The biological machinery- protein is intrinsically fine-tuned by molecular recognition driven from physical forces. To date, it is well-known that such biological macromolecules participate in signaling pathway synchronizing fundamental physiologic function of all living organisms on earth, capability of predicting such behavior of specifically functionalized entities irreplaceable for cooperative biological function network has become one of the most integral part in developing strategies for handling diseases with identified molecular targets.
In this study, a series of 2-aminopyrimidine-based compounds is designed and synthesized for exploring and probing available space in binding pocket of MTH1 protein.
Firstly, binding affinity profile of the compound series is determined in enzyme inhibition assay which unraveled tremendous deviation by a factor of 106 in biochemical potency of 2-aminopyrimidine-based compounds. Propagated by this experimental observation, protein-ligand complex structures determined to atomic resolution is further obtained via x-ray crystallography. Consequently, counterintuitive results were found in crystal structure of MTH1 in complex of an array of 2-aminopyrimidine-based compounds. Thus, it was confirmed that similar binding mode is adopted by protein-ligand complex structures determined in this study.
From above-mentioned experimental results, it appears to be difficult to rationalize the deviated binding affinity profiles determined for 2-aminopyrimdine-based compound series. To gain more insights into how two binding partners may interact, isothermal titration calorimetry was performed to characterize thermodynamic profiles of scaffold 2-derived compounds. Intriguingly, profound EEC (Enthalpy-Entropy compensation) phenomenon was notified which may be interpreted in terms of previously proposed
physical theory. Nevertheless, it could be somehow just an experimental artifact as described in research articles with focus on the issue of EEC observed in isothermal titration calorimetry.
Drawing a conclusion on current experimental results would be compromising partly attributed to the complexity of protein-ligand interaction and surrounding solvent environment. Nevertheless, in experimental analysis performed on protein-ligand complex structure and related binding profiles several notable points were found which is formulated as followed, 1. The impact of replacement of substituent in R2-position is far more significant than R1-position of scaffold, 2. Compound series derived from scaffold 1 is less active in enzyme inhibition assay against MTH1 which is attributed to incorporation of ethyl group at C5 of the pyrimidine moiety, 3. Overlap of R2-substituent with α-phosphate of substrate in the catalytic site for hydrolysis of triphosphate group appears to be related to loss of affinity in compound 12, 22, 29 and 30.
In order to complement the initial objective of optimizing biochemical potency of hit compound from HTS, substituents were utilized to probe the adaptability of MTH1 toward an array of structurally-related compounds. For optimal filling of minor pocket adjacent to R1-substituent, extensive hydrophobic interaction with phenyl cluster in the pocket is established with incorporation of N-cyclopropyl group. While for region occupied by R2-substituent, divergence in binding interaction is accompanied by increase in the size of substituent and this trend is also observed in thermodynamic study.
Opportunities for further optimization campaign is provided based on the proposition that integral element in binding pocket is thoroughly characterized in terms of molecular dynamic properties which involves solvation and desolvation during complex formation. To this point, more compiled research works are left to be conceptualized and accomplished.
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Tables and Figures
Table 1. Structure and related biochemical data of scaffold 1-derived compoundsa
Scaffold 1 ID R1= R2= Structure IC50 (nM)1 Ki (nM)2
1 57300 36200
3 261000 165200
6 71100 45000
13 263900 167000
14 >106 675300
16 4800 3000
a1Biochemical potency of scaffold 1 derivatives was determined in enzyme inhibition assay using PPiLight inorganic pyrophosphate assay kit from Lonza which detects inorganic phosphate released from hydrolysis of substrate 8-oxo dGTP catalyzed by MTH1.
2Inhibitiory constant Ki was calculated from IC50 determined in enzyme inhibition assay using formula IC50=(1 + [𝑆]/𝐾𝑚 ) × 𝐾𝑖.
Table 2. Structure and related biochemical data of scaffold 2-derived compoundsa
Scaffold 2 ID R1= R2= Structure IC50 (nM)1 Ki (nM)2
12 310 190
20 10 6.3
22 1500 949.3
24 6.3 4.0
25 41 25.9
26 22.6 14.3
a1Biochemical potency of scaffold 2 derivatives was determined in enzyme inhibition assay using PPiLight inorganic pyrophosphate assay kit from Lonza which detects inorganic phosphate released from hydrolysis of substrate 8-oxo dGTP catalyzed by MTH1.
