The development of a novel chemical tool was accomplished against NanB. This “relaxed” chemical tool works by negative allosteric modulation. Orthosteric sites are conserved within the sialidase family and allosteric site modulation provides an alternative route to the development of specific modulators. Optactamide was determined to be specific to NanB when tested against NanC, a sialidase with the highest sequence homology to NanB at 46%. However, Optactamide did inhibit NanA with approximately 50% inhibition at 100µM despite a lower sequence homology of 24%. The allosteric site of NanA contains a very similar binding site to NanB. Further optimisation of
Optactamide against NanB should focus on achieving specificity as well as potency. Firstly, an Optactamide-NanA crystal complex should be solved or alternatively, in the absence of an Optactamide-NanA crystal structure, a CADD approach could be used to determine the binding mode of Optactamide within NanA. This will provide valuable information for SBDD assumptions in achieving NanB selectivity in future Optactamide analogues as well as providing an opportunity for the development of a specific NanA inhibitor. Overlay of the structure of NanA (PDB: 2YA4) with Optactamide-NanB crystal structure (PDB: 4XYX) suggests that the binding site maybe similar with only minor positional changes of the key protein side groups involved in van der Waal interactions with Optactamide (structural RMSD: 1.8 Å). In NanA the backbone carbonyl of threonine 251 is replaced with a backbone carbonyl of a lysine (residue 338). Glutamine 494 in NanB (a residue that contributes to direct hydrogen bonding with one of the diols of Optactamide) is replaced with a glycine (residue 556) in NanA. However, glutamine 741 within NanA is positioned 3.7 Å away from the position of glutamine 494 within NanB. This results in a slight steric clash with an OH from the diol of Optactamide (Figure 56). It is unlikely this would render Optactamide inactive, but would change the binding position of Optactamide slightly. Glutamic acid 658 in NanB is thought to contribute to electrostatic interactions with the arene of Optactamide. This glutamic acid is also in a slightly different position in NanA (2.1 Å further away, residue 742). It is possible the flexibility within Optactamide might still accommodate the positional change of the glutamic acid, but both positional changes of the glutamic acid and glutamine would result in sub-optimal binding of Optactamide within NanA (Figure 56). This could explain the reduced potency of Optactamide observed against NanA in the 4-Munana assay (see Chapter 3.5, Activity assays and synergistic inhibition).
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Figure 56. Superimposed structure of NanA (PDB: 2YA4) against Optactamide-NanB (PDB: 4XYX) crystal structure (RMSD: 1.8 Å). Electrostatic surface representation shown in each image. A. Optactamide (green) binding position within NanB (blue) overlaid in the same position within NanA (pink). B. Close up image of the cyclopentane diol binding position of Optactamide. Changes in the position of the glutamine results in a slight clash with Q741 (NanB residue at this position is Q494). C. Close up image of the arene binding position of Optactamide. Changes in the position of glutamic acid (NanA: E742 and NanB: E658) results in slightly suboptimal binding for an electrostatic interaction with the arene.
Adaption of Optactamide to a NanA chemical tool could be possible. Improvements in potency and selectivity would include the change of the amide to an amine within Optactamide as this would likely generate a favourable electrostatic interaction with glutamine 742. Addition of a CH2 to move
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interaction. Further improvements in potency against NanA could be generated by: 1) modification of the benzyl ring using the Topliss series or 2) complete replacement of the arene with another functionality guided by CADD. The analogues sythesised would need to be screened in parallel against NanB using the 4-Munana assay to exclude functionality that cause reduced selectivity.
Comparison of Optactamide’s binding position within NanB (PDB: 4XYX) with a structural overlay of NanC (PDB: 4YZ1) confirms that binding within NanC would be unfavourable (structural RMSD is 1.7 Å). Firstly, glutamine 494 within NanB (a residue that contributes to direct hydrogen bonding with one of the diols of Optactamide) is not present in NanC. Instead arginine 538 within NanC occupies this position resulting in a change in the electrostatic and accessible surface of the binding site. The electrostatic surface of this arginine directly clashes with an OH from the diol of
Optactamide rendering Optactamide unlikely to bind at this position (Figure 57). Mutation of arginine 538 into a glutamine to develop a NanCR538Q mutant might result in Optactamide efficacy.
The generation of bacterial resistance to antibiotics is a major health concern (see Chapter 1.42, Antibiotics). The generation of a NanBQ494R would likely result in reduced Optactamide activity and
could be a mechanism of bacterial resistance against Optactamide. The antibiotic clock (see Chapter 1.42, Antibiotics) dictates how useful an antibiotic will be. If a NAM of NanB or NanA was to be developed as an antibiotic the potential routes of resistance would need to be investigated and evaluated. Other residues that likely contribute to a reduced binding ability include: serine 642, glutamine 700 and histidine 704. These residues are positioned further into the pocket resulting in a steric clash with the arene of Optactamide.
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Figure 57. Superimposed structure of NanC (PDB: 4YZ1) against Optactamide-NanB (PDB: 4XYX) crystal structure (RMSD: 1.7 Å). Electrostatic surface representation shown in each image. A. Optactamide (green) binding position within NanB (blue) overlaid in the same position within NanC (gold). B. Close up image of the cyclopentane diol binding position of Optactamide. A direct clash with R538 is observed (NanB residue at this position is Q494). C. Close up image of the arene binding position of Optactamide. Changes in the position of the residues that exist in the arene binding pocket (serine, histidine and glutamic acid) are observed.
