3-1 MTH1 protein purification
The purification of target protein from cell lysate is intended to obtain research material for subsequent experimental trials with high purity. The complete procedure is composed of three individual parts which involves affinity column purification of his-tagged target protein followed by removing purification tag with thrombin digestion at interval stage before the final purification procedure of size-exclusion chromatography is performed.
3-1-1 Affinity Chromatography
The purified target protein MTH1 with his-tag was eluted from his-tag affinity column during wash volume 550-700 ml (Figure 2A). The purity of his-tagged MTH1 protein was examined in the following SDS-PAGE analysis (Figure 2B). In regard of band shown on SDS-PAGE gel which is indicative of protein degradation, experimental condition including incubation time and temperature during large-scale protein expression was revised and modified.
As a result, three independent experimental trials for large-scale protein expression were performed under condition of incubation temperature 22℃ for 14﹑16 hours and 37℃ for 2 hour. From Figure 2B it is demonstrated that protein purified from lysate of E.
coli cells incubated at 37℃ retains the integrality of his-tagged MTH1 protein compared to 22℃ which indicates incubation time and temperature might be the influential factors in protein degradation for this case.
3-1-2 Size-exclusion chromatography (SEC)
After purified his-tagged MTH1 protein has been obtained, his-tag for affinity chromatography was removed with thrombin digestion. To separate cleaved his-tag and MTH1 protein, size-exclusion-chromatography was performed on Superdex75 Gel Filtration column (GE healthcare) equilibrated with gel filtration buffer (20 mM Tris pH 7.4, 150 mM NaCl, 2 mM TCEP, 5% Glycerol). As the separation of MTH1 protein from other undesired substances present in sample solution depends on size of molecules, the standardized elution volume corresponding to representative molecular weight is given in the form of referential parameter as shown in Figure 2C. After the final stage of purification, the separated MTH1 protein was examined on SDS-PAGE gel which shifts from the position of molecular weight 20 kDa to approximately 18 kDa compared to his-tagged MTH1 protein (Figure 2D).
Consequently, purified MTH1 protein with his-tag removed (Figure 2E) serves as experimental material in later crystallization trial for x-ray diffraction experiment (Figure 2F).
3-2 Determination of binding affinity profiles and structure-activity relationship (SAR) 3-1 Fragment-based drug screening and optimization performed with 2-aminopyrimidine-based scaffold
In searching for scaffold compound with moderate binding affinity toward MTH1, high-throughput screening (HTS) on 2313 structurally irrelevant fragments was performed by Ph.D. candidate Cheng Peng from our lab. Consequently, four compounds were identified to be micromolar binders in the enzyme inhibition assay. From these identified hits, compound 6077639 was selected as structural skeleton for further optimization campaign in regard of its inhibitory activity on prostate cancer cell lines.
Provided that x-ray structure of MTH1 bound with compound 6077639 has been
solved by Ph.D. candidate Cheng Peng, notable interaction pattern which may contribute to favorable binding interaction are characterized. Attributed to observation from x-ray complex structure, systematical exploration of binding pocket using 2-aminopyrimidine scaffold was further performed to elucidate the correlation between interaction pattern of individual functionality and its contribution to binding affinity profile of inhibitor candidates.
For simplicity of description, the synthesized compound series in this study is categorized into two separate groups according to the presence or absence of ethyl group at C5 of the pyrimidine moiety. The parent compounds of these 2-aminopyrimidine derivative compound series are referred to as scaffold 1 and 2 in the following text.
3-2-2 Binding affinity profile and SAR study of scaffold 1-derived compounds
For compounds optimized using scaffold 1 which has ethyl group incorporated at C5 of the pyrimidine ring (Figure 3A), dramatic loss in binding affinity was observed compared to hit compound 6077639 (Figure 3A).
In the six compounds derived from scaffold 1 with biochemical potency distributes unevenly in sub-millimolar IC50 range (Fig 3B-E), variation in binding affinity profile is noteworthy which approximately differentiates by a factor of 200 for the weakest and the most potent binder in this compound series.
