar binding affinities (Figure 5
]. Contributions from many groups in the development of α-mannosides and oligomannosides have improved FimH antagonists even further [57
The targeting of neighboring residues has also been used in the development of antagonists for FimH-like adhesin (FmlH) [31
]. FmlH is a pilus adhesin which binds galactosides and N
-acetyl-galactosaminosides presented on bladder and kidney tissue, facilitating the adhesion of E. coli
to these surfaces. In efforts to inhibit this interaction, aryl galactosides and N
-acetyl-galactosaminosides were designed which facilitated several key protein interactions: a π-π interaction with Tyr46, a salt bridge between the carboxylate and Arg142, and a H2
O-mediated H-bond between the N
-acetyl group and Lys132. The best inhibitor (14
) displayed a Ki
of approximately 90 nM and upon administration in a mouse model was able to reduce the bacterial load in both the kidney and bladder (Figure 6
). Co-treatment with a FimH antagonist further improved bacterial elimination.
In addition to the aforementioned glucosylceramide synthase inhibitor miglustat, iminosugars have also been developed as protein chaperones with picomolar affinities for the treatment of Gaucher disease, the most prevalent lysosomal storage disease (LSD) [63
]. LSDs ultimately result from a glycosidase deficiency, as glycosidases are important for the break-down of lysosomal glycosphingolipids. In LSDs such as Gaucher disease and Fabry disease, genetic mutations result in the misfolding of proteins, which are then targeted for degradation in the endoplasmic reticulum instead of being trafficked to the lysosome, resulting in significantly reduced lysosomal concentrations of protein. In pharmacological chaperone therapy, sub-inhibitory concentrations of a protein ligand can be used to stabilize the protein conformation, enabling successful trafficking of the protein to the lysosome; if designed appropriately, upon reaching the lysosome the protein should bind with higher affinity to its native ligand (also present in larger excess), thereby still retaining its native activity. In order to be effective as molecular chaperones, these glycomimetics should both be selective for their target, as well as reach the endoplasmic reticulum. This approach has previously been demonstrated for the glycomimetic 1-deoxygalactonojirimycin (Migalastat®
; Figure 7
), an inhibitor of α-galactosidase in vitro, which has been successfully used as a pharmacological chaperone in the treatment of Fabry disease [64
]. Alternatively, glycomimetic inhibitors based on 1-deoxynojirimycin (DNJ) have been developed by Mena-Barragán et al. and García-Moreno et al. in the development of a therapy against Gaucher disease (Figure 7
), which results from a β-glucocerebrosidase deficiency [63
]. Modification of DNJ to form sp2
-iminosugars significantly enhanced targeting to the endoplasmic reticulum, and even more fortunately the ligands were found to have enhanced binding at neutral pH over acidic pH, which suggests that their affinity will decrease after entering the lysosome which should aid in protein dissociation and reduce competition with its native substrates. The iminosugars were found to successfully act as molecular chaperones for proteins expressed in mutated G188S/G183W fibroblasts (a disease-associated genetic mutation); for example, structure 20
afforded a more than 70% increase in protein activity at only 20 pM concentration, and a 300% improvement at a 2 nM concentration.
Several other successful examples of ligand modification have been used to enhance the affinities of glycomimetics for their protein target. For example, Siglec-7 inhibitors have been synthesized which contain C-9 aromatic modifications (also targeting a ‘hydrophobic gate’ observed in the crystal structure) and/or triazole-containing hydrophobic groups at C-2 of Neu5Ac, in an effort to develop inhibitors which could prevent immune evasion by cancer cells (Figure 8
]. Similar in structure, Siglec-2 (also known as CD22) Neu5Ac glycomimetics containing a C-9 N
-aromatic moiety, C-4 N
-acyl derivative, and C-2 n
-alkyl group have been used as inhibitors and towards drug conjugates to specifically target uptake into specific subsets of immune cells via Siglec-2-binding clathrin-mediated endocytosis (Figure 9
]. Pseudomonas aeruginosa
lectin B (LecB) inhibitors have been developed in an effort to tackle biofilm formation: low molecular weight, nanomolar affinity ligands with good kinetic and thermodynamic properties were developed by targeting a hydrophobic patch on the protein [72
]. Additionally, much work has been focused on DC-SIGN antagonists as anti-adhesives, by targeting a hydrophobic groove on the protein [73
A novel approach which also targets neighboring residues of a lectin binding site has been the development of a covalent lectin inhibitor against LecA of Pseudomonas aeruginosa
]. Both LecA and LecB virulence factors have been associated with biofilm formation; although high affinity inhibitors against LecB have been developed, LecA has proven a more challenging target. In order to overcome the large koff
associated with LecA-ligand interactions, thereby enhancing affinity, a covalent inhibitor was developed which targets a nearby cysteine (Cys62) residue (Figure 10
). This use of a covalent inhibitor attempts to circumvent the inherently weak affinities which arise from the short lifetimes of lectin-ligand complexes by permanently appending the ligand to the protein.
