Crystallographic and thermodynamic characterization of phenylaminopyridine bisphosphonates binding to human farnesyl pyrophosphate synthase

Human farnesyl pyrophosphate synthase (hFPPS) catalyzes the production of the 15-carbon isoprenoid farnesyl pyrophosphate. The enzyme is a key regulator of the mevalonate pathway and a well-established drug target. Notably, it was elucidated as the molecular target of nitrogen-containing bisphosphonates, a class of drugs that have been widely successful against bone resorption disorders. More recently, research has focused on the anticancer effects of these inhibitors. In order to achieve increased non-skeletal tissue exposure, we created phenylaminopyridine bisphosphonates (PNP-BPs) that have bulky hydrophobic side chains through a structure-based approach. Some of these compounds have proven to be more potent than the current clinical drugs in a number of antiproliferation assays using multiple myeloma cell lines. In the present work, we characterized the binding of our most potent PNP-BPs to the target enzyme, hFPPS. Co-crystal structures demonstrate that the molecular interactions designed to elicit tighter binding are indeed established. We carried out thermodynamic studies as well; the newly introduced protein-ligand interactions are clearly reflected in the enthalpy of binding measured, which is more favorable for the new PNP-BPs than for the lead compound. These studies also indicate that the affinity of the PNP-BPs to hFPPS is comparable to that of the current drug risedronate. Risedronate forms additional polar interactions via its hydroxyl functional group and thus exhibits more favorable binding enthalpy; however, the entropy of binding is more favorable for the PNP-BPs, owing to the greater desolvation effects resulting from their large hydrophobic side chains. These results therefore confirm the overall validity of our drug design strategy. With a distinctly different molecular scaffold, the PNP-BPs described in this report represent an interesting new group of future drug candidates. Further investigation should follow to characterize the tissue distribution profile and assess the potential clinical benefits of these compounds.

The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density.The red, orange, yellow and green segments on the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria.The upper red bar (where present) indicates the fraction of residues that have poor fit to the electron density.

Mol Chain Length
Quality of chain 1 F 375 The following table lists non-polymeric compounds that are outliers for geometric or electrondensity-fit criteria: Mol Type Chain Res Geometry Electron density 4 GOL F 405 -X 2 Entry composition i O There are 5 unique types of molecules in this entry.The entry contains 3091 atoms, of which 0 are hydrogen and 0 are deuterium.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
Molecule 1 is a protein called Farnesyl pyrophosphate synthase.

O
In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues.

O
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues.

O
There are no RNA chains in this entry.

O
There are no non-standard protein/DNA/RNA residues in this entry.

O
There are no carbohydrates in this entry.

O
Of 5 ligands modelled in this entry, 3 are monoatomic -leaving 2 for Mogul analysis.
In the following table, the Counts columns list the number of bonds (or angles) for which Mogul statistics could be retrieved, the number of bonds (or angles) that are observed in the model and the number of bonds (or angles) that are defined in the chemical component dictionary.The Link column lists molecule types, if any, to which the group is linked.The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value.A bond length (or angle) with |Z| > 2 is considered an outlier worth inspection.RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).There are no chirality outliers.

Mol Type Chain
There are no torsion outliers.
There are no ring outliers.

O
There are no such residues in this entry.

Polymer linkage issues
There are no chain breaks in this entry.
6 Fit of model and data i O 6.1 Protein, DNA and RNA chains i

O
In the following table, the column labelled '#RSRZ> 2' contains the number (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution.The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue.The column labelled 'Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9.

O
There are no non-standard protein/DNA/RNA residues in this entry.

O
There are no carbohydrates in this entry.

O
In the following table, the Atoms column lists the number of modelled atoms in the group and the number defined in the chemical component dictionary.LLDF column lists the quality of electron density of the group with respect to its neighbouring residues in protein, DNA or RNA chains.The B-factors column lists the minimum, median, 95 th percentile and maximum values of B factors of atoms in the group.The column labelled 'Q< 0.9' lists the number of atoms with occupancy less than 0.9.

O
There are no such residues in this entry.

O
These plots are drawn for all protein, RNA and DNA chains in the entry.The first graphic for a chain summarises the proportions of errors displayed in the second graphic.The second graphic shows the sequence view annotated by issues in geometry and electron density.Residues are colorcoded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more.A red dot above a residue indicates a poor fit to the electron density (RSRZ > 2).Stretches of 2 or more consecutive residues without any outlier are shown as a green connector.Residues present in the sample, but not in the model, are shown in grey.
Bond lengths and bond angles in the following residue types are not validated in this section: GOL, MG, JD1The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value.A bond length (or angle) with |Z| > 5 is considered an outlier worth inspection.RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).In the following table, the Non-H and H(model) columns list the number of non-hydrogen atoms and hydrogen atoms in the chain respectively.The H(added) column lists the number of hydrogens added by MolProbity.The Clashes column lists the number of clashes within the asymmetric unit, and the number in parentheses is this value normalized per 1000 atoms of the molecule in the chain.The Symm-Clashes column gives symmetry related clashes, in the same way as for the Clashes column.Clashscore is defined as the number of clashes calculated for the entry per 1000 atoms (including hydrogens) of the entry.The overall clashscore for this entry is 2. All (5) close contacts within the same asymmetric unit are listed below.
Xtriage's analysis on translational NCS is as follows: The largest off-origin peak in the Patterson function is 3.98% of the height of the origin peak.No significant pseudotranslation is detected.1Intensities estimated from amplitudes.
In the following table, the Chirals column lists the number of chiral outliers, the number of chiral centers analysed, the number of these observed in the model and the number defined in the chemical component dictionary.Similar counts are reported in the Torsion and Rings columns.'-' means no outliers of that kind were identified.