In the 2-aminopyrimidine-based compound series optimized using scaffold 1, exploration of structure-activity relationships (SARs) was performed on the substituents in the R1- and R2-position. To complement this objective, compound 1, 3, 6, 13, 14 and 16 was designed and synthesized by our colleague Zhe-Hwa Cheng from graduate institute of pharmacy. In biochemical assay, compound 14 with 1-methylpiperazinyl incorporated in the R2-position exhibits millimolar activity toward MTH1 and is the
weakest binder in this structurally-related compound series. Whilst micromolar potency is determined for compound 16 substituted with piperidinyl group in R2-position. In summary, the observation described above suggests that influence of substituent in the R2-position is profound (Fig 3B-E). Nevertheless, in the case of substituent replaced in the R1-position which are N-methyl and N-cyclopropyl in this compound series, the impact on binding affinity profile is rather insignificant as observed in compounds pairs 1 and 13; 3 and 14; 6 and 16 which differentiate by a factor of 4 to 15 depending on substituent incorporated in the R2-position.
3-2-3 Binding affinity profile of scaffold 2-derived compounds
Followed by biochemical assay for characterization of binding affinity profiles of the 2-aminopyrimidine-based compound series derived from scaffold 1. It was notified that using scaffold 1 for optimization appears to hinder the enhancement of binding affinity toward MTH1 (Figure 3). In regard of this obstacle in the optimization campaign, ethyl group at C5 of the pyrimidine moiety was speculated to be the underlying cause for the dramatic loss in biochemical potency. To confirm the assumption that ethyl group at C5 of the pyrimidine moiety should be the underlying cause for weak activity of scaffold 1-derived compounds toward MTH1, A series of structurally-related compounds based on scaffold 2 (Figure 4A-B) with ethyl group at C5 of the pyrimidine moiety removed was designed and synthesized.
Expectedly, the removal of ethyl group indeed contributed to enhancement of the binding affinity profiles of 2-aminopyrimidine-based compound series derived from scaffold 2 (Figure 4C-F). The overall binding affinity profile of the compound series was determined in the nanomolar IC50 range. Nevertheless, despite of improved binding affinity toward MTH1, significant deviation in binding affinity profile within the
compound series is notified which differentiates by three orders of magnitude.
Among the substituents incorporated at R2, (oxetan-3-yl) piperazinyl and 1-methylpiperazinyl group apparently prohibits the elevation of binding affinity of compound 12, 22, 29 and 30 which are replaced with substituents referred above. While compounds incorporated with either piperidinyl or morpholine substituent were found to be nanomolar binders in enzyme inhibition assay (Figure 4D,F).
Consequently, the comparison of binding affinity profiles in regard of the influence of substituents at either R1 or R2 reveals that the modulation of binding affinity is more correlated to substituents incorporated in the R2-position rather than R1-position. Of note, this finding is in coherence with the observation made in the 2-aminopyrimidine derivative compound series optimized based on scaffold 1 discussed previously (Fig 3).
3-3 Thermodynamic study on 2-aminopyrimidine-based compounds derived from scaffold 2
For establishment of complementary affinity profiles of optimized compound series, binding affinity of ligand toward MTH in the absence of substrate was measured using isothermal titration calorimetry (Figure 5A). In consideration of the detection limit of isothermal titration calorimetry (ITC) which lies within the range of 10-2-10-9 M according to instruction of manufacturer, the determination of dissociation constant (Kd) including related thermodynamic parameters was performed on 2-aminopyrimidine-based compound series optimized using scaffold 2 (Figure 4A,B) with weak binders derived from scaffold 1 excluded.
3-3-1 Enthalpy-driven binding of 2-aminopyrimidine-based compounds to MTH1 To determine the thermodynamic profile of the present compound series modified
using scaffold 2 with profound improvement in biochemical potency, isothermal titration calorimetry (iTC) was applied to record signals of heat released from chemical reaction involving breakage and formation of chemical bonds which allows calculation of thermodynamic parameters ∆H and -T∆S from titration curve fitted using 1:1 binding model (Appendix 10) . Consequently, an array of thermodynamic profiles for the 2-aminopyrimidine-based compound series binding to MTH1 was established (Figure 5B-I). Meanwhile, it is notable that binding affinity profile measured using iTC is in consistency with previously determined biochemical potency in enzyme inhibition assay (Figure 5J). In spite of similar structural skeleton, deviated binding mechanism is revealed in compound 12, 22, 29 and 30 (Figure 5K). While for compound 20, 24, 25 and 26 which are the most potent binding partners for MTH1 in this study, deviation in binding mechanism is absent (Figure 5L).