2Inhibitiory constant Ki was calculated from IC50 determined in enzyme inhibition assay using formula IC50=(1 + [𝑆]/𝐾𝑚 ) × 𝐾𝑖.
Table 2. Structure and related biochemical data of scaffold 2-derived compoundsa (continued)
Scaffold 2 ID R1= R2= Structure IC50 (nM)1 Ki (μM)2
29 2030 1284.8
30 140 88.6
a1Biochemical potency of scaffold 2 derivatives was determined in enzyme inhibition assay using PPiLight inorganic pyrophosphate assay kit from Lonza which detects inorganic phosphate released from hydrolysis of substrate 8-oxo dGTP catalyzed by MTH1.
2Inhibitiory constant Ki was calculated from IC50 determined in enzyme inhibition assay using formula IC50=(1 + [𝑆]/𝐾𝑚 ) × 𝐾𝑖.
Table 3. Thermodynamic profiles of scaffold 2-derived compounds determined with iTC
ID N
(sites)
Kd
[× 109 M-1]*
∆G [kcal/mol]
∆H [kcal/mol]
-T∆S [kcal/mol]
6077639 1.69 199 ± 12 -9.16 ± 0.16 -7.34 ± 0.08 -1.8 ± 0.08
20 0.89 6.2 ± 4.8 -11.3 ± 0.69 -20.66 ± 1.6 9.3 ± 2.2
22 0.78 64.9 ± 16.2 -9.76 ± 0.25 -15.43 ± 0.05 5.7 ± 0.3
24 0.92 2.9 ± 0.1 -11.69 ± 0.07 -18.7 ± 0.28 4.7 ± 0.2
25 1.01 9.6 ± 5.3 -11.05 ± 0.41 -14.83 ± 0.86 3.8 ± 0.5
26 1.01 3.1 ± 0.5 -11.59 ± 0.21 -18.3 ± 0.42 6.7 ± 0.6
29 1.25 129 ± 53 -9.75 ± 0.81 -7.95 ± 2.47 -1.8 ± 1.7
30 1.12 16.1 ± 3.9 -10.66 ± 0.15 -11.7 ± 0.36 1.0 ± 0.2
*Dissociation constant Kd of test compounds was calculated from equation Kd= 1/Ka where Ka is the association rate constant given in M-1s-1.
Table 4. Data collection and refinement statistics for MTH1 crystal structure complexed with 11 structurally-related 2-aminopyrimidine-based compounds
Complex structure MTH1-6 MTH1-12 MTH1-14
Beamline
Reflections (total/unique) 218220/14689 474823/19253 359134/25855 Refinement
Rwork/Rfree 19.2/26.0 19.0/25.3 19.3/24.7
Reflections (factor/test) 14317/1347 18634/1738 25104/2316
No. of protein atoms 2476 2476 2476
B-factor (macromolecules ) 27.8 24.1 27.3
B-factor (ligands) 29.8 23.1 48.5
* values for highest-resolution shell are shown in parentheses.
Table 4. Data collection and refinement statistics for MTH1 crystal structure complexed with 11 structurally-related 2-aminopyrimidine-based compounds (continued)
Complex structure MTH1-16 MTH1-20 MTH1-22
Beamline
Reflections (total/unique) 111364/19234 219912/19298 363668/25653 Refinement
Rwork/Rfree 19.8/26.3 19.0/24.6 19.0/24.8
Reflections (factor/test) 18526/1726 18970/1842 25018/2329
No. of protein atoms 2476 2475 2476
B-factor (macromolecules ) 32.2 27.0 29.3
B-factor (ligands) 41.0 17.2 23.6
* values for highest-resolution shell are shown in parentheses.
Table 4. Data collection and refinement statistics for MTH1 crystal structure complexed with 11 structurally-related 2-aminopyrimidine-based compounds (continued)
Complex structure MTH1-24 MTH1-25 MTH1-26
Beamline
Reflections (total/unique) 403919/16640 463061/16988 453854/14867 Refinement
Rwork/Rfree 19.3/25.6 19.5/26.6 19.8/26.6
Reflections (factor/test) 16173/1483 16090/1517 13688/1147
No. of protein atoms 2476 2476 2476
B-factor (macromolecules ) 30.6 39.1 43.6
B-factor (ligands) 20.0 30.6 34.5
* values for highest-resolution shell are shown in parentheses.
