Example Module. Exam mple emo. Modu dule. Example Module. From Concept to Pharmacy. The Pharmacophore and Molecular Recognition. Lead Optimizationtion

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1 From Concept to Pharmacy The Pharmacophore and Molecular Recognition One of many mos taught in the course, Medicinal Chemistry (CHEM-4300 and CHEM-6300) 0) at Rensselaer Polytechnic Institute by Mark P. Wentland Lead Optimizationtion Screen to identify hit/lead Molecular target discovery & validation; Payer Exam mple emo Modu Cellular +/or functional activity Discovery ADME/Tox SAR-potency-selectivity FDA Clinical Drug delivery Development ADME/Tox Scale-up/cost Patent In vivo efficacy uleedic Medicinal Chemistry: - What to make - How to make it Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 2 Mo - The Pharmacophore and Molecular Recognition Protein Molecular Targets for Small Molecule Drug Discovery 5' 3' 3' 5' DNA Nucleus Transcription Cytoplasm Example ple Mo Mo 5' 3' mrna Translation (protein synthesis) Druggability ity of MTs: Link to disease Amenable to: - Screening (HTS) - Small molecule - Selectivity - Structure-based design Enzyme GPCR Ion channel Other protein molecular targets: Growth factor receptors, nuclear receptors, biogenic amine transporters, transcription factors, numerous protein-protein and DNA-protein interactions, etc. 5' 3' S Molecular Targets of FDA-Approved Drugs Santos, R., et al, Nat. Rev. Drug Disc. 207, 6, 9. Of the,348 US FDA-approved small molecule drugs for which the biological target is known: Proportion of smallmolecule drugs that target major families: 999 target 549 human proteins 25 target 84 pathogen proteins 63 target 9 other human biopolymers 7 target 7 other pathogen biopolymers Other 30% Nuclear receptors 6% 3 GPCRs 33% Ion Channels 8% GPCRs (7TM) uon ion channels Kinases Nuclear receptors Other Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 3 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 4 Mo - The Pharmacophore and Molecular Recognition

2 Druggability of Molecular Targets for Small Molecule Drug Discovery Screening Small Structure- Link to Molecular target (MT) (HTS) molecules Selectivity based design disease Example em Mo M Enzymes GPCRs + + am+ Ion channels + + RNA DNA Protein-protein interactions + Biogenic amine transporters Nuclear hormone receptors MT is generally amenable to this property/technology MT is somewhat amenable to this property/technology MT is not very amenable to this property/technology d The Pharmacophore The functional groups (ionization considered) of a drug and the bioactive conformation they must adopt to sustain high-affinity and specific non-covalent interactions with the molecular target. Example Binding forces responsible for this molecular recognition are the same that stabilize protein tertiary structure: Hydrophobic interactions Hydrogen bonds Gleevec bound Numerous other polar interactions to Abl-TK Gleevec bioactive conformation Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 5 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 6 Mo - The Pharmacophore and Molecular Recognition Enzyme-Ligand Non-Covalent Interactions: Competitive Inhibition Lineweaver-Burk Plots for Determination of K i of a Competitive Inhibitor E + S H2O Inhibitor Exam ample M Mrthos E S [E S] E P(s) NCC (non-covalent complexes) Inhibitor Orthosteric site Substrate P(s) + E Substrate E I E + I K k i = [E] [I] on [E e I] NCC k off MoE [v] (min/mm) Example ple e Mo [I] = 3X μm [I] = 2X μm [I] = X μm [I] = 0 μm 0.2 Inhibitor design strategies: Mimic S, TS or P(s) k off = kon K m app K m 0. { K m ( + [I]/K i ) [S] ule (mm - ) Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 7 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 8 Mo - The Pharmacophore and Molecular Recognition