The plot of thermodynamic signatures demonstrates that the binding interaction between target molecule MTH1 and 2-aminopyrimidine based compound series is dominated by favorable enthalpic component of Gibbs free energy (Figure 6A). From another aspect, despite of significant enthalpic signal measured directly in biophysical analysis using iTC, the enthalpic contribution is always counteracted by a minor entropic component which eventually leads to similar binding affinity profile observed across the investigated compound series.
3-3-2 Phenomenon of Enthalpy-Entropy Compensation (EEC)
Within the 2-aminopyrimidine-based compound series which bind to MTH1 in an enthalpy-driven fashion, profound enthalpy-entropy compensation (EEC) was unraveled (Figure 6A,B) when enthalpic component (∆H) was plotted against entropic component (-T∆S) with correlation coefficient value R2= 0,94 (Figure 6B). Whereas, the correlation
which is indicative of enthalpy-entropy compensation is questionable in regard of numerous published research articles debating over the issue of empirical error present in the calculated thermodynamic parameters ∆H and -T∆S. To decipher the observed phenomenon which is often masked by errors rooted in the equation ∆G=∆H-T∆S applied in calculation of the entropic component ∆S which could not be measured experimentally, it was suggested that thermodynamic data should be handled with carefulness in respect to susceptible error in the experimental workflow described above.
3-3-3 Rationalization of observed EEC phenomenon through insights provided by atomic model of MTH1 in complex with 2-aminopyrimidine-based compounds
Assuming that behind the phenomenon of EEC there is certain physical mechanism modulating such intrinsic interplay between binding partners and solvent molecules in surrounding environment as proposed in numerous research works, the counteracting effect between thermodynamic parameters enthalpy and entropy (Figure 6C) may somehow be investigated with insights from atomic structure of protein-ligand complex which uncovers interaction pattern of binding partners.
Thus, to clarify the complex formation process described using thermodynamic parameters ∆H, -T∆S and Gibbs free energy ∆G which is the summation of enthalpy (∆H) and entropy (-T∆S), schematic illustration is presented in Figure 6D-F. Elaborately, enthalpic component of binding free energy involves formation of H-bonds and van der Waals contacts while entropic component is involved in conformational change and hydrophobic interaction which can hardly be measured using present experimental techniques.
3-3-3-1 Thermodynamic profiles of structurally-related compounds with substituent
explored on R2-position
As revealed in bar diagram from Figure 6A which demonstrates typical phenomenon of EEC observed frequently in compound series stem from optimization campaign. The relation between structural feature of structurally-related compounds and the effect of EEC is further investigated as the present compound series is systematically explored on R1 and R2-position with substituents.
Firstly, thermodynamic profiles of scaffold 2-derived compounds binding to MTH1 was categorized according to R1-substituent, which is underrepresented by compound 12, 20, 26, 30 and compound 22, 24, 25, 29 depicted as bar diagram displayed in Figure 7A-B. Subsequently, the thermodynamic parameters ∆H and -T∆S was plotted in diagram with correlation coefficient R2 indicated (Figure 7B-C). From the results of linear regression analysis on thermodynamic parameters of scaffold 2-derived compounds categorized based on substituent incorporated in R1-position, the R2 value for compounds with N-methyl group incorporated in R1-position is 0.99 while the presence of additional cyclopropane ring lead to decreased correlation coefficient value of R2= 0.86. For rationalization of this paradox, visualization of atomic details of protein-ligand complex is performed with x-ray crystallography. In Figure 7D, structural alignment between compound 20 (colored magenta) and 30 (colored yellow) unraveled deviated positioning of R2-substituent in MTH1 binding pocket. Meanwhile, this tendency was also observed in structural alignment of compound 25 (colored blue) and 29 (colored violet) in Figure 7E.