Table 4. Data collection and refinement statistics for MTH1 crystal structure complexed with 11 structurally-related 2-aminopyrimidine-based compounds (continued)
Complex structure MTH1-29 MTH1-30
Beamline
Reflections (total/unique) 332824/19399 342060/30306
Refinement
Rwork/Rfree 19.9/25.2 18.8/23.1
Reflections (factor/test) 19046/1802 29190/2580
No. of protein atoms 2476 2476
B-factor (macromolecules ) 37.0 21.1
B-factor (ligands) 35.9 30.0
* values for highest-resolution shell are shown in parentheses
Figure 1. Schematic depiction of simplified work flow and objectives of this study (A) Brief overview of experimental work flow and objectives in this study (the concept
of the study is depicted illustratively which resembles designed ligand which plays as a key (referred to lock and key model) and interacts with a given target molecule.
In this study, to investigate interaction pattern of 14 small molecule compounds (at the stage of hit-to-lead optimization) in the binding pocket of target molecule, biochemical and biophysical assays such as enzyme activity analysis and isothermal titration calorimetry (iTC) were performed. For visualization of ligand molecule in binding pocket, crystals of target molecule in complex with ligand were obtained via soaking and complex structures were subsequently determined with x-ray diffraction.
(B) Simplified work flow of drug discovery campaign which is comprised of three integral stages. Starting from high-throughput screening performed on compound libraries, hits with moderate affinity toward a given target are identified. Through optimization of hit compounds by means of biochemical and biophysical techniques, leads with elevated binding affinity and improved selectivity profiles are generated and serve as drug candidates for further evaluation of in vitro and in vivo efficacy.
(C) Off-target effects which is related to lack of specificity and selectivity of a given ligand have to be taken into consideration which is influential in establishment of efficacy of designed drug candidates.
Figure 2. Purification of MTH1 protein from E.coli cell lysate
(A) purification of his-tagged MTH1 with affinity chromatography from cell pellet of E.coli incubated under three independent experimental conditions during large-scale expression of target protein.
(B) the quality of purified his-MTH1 protein was subsequently checked on SDS-PAGE gel which suggests protein degradation under condition of incubation time 14 and 16 hour at 22℃. Whilst his-MTH1 protein purified from cell lysate of E.coli incubated at 37℃
for two hours remains intact after purification using affinity column.
(C) Size-exclusion chromatography is applied for separation of his-tag and MTH1 protein.
MTH1 protein (18 kDa) was eluted out of column at elution volume 60-80 ml.
(D) SDS-PAGE analysis for confirming separation of MTH1 protein from cleaved his-tag.
(E) Schematic illustration of work flow of recombinant protein purification
(F) Purified protein sample serves as experimental material in biochemical, biophysical assays, and protein crystallization.
Figure 3. Binding affinity profile of scaffold 1 derivative compounds (A) Structural architecture of scaffold 1 with R1 and R2-substituent marked
(B) Structural architecture of six structurally-related compounds derived from scaffold 1 with ID indicated below
(C-E) dose-response curve of scaffold 1 derivative compounds measured in enzyme inhibition assay, IC50 value is calculated from fitting curve and Ki inhibition constant is then empirically converted from IC50 value. It is remarkable that comparison of Ki of scaffold 1 derivatives according to substituents incorporated in the R1 and R2-position demonstrates significant fluctuation in biochemical potency upon replacement of substituents in R2-position. In opposite, the effect of R1-substituent is less prominent.
Figure 4. Binding affinity profile of scaffold 2 derivative compounds (A) Structural architecture of scaffold 2 with R1 and R2-substituent marked
(B) Structural architecture of eight structurally-related compounds derived from scaffold 2 with ID indicated below
(C-F) dose-response curve of scaffold 2 derivative compounds measured in enzyme inhibition assay, IC50 value is calculated from fitting curve and Ki inhibition constant is then empirically converted from IC50 value. It is remarkable that comparison of Ki of scaffold 2 derivatives according to substituents incorporated in the R1 and R2-position demonstrates significant fluctuation in biochemical potency upon replacement of substituents in R2-position. In opposite, the effect of R1-substituent is less prominent.
Figure 5. Thermodynamic study of scaffold 2 derivatives binding to MTH1 protein (A) Schematic representation of two analytical methods for characterization of binding affinity profiles of 2-amino pyrimidine-based compound series in this study. In enzyme inhibition assay, ligand and substrate compete for the same binding site on MTH1 protein.
Oppositely, binding affinity between protein and ligand is measured in thermodynamic study using isothermal titration calorimetry (iTC).
(B-E) Experimental data of scaffold 2 derivative compounds with pKD value lower than 8.0 (classified as group i shown in diagram (J)) recorded using isothermal titration calorimetry.