The results obtained from the S. pneumoniae infection/invasion assay suggest that NanB has only a minor role in the invasion of S. pneumoniae into lung epithelial cells. At high concentrations of Optactamide that would cause full inhibition of NanA, lung epithelial cell protection is observed. Using a specific chemical tool that is selective for NanA would confirm these results. Despite protection being observed in an in vitro set up, these results may not correlate with in vivo
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experiments. Optactamide would not be useful for in vivo experimentation as its solubility is low and its toxicity is high (as observed in a MTT toxicity assay) (see Chapter 3.7, Cell and bacterial assay). The limitations of a “relaxed” chemical tool design are evident in this situation. Further optimization of Optactamide will need to follow the “constrained” chemical tool design with the future aim of in vivo experimentation. Furthermore, full protection of the lung epithelial cell line against S.
pneumoniae was not observed with NanA and NanB inhibition suggesting that NanC may also need to be inhibited. Alternatively, these results could indicate that these sialidases are multifaceted and catalytic activity is not the only important function these proteins have in S. pneumoniae virulence. The carbohydrate binding domains of these proteins may also have an important role in S.
pneumoniae virulence. The carbohydrate binding domain in particular, is present in NanA, NanB and NanC and binds to terminal sialic acid of glyconjugates (Owen et al., 2015, Yang et al., 2015, Xu et al., 2008). Development of chemical tools that inhibit sialic acid binding within the carbohydrate
binding domains could prove challenging, but inhibition of both the catalytic activity and carbohydrate recognition/binding from the CBM domains could result in full protection from S. pneumoniae invasion and adhesion. Alternatively, use of an engineered multi-valent CBM with improved affinity as a competitive inhibitor of S. pneumoniae sialidase CBM could provide a novel approach to limiting S. pneumoniae invasion and adhesion. Similarly the influenza virus binds to sialic acid present on host cell surfaces (Connaris et al., 2014). A multi-valent CBM has been used in
in vivo studies and shown to provide mice with complete protection from a lethal challenge of a 2009 pandemic H1N1 influenza virus (Connaris et al., 2014).
Other GH-33 sialidases that allosteric sites could be located in include TcTS (PDB: 1MR5), NanI (PDB: 2VKs), NedA (PDB: 1EUR), VCS (PDB: 1KIT), Neu2, TrSA, STNA, PaNa, Btsa, BDI-2946,
Baccac_01090 and RgNanH (PDB: 4X47) (see Chapter 1.59, Conservation). Further glycosidic hydrolases that allosteric sites could exist in include the GH-1 sialidases as the water channel is conserved with seven internal water molecules conserved across 90% of the known published structures (see Chapter 1.59, Conservation). Direct disruption of one of these conserved waters could result in reduced enzymatic activity. It is also likely allosteric sites exist within water channels of other protein types including: serine proteases, kinases, cytochrome P450 and ATP-synthase (Teze et al., 2013, Knight et al., 2009, Meyer, 1992, Gohlke et al., 2012, Oprea et al., 1997).
It is possible that the PDB provides an as of yet unmined wealth of information of novel
unexplored small molecule binding sites in other protein families. Hardy and Wells were the first to identify that small molecules present within crystallization conditions could “serendipitously” bind to
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the protein within the crystal structure and identify pockets amenable to small molecule binding and modulation (see Chapter 1.35, Fragment screening and serendipitous binders).
Figure 58. Examples of CHES serendipitously bound within crystal structures of: A. the NK1 fragment of HGF/SF complexed with CHES (PDB: 5CT1) and B. phosphoribosylglycinamide formyltransferase (purN) from Coxiella burnetii (PDB: 3TQR). CHES is found bound within a pocket in A. CHES is found bound on the surface of B.
However, care will need to be taken with this approach as it is unlikely that all pockets
containing serendipitous small molecule binders in the PDB would be novel allosteric sites. It is likely small molecules bind at the surface and contribute to crystal-crystal contacts (Figure 58). The
presence of these small molecules in high millimolar concentrations and errors within crystal structures due to low resolution coordinate errors or density misinterpretation could lead to incorrect assumptions. Additionally, the active sites of enzymes are rich with polar residues and would be more amenable to small molecule binding. A large number of ligands are found bound within structures in the PDB. Evaluating each small molecule bound within each structure is no small task. Computational tools that can screen and identify serendipitous binders within pockets,
evaluate their binding and determine if this site is allosteric would be useful in reducing this workload.
The design of a computational tool that screens for allosteric pockets using a machine learning approach was developed by Chen et al, 2016. This computational tool uses structural information from previously determined orthosetric sites (159 protein-ligand complexes from PDBbind), allosteric sites (59 protein-ligand complexes from literature and the AlloSteric Database) and
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miscellaneous (99 protein-ligand complexes, where the ligand is neither defined as an orthosteic or allosteric binder and deemed to have no modulatory activity) to rank protein cavities into categories (allosteric, regular, orthosteric)(Chen et al., 2016). A validation test was carried out on CHES, in collaboration with our work. In the PDB 158 CHES-protein binding sites were identified. Only 14 of these sites were defined as buried in pockets and from this only one was identified as an allosteric site (NanB) (Chen et al., 2016).
To further improve the success of a computational screen a direct comparison of multiple structures of the same protein (to evaluate structural conformational shifts in the presence of small molecule binding) should be including in the evaluation. Additionally, the importance of the pocket (identified to serendipitously bind a small molecule) to the protein’s function should be examined and included in the evaluation. Water channels within proteins are thought to have important structural and functional roles (see Chapter 1.59, Conservation). Small molecules that bind to important structural features of proteins would be more likely to have a modulatory impact. Research groups depositing protein structures to the PDB should be aware of serendipitous small molecule binders and its potential for allosteric site discovery.
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