3-3-3-2 Thermodynamic profiles of structurally-related compounds with substituent explored on R1-position and structural relevance of EEC phenomenon
Opposed to arrangement of the scaffold-2 derived compounds in Figure 7 according
to substituent incorporated in R1-position as described above. The compound series is categorized into four groups according to substituent incorporated in R2-position as illustrated in Figure 8. Rearrangement of thermodynamic profiles reveals profound EEC upon replacement of R1-substituent in scaffold-2 derived compounds incorporated with four variants of substituents in R2-position. As plausible explanation for this intrinsic phenomenon, structural alignment of compounds from Figure8 A-D provides insights into binding mode adapted by these compounds in MTH1 binding pocket which suggests more extensive hydrophobic interaction between R1-substituent and phenyl cluster made up of Phe27, Phe72, Phe74, Phe139 upon replacement of N-methyl group with N-cyclopropyl group (Figure 8E-H).
3-3-3-3 The influence of size of substituent in the aspect of Enthalpy-Entropy Compensation
For scaffold-2 derived compounds investigated in thermodynamic study using iTC.
Comprehensive overview of thermodynamic profiles of representative compounds in combination with structural insights is presented in Figure 9 which is compiled of three individual parts. In this compact figure, thermodynamic profiles illustrated in bar diagram for compound 24, 29, 12, 30 (Fig 9E-H) reveal deviated pattern of thermodynamic signatures within the structurally-related compound series. In attempt to uncover the correlation between structural features of compound and deviated thermodynamic profiles, insight into the structural architecture of MTH1 binding pocket (shown in surface representation) bound with compound 24, 29, 12, 30 (shown in stick representation) is presented in Figure 9 A-D. To formulate conclusion for the compensation effect based on information from structural details of protein-ligand complex and related thermodynamic profiles, it should be first notified that as the size of
R2-substituent increases, less enthalpically favorable and more entropy-driven is the binding interaction which is observed in compound 29 and 30 in comparison to compound 24 and 12 (Figure 9I-L). Therefore, as already demonstrated in Figure 8, incorporation of N-cyclopropyl group in R1-position leads to more entropically favorable binding interaction.
34 Structural analysis of MTH1 complex structure bound with 2aminopyrimidine -based compounds
Structural analysis of binding interaction was performed on eleven MTH1-inhibitor complex structures obtained via soaking preformed MTH1 crystals in ligand dissolved in DMSO and further diluted in reservoir solution (1:10). Similar binding pattern in above-mentioned protein-ligand structures is unraveled. For elucidation of underlying mechanism of the varying binding affinity profiles revealed in enzyme inhibition assay for determination of inhibitory activity of 2-aminopyrimidine-based compound series comprising fourteen structurally-related compounds, thorough inspection of interaction pattern in an array of structurally-related ligands binding to target molecule MTH1 protein provides plausible explanations for the structure-activity relationship (SAR) uncovered in biochemical assay (Figure 10).
3-4-1 Conserve binding mode of pyrimidine-based compounds in the binding site of MTH1
Upon inspection of protein-ligand complexes solved by x-ray crystallography, it was found that conserve binding mode is adopted by 2-aminopyrimidine-based compound series bound in the binding pocket of MTH1 (Figure 10B). To illustrate the details of general binding geometry and orientation of the 2-aminopyrimidine-based
compounds, common features of structural architecture of the 2-aminopyrimidine compound series are decomposed into (i) pyrimidine moiety and (ii) R1 and R2-substituent for ease of description.
3-4-2 Pyrimidine moiety
From previous results of high-throughput screening (HTS) performed on fragment library composed of 2313 compounds of divergent structural architecture purchased from CHEMBRIDGE, one bioactive hit compound was identified which is referred to as compound 6077639 in respect of ID code assigned by manufacturer. Followed by the discovery of this HTS hit compound, cocrystal structure of MTH1 bound with compound 6077639 determined to a resolution of 1.8 Å was resolved by Ph.D. candidate Cheng Peng which sheds light on the intrinsic pattern of protein-ligand interaction (Figure 10A).