(F-I) Experimental data of scaffold 2 derivative compounds with pKD value higher than 8.0 (classified as group ii shown in diagram (J)) recorded using isothermal titration calorimetry.
(J) Plotting pKi value determined in enzyme inhibition assay against thermodynamically measured pKD gives a regression coefficient value of 0.92 (K) binding isotherms calculated with one-site fitting model using raw data measured in iTC experiment from (B-E), significant deviation in the magnitude of exothermic signal recorded is notified (circled and indicated with arrows). (L) binding isotherms calculated with one-site fitting model using raw data from (F-I), relatively averaged magnitude of exothermic signal is characteristic for compounds from group ii (circled).
Figure 6. Phenomenon of EEC observed in scaffold 2 derivatives binding to MTH1 protein
(A) Diagram of thermodynamic profiles determined with isothermal titration
calorimetry for 2-aminopyrimidine-based compounds derived from scaffold 2. Profound enthalpy-entropy compensation (EEC) is observed in the structurally related compound series. In the diagram, thermodynamic parameters ∆G, ∆H and -T∆S are depicted as bar in color white, blue and orange, respectively. Substituents incorporated in R1 and R2-position of each scaffold 2 derivative compounds are displayed at top of the bar diagram.
(B) Plot of correlation between two thermodynamic parameters ∆H and -T∆S. (r2= 0.94) (C) Schematic draw of working mechanism underlying protein-ligand complex
formation which is analyzed using isothermal titration calorimeter. On the right side of arrow, mutually counteracting thermodynamic parameters ∆H and -T∆S which
contribute to binding free energy (∆G) are illustrated schematically.
(D-F) Schematic representation of protein-ligand complex formation process illustrated with thermodynamic parameters notified. Firstly, as the binding partners are both solvated before the formation of complex, (D) driving force (∆G, binding free energy) of complex formation propagate ligand molecule to enter the binding pocket.
The binding of two independent binding partners generally involves (E) formation of h-bonds and van der Waals contacts which are detected in the form of heat signal from iTC experiment. (F) While almost impossible to be observed in present experimental techniques, the conformational change of protein and ligand molecules during formation of complex or release of solvent molecules into bulk solvent and hydrophobic
interaction which does not involve formation and breakage of chemical bonds would be attributed to entropic component (-T∆S) of binding free energy (∆G).
Figure 7. Comparison of thermodynamic profiles from structurally related compounds with different substituents incorporated in the R2-position
(A,B) eight structurally-related compounds are grouped according to substituents incorporated in R1-position as shown in bar diagram (A) and (B). Subsequently, thermodynamic profiles of the 2-aminopyrimidine-based compound series are plotted in bar diagram which stand for calculated thermodynamic parameters ∆G, ∆H and -T∆S.
Four variants of R2-substituents corresponding to each compound are displayed above the bar diagram (A) and (B).
(C), (D), profound enthalpy-entropy compensation (EEC) is demonstrated in (C) which represents result from correlation analysis of thermodynamic parameters ∆H and -T∆S derived from structurally-related compound series illustrated in (A) with a R2 value of 0.993. While with N-cyclopropyl incorporated in R1-position, compound series with an array of identical R2-substituents from (B) result in a less prominent value of R2 which dropped to 0.858 in the correlation analysis of thermodynamic parameters ∆H and -T∆S as indicated in diagram (D).
(E), (F), Representative compounds with an array of substituents explored in the R2-position are superimposed in (E) and (F), separately. In (E), compound 20 and 30 from (A) are shown in stick representation colored in magenta and yellow, respectively. While with the same R2-substituents as compounds referred above, compound 25 and 29 from (B) with different R1-substituent incorporated are shown in stick representation colored in cyan and violet, respectively.
Side chains of adjacent residues in S2 pocket are shown in line representation colored in purple.
Figure 8. Comparison of thermodynamic profiles from structurally related compounds with different substituents incorporated in the R1-position
(A-D), thermodynamic parameters are plotted as bars colored in white, blue and orange for ∆G, ∆H and -T∆S, respectively. In each bar diagram, thermodynamic profiles of structurally related compounds which differentiate in R1-substituent are categorized into
(A-D), thermodynamic parameters are plotted as bars colored in white, blue and orange for ∆G, ∆H and -T∆S, respectively. In each bar diagram, thermodynamic profiles of structurally related compounds which differentiate in R1-substituent are categorized into