A Combination of Thermodynamic and Structural Information Guides Optimization of Substituted

Diaminopyrimidine Renin Inhibitors

Synopsis of a presentation from the 2007 Trends in Microcalorimetry Conference presented by:

Ronald W. Sarver Pfizer Global Research & Development

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Renin is an aspartyl protease involved in the production of angiotensin II, a potent vasoconstrictor. Renin in-hibitors can prevent blood vessel constriction and could therefore be useful for the treatment of hypertension. High throughput screening efforts identified a small molecule renin inhibitor.

A combination of biological screening assay data, X-ray structure determinations, thermodynamic binding data and medicinal chemistry was used to advance the original HTS hit to a highly potent and selective inhibitor.

In his presentation, Dr. Sarver outlined the advantages of measuring binding thermodynamics including:

• Aid in template selection (enthalpic vs entropic) • Differentiation of agonists and antagonists • Comparison of inhibitor binding with designed structural changes to

∗ Estimate binding strength of specific binding interactions ∗ Provide a quick guide to whether an H-bond was formed with the protein ∗ Identify thermodynamic hot-spots in the active site (areas that provide good binding enthalpy or entropy) ∗ Provide detailed information on contributions of specific interactions to binding strength when co-structures are available

• Aid in design of optimal interactions ITC Data Guides the Lead Evaluation and Optimization of Renin Inhibitors

Thermodynamic data for inhibitor binding to renin were collected using isothermal titration calorimetry (ITC). These data were used to help guide inhibitor optimization by suggesting molecular alterations to improve bind-ing affinity from both a thermodynamic and structural perspective.

Traditional lead optimization can lead to drug binding that is entropically driven. For example:

• Designing ligands with restricted conformations pre- fit to the active site pocket and adding hydrophobicity to make interactions with solvent less favorable • Entropically driven binding can be detrimental since protein mutations can result in large decreases in activity - small changes in the active site are not well accommodated by a rigid inhibitor. Enthalpically driven binding is advantageous for several reasons including:

• Lead compounds that exhibit favorable binding enthalpy provide several advantages.

∗ More flexible ligands can better accommodate changes (mutations) in the active site (important for antibacterials and antivirals).

∗ Favorable ∆H indicates good electrostatic interactions of compound with target.

∗ Binding affinity of an enthalpic lead can be enhanced by introducing conformational restraints, hydropho-bicity, or additional enthalpic interactions.

• Literature suggests it is more difficult to increase ligand affinity when binding interactions are entropically driven. Such ligands are highly hydrophobic and rigid*

*Velazquez-Campoy, et.al. (2001) Therm. Acta. 380, 217-227

High throughput screening efforts identified a small molecule renin inhibitor with a core substituted diaminopyrimidine ring. Parallel medicinal chemistry efforts based on this lead resulted in the compound shown below, and a complex of this compound bound to renin was crystallized and structural data obtained by X-ray diffraction.

Crystal Structure of HTS Hit Bound to Renin

The structure indicated there were adjacent unoccupied binding pockets. Synthetic efforts were initiated to extend functionality into these pockets to improve affinity and adjust pharmacokinetic parameters.

Initial additions to the pyrimidine ring template that extended into the large hydrophobic S2 pocket did not improve af-finity and dramatically altered the thermodynamic driving force for the binding interaction.

Binding of the core template was enthalpically driven whereas binding of initial inhibitors with S2 extensions were both enthalpically and entropically driven but lost significant binding enthalpy. Additional electrostatic interactions were then incorporated in the S2 extension to improve binding enthalpy while taking advantage of the favorable entropy.

Structure data and molecu-lar modeling suggested that addition of a methoxy-propyl group extending into the S3 subpocket would improve inhibitor affinity and result in greater binding enthalpy.

Co-structure of HTS hit bound to renin showing the Connelly surface of the binding site color coded according to electro-static potential. Red indicates negatively charged and blue positively charged surface area. A large hydrophobic pocket S2 and the mouth of the smaller hydrophobic S3 subpocket are identified.

The inhibitor containing the added methoxy-propyl group bound as predicted into the S3 subpocket.

ITC data confirm the predicted improvement in inhibitor affinity and improved binding enthalpy upon addition of the methoxy-propyl group extending into the S3 subpocket.

Binding Thermodynamics of Renin Inhibitors Obtained by

Isothermal Titration Calorimetry

Thermodynamics Suggested Structural Alterations of S2

Substituent to Improve Renin Affinity

Calorimetry indicated ~7x improved affinity and 5 kcal/M more favorable enthalpy but 4 kcal/M more unfavor-able entropy (restricting ether ap-pendage).

aIC50 ’s determined in duplicate using a GFP assay, Holsworth, et al. Bioorg. Med. Chem. 13 (2005) 2657-2664

aIC50 ’s determined in duplicate using a GFP assay, Holsworth, et al. Bioorg. Med. Chem. 13 (2005) 2657-2664

Calorimetry suggested main binding force was mainly hydro-phobic effect and change in pro-tein to flap-open conformation, no significant H-bonds were formed in the S2 pocket, there-fore, need to correctly position polar functionality in S2; achieved ~2x improved affinity, significant loss in enthalpy but large favorab le ent ropy (expulsion of ordered H2O).

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Dr. Sarver summarized this presentation as follows:

• Renin inhibitor affinities improved > 6000 fold from initial 6 µM lead

• Appendage for S2 pocket identified from NMR screen

• Combination of thermodynamic and structural data provided unique insights into inhibitor design not obtained by either technique alone

∗ S3sp extension provided improved affinity due mainly to increased binding enthalpy from van der Waals interactions but additional gains could be obtained by restricting rotation of the ether appendage

∗ Insight into lack of improvement in affinity with initial aryl-benzamide extension into S2, mainly hydropho-bic effect and lack of H-bonds

∗ H-bonding of arylsulfonamide improved binding enthalpy of S2 extension and retained entropic advantage detected with S2 pocket derivatives

The results from the synthetic efforts to improve H-bonding and the resultant affinities were monitored and further guided using the ITC data.

Lead optimization yielded an overall, 45X increase in affinity, similar ∆H and improved binding entropy.

Aryl Sulfonamide Formed H-bonds with Ser219, Tyr220, and Thr77 in S2 Pocket Improving Affinity

Improved enthalpy compared to aryl benzamide S2 extension that didn't form productive H-bonds and now gained binding entropy

The Uses of Calorimetry in Drug Discovery From the 2007 Trends in Microcalorimetry Conference

Hit Stage

1. Kd and stoichiometry (the gold standard)

2. Eliminate false positives

3. Target selectivity

4. Screen detergents for best co-crystallization conditions

5. Test compounds for true binding before X-ray structure determination

6. Fragment based drug discovery

Lead Stage

1. Hydrogen binding alignment

2. Hydrophobic interactions

3. Enantiomer binding

4. Molecule flexibility

Drug Candidate

1. Test preclinical and clinical candidates for thermodynamic properties

2. Test backup and failed candidates for thermodynamic properties

3. Test competitor’s compounds for thermodynamic properties

For more information about the use of ultrasensitive microcalorimetry in the drug discovery and de-velopment processes, copies of review articles on lead optimization or other reviews from the 2007

Current Trends in Microcalorimetry Conference, please contact MicroCal.

MicroCal, LLC www.microcal.com

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