At first glance, the binding mode is well-characterized which is mainly attributed to the pyrimidine moiety of ligand molecule which is held firmly in the bottom of binding pocket by the indole ring of Trp117 via π-π stacking interaction. H-bonds formed between recognition element Asp119-Asp120 binding motif and amino group of hit compound 6077639 also contribute to the stabilization forces in the protein-ligand complex (Figure 10A).
In the present optimized compound series, the pyrimidine moiety serves as anchor group which strengthens and rigidifies the orientation of ligand molecule which is related to restriction on bond angle of H-bonds. Simultaneously, systematical exploration with substituents on the pyrimidine moiety allows tracing of structural changes upon binding of the pyrimidine derivative compounds in the binding pocket of MTH1 in a rational fashion.
3-4-3 R1 and R2-substituent
Chosen for the subsequent optimization campaign from HTS, compound 6077639 serves as structural skeleton for the present 2-aminopyrimidine-based compound series (Figure 10A). For modification of hit compound 6077639, C4 and C6 of the pyrimidine moiety are assigned as R1 and R2-substituent in the following passages and explored with several functionalities. In the case of R1-substituent, which occupies the cavity formed by phenyl rings of Phe27, Phe72, Phe74 and Phe139, interacts with these side chains through hydrophobic contacts. On the other hand, from the pyrimidine moiety anchored by indole ring of Trp117, substituent at R2-position expends the skeleton of small molecule compound toward the entrance of binding pocket and forms either hydrophobic interaction or van der Waals contact with several rigid components of binding pocket made up of side chains of Tyr7, Thr8, Leu9, Lys23 and Asn33. (Figure 11C).
After thoroughly inspecting the structural details revealed in binding pocket of MTH1 bound with different ligand molecules obtained via x-ray crystallography. The high similarity of binding mode observed in protein-ligand complexes determined in this study further hampers the formulation of rationales for the largely deviated binding affinity profiles of the 2-aminopyrimidine-based compound series. Nevertheless, there remains some notable features to be explored and may provide plausible explanation for the varying inhibitory activity of the present 2-aminopyrimidine-based compounds against MTH1.
3-5 Superposition of MTH1 complexed with product 8-oxo dGMP and compound 29, 30 In the binding pocket of MTH1, compound 29 and 30 overlay well with MTH1 product 8-oxo dGMP which serves as natural ligand exploited for drug design (Figure 14A-B). To dissect the interaction pattern of above-mentioned ligands, contact between
ligand moieties and nearby residues was analyzed. From the structural analysis it was noted that oxetane ring incorporated at N4 of compound 30 lends itself toward loop-A sit in S2’ pocket (Figure 14C) and consequently lesser interaction with side chains of Tyr7, Thr8, Leu9, Met81 and Val83 is formed. In regard of this observation, recalls that in MTH1 x-ray structure complexed with product 8-oxo dGMP the α-phosphate is overlaid with oxetane ring from both compound 29 and 30 which otherwise adopted an alternative binding pattern in catalytic region with loop-A involved. With information regarding binding pattern of triphosphate group in MTH1 binding pocket which is bound in coordination with water molecule network intended for hydrolysis of substrate 8-oxo dGTP. Modification of functionality in this position should take this point into consideration. Further, based on this observation, exploration of region occupied by deoxyribose and α-phosphate with substituents requires more attention to be paid in respect to the hydrophobic side chains and several structural water molecules which are likely the key factors in modulation of optimal ligand binding.
3-5-1 Absence of affinity gain with extended substituent toward the entry of binding pocket
The initiative of growing structural skeleton selected from high-throughput screening (HTS) toward entry of binding pocket is complemented by design and synthesis of compound 29 and 30. However, enhancement in binding affinity is not observed in
The initiative of growing structural skeleton selected from high-throughput screening (HTS) toward entry of binding pocket is complemented by design and synthesis of compound 29 and 30. However, enhancement in binding affinity is not observed in