2.4. Conformational Pre-organization
Improvements in binding affinity through pre-organization have been successful in a number of glycomimetics [75
]. Pre-organization reduces the entropic penalties associated with binding and additionally tends to reduce polar surface area since internal polar groups interact amongst each other, effectively shielding them from bulk solvent. Molecules have inherent entropy when free in solution, related to both translation and rotation (including internal rotation at single bonds). Entropic costs are associated with the binding of ligands, since a restriction of motion occurs through both a loss of rotational and translational entropy (for both ligand and protein); the greater the rigidity of the formed complex, the higher the entropic penalty of binding [77
As mentioned previously, productive binding can only occur with a negative free energy; this requires that the unfavorable entropic costs from restriction of the binding site be offset by favorable intermolecular interactions of ligand binding, considering both enthalpic contributions (e.g., H-bonding, van der Waals, etc.) and entropic contributions (e.g., release of water molecules from the binding site). Flexible receptors which require an ‘induced fit’ binding mode suffer from even greater entropic binding penalties, since the protein loses much of its conformational flexibility, therefore requiring even greater enthalpic compensatory interactions to enable productive binding events.
Pre-organization has been shown to play an important role in the development of glycomimetic inhibitors. In the amino-glycosides, distortion of the pyranose ring has been used in efforts to mimic the flattened shape of the enzymatic transition state. This conformational distortion can be accomplished using a variety of approaches, such as the introduction of an sp2
-hybridized center, modification of the ring size, or by generating bicylic or bridged systems [13
The importance of conformational pre-organization has also been observed in the generation of LecB inhibitors. Glycan screening indicated that the Lewis A (Lea
) trisaccharide, β-d
-GlcNAc, bound LecB with a Kd
of 220 nM [79
]. Attempts to simplify the structure eliminated the d
-galactose moiety entirely to afford the disaccharide α-l
-GlcNAc, but unfortunately isothermal titration calorimetry (ITC) experiments indicated a significantly reduced binding affinity resulting from an increased entropic penalty [80
]. To further simplify the construct and reduce flexibility, α-l
-fucosides were modified with heterocyclic aglycone substituents to afford substrates which, in some cases, could bind with affinities similar to those of Lea
Another successful example of pre-organization was illustrated in the development of an E-selectin antagonist. The native ligand of E-selectin, sialyl Lewis X (sLex
), binds with six solvent-exposed H-bonds and a salt bridge [11
]. In efforts to improve the affinity of sLex
, a glycomimetic antagonist was developed which could be pre-organized into the binding-site conformation, minimizing the entropic penalties associated with binding. Based on the crystal structure, it was observed that the N
-galactosamine moiety does not form direct contacts with the protein, but instead only acts as a linker between the other residues; therefore, it was replaced by a non-carbohydrate moiety that linked the d
-galactose and l
-fucose residues in a correct spatial orientation [84
]. By strategically placing substituents on the linker, the structure could be even more rigidified to further improve pre-organization and thereby also antagonist affinity [85
]. With later iterations, the Neu5Ac moiety was replaced by (S
)-cyclohexyl lactic acid which even further rigidified the glycomimetic conformation [84
The importance of pre-organization has also been demonstrated in the development of FimH antagonists, upon comparing septanose versus pyranose glycomimetic scaffolds (Figure 11
]. In an examination of binding to the conformationally rigid FimH lectin domain, the highly flexible septanose derivative resulted in a 10-fold affinity loss. NMR, X-ray crystal structure, and molecular modeling all indicated that the related septanose and pyranose derivatives formed a superimposable network of H-bonds, yet the septanose displayed lower affinities; ITC confirmed that this loss of affinity resulted from an entropic penalty arising from flexibility of the septanose core.
Numerous glycomimetics have incorporated multivalency in order to better mimic the multivalent presentation of native ligands [81
]. Multivalency can improve binding affinities in several ways: (i) chelation; (ii) statistical rebinding effects; or (iii) clustering of soluble binding partners [92
]. The design of multivalent scaffolds must be carefully considered in order to incorporate proper spacing and flexibility, enabling a correct fit of the ligand into the binding site, yet concomitantly minimizing the entropic costs of binding. In general, flexible scaffolds are often more forgiving if poorly designed, but suffer from much greater entropic penalties upon binding. A recent study from the Hartmann and Lindhorst groups has also nicely demonstrated that tuning scaffold hydrophobicity can also play a significant role in the affinity of multivalent constructs [94
In an elegant study, DC-SIGN glycomimetic antagonists were conjugated to oligovalent molecular rods and used to study multivalency effects of binding, affording nanomolar antagonists (Figure 12
]. The constructs contained a rigidified core based on phenylene-ethynylene units (previously used in the generation of P. aeruginosa
LecA inhibitors), and were designed to be an ideal length (approx. 4 nm) for chelation to bridge carbohydrate recognition domains on neighboring DC-SIGN subunits. The length of the rigid core could be controlled, with the rigidity effectively reducing entropic binding penalties, while the ends of the rods contained trivalent constructs which had been assembled using short, more flexible linkers. The incorporated trivalent groups were intended to address favorable statistical rebinding, with the flexible linkers aimed at facilitating a better fit of ligand into the binding site (at a minor entropic cost). This intelligent design (with appropriate control compounds) was able to probe the different effects of ligand, rigid rod, and proximity effects individually.
Various other multivalent constructs have been generated in the development of carbohydrate-based pharmaceuticals, often with a lead monovalent glycomimetic being incorporated into a polyvalent construct at a later stage of project development. Multivalent constructs have targeted fucose-binding pathogenic soluble receptors, in efforts to improve the outcome of patients with cystic fibrosis, or alternatively to generate simplified mimetics of sLex
that can mimic its native structure yet are easier to access synthetically [87