3 Enzyme inhibition (%) IC 50 Value and Dose Response Curves Example ple Mo IC 50 =.0 μm Inhibitor concentration - μm IC 50 = K i + IC 50 = 0 μm [S] K m IC 50 = [Inhibitor] that reduces product formation by 50% Both inhibitors are equally active; one is 0-fold more potent Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 9 Mo - The Pharmacophore and Molecular Recognition In vitro (outside the body) Acellular assays: Ki - inhibition constant Kd - dissociation constant nt Quantification of Biological Activity IC50 - concentration of drug responsible for 50% of maximal effect or inhibition pic50 = - log IC50 in molar Cell-based assays: EC50 - concentration of drug which produces 50% of the maximum possible effect or response MIC (Minimum Inhibitory Concentration) n) - lowest concentration of drug to inhibit the growth of, for example, bacteria In vivo (inside an intact living organism) ED50 (Median Effective Dose) - median dose of drug effective in 50% of the animals or a 50% response in a biological system. LD50 (Median Lethal Dose) - median concentration of drug that will kill 50% of the test animals within a designated period TI (Therapeutic Index) - LD50 ED50 MTD (Maximum Tolerated Dose) - highest dose of compound that did not show any overt signs of toxicity. PD50 - median dose to protect 50% of animals from an otherwise lethal infection. IUPAC Compendium of Chemical Terminology ( Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 0 Mo - The Pharmacophore and Molecular Recognition Protein Ligand Binding: A Closer Look Protein Flexibility and Induced Fit Protein Ligand Binding: A Closer Look Protein Flexibility and Induced Fit Lock and key (Fisher, 894): L + L 894am Induced fit (Koshland, 958): + L 2 a Mod ule ModL L 2 L 2 Teague, S. J. "Implications of Protein Flexibility for Drug Discovery." Nature Rev. Drug Disc. 2003, 2, 527. Cozzini, P.; et al. Target Flexibility: An Emerging Consideration in Drug Discovery... J. Med. Chem. 2008, 5, Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 2 Mo - The Pharmacophore and Molecular Recognition

4 X-Ray Crystal Structures of HIV-PR/Inhibitor Complexes Allosteric Enzyme Modulation Negative Allosteric Modulator: A drug that binds to the enzyme at a different (allosteric) site than substrate and stabilizes a conformation having poor substrate recognition Inhibition Allosteric site NAM Orthosteric site Exampl NAM Mo Saquinavir Ritonavir Indinavir Nelfinavir (HXB) (HXW) (HSG) (OHR) Positive Allosteric Modulator: A drug that binds to an enzyme at an allosteric site and stabilizes a conformation having good substrate recognition Activation ule odam PAM PAM Abood, M. E. JMC 206, 59, 42. Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 3 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 4 Mo - The Pharmacophore and Molecular Recognition Irreversible Enzyme Inhibition Non-Superimposable Mirror Images Example ple le Mo Irreversible inhibition: n: Covalent bonding of inhibitor to enzyme Br SH Inh* reversible SH Inh* irrev. S Inh* + Br E I* E I* Ex ule - Singh, J.; et al. The resurgence of covalent drugs. Nat. Rev. Drug Disc. 20, 0, Wilson, A. J. et al. Keep Calm, and Carry on Covalently. J. Med. Chem. 203, 56, sc8i Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 5 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 6 Mo - The Pharmacophore and Molecular Recognition

5 Drugs Marketed as Racemates over Time Nat. Rev. Drug Disc. 2003, 2, 424. Frequency (%) Exa mp SE Racemates Single Enantiomers Achiral xam 5 R SE A R A e du ul Update: Agranat, I.; et al. The predicated demise of racemic new molecular entities is an exaggeration. Nat. Rev. Drug Disc. 202,, 972. Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 7 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 8 Mo - The Pharmacophore and Molecular Recognition Chirality Effects on Drug Binding High ER's: 3/4 Points of complementarity (See: Crossley, R. Tetrahedron, 992, 48, 855) From one Extreme to Another EBS R BS R R4 Exa Ex mpl mr R2 R2 R4 BS 4 R3 SpBS 2 BS 4 R3 BS 2 ple M BS 3 High affinity ligand BS 3 Low affinity ligand Both enantiomers have similar contacts with MT, however, the distomer has an unfavorable conformation (See: Huai, Q.; et al, JMC 2006, 49, 867) ER's near unity: Groups attached to center of chirality contact bulk water rather than MT Binding Sites are large enough to accommodate different size groups Conformational effects of molecular target (i.e., induced fit) and ligand Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 9 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 20 Mo - The Pharmacophore and Molecular Recognition

6 Lead Optimization Overall goal: Identify and correct the deficiencies of a lead molecule in order to advance a candidate to the clinic. In general, leads come Efrom high-throughput screening. i.e., HTS Hits Leads amp e M HTL* Lead Optimization Overall goal: Identify and correct the deficiencies of a lead molecule in order to advance a candidate to the clinic. Exam Design/Synthesis-Biological Evaluation Iterations: Potency selectivity Information Cellular activity Molecular target-based or Phenotypic-based screening *Provided the following are confirmed +/or resolved during Hit-To-Lead activities: - Structure and purity - Activity is reproduced and dose-related - Potential for an SAR to develop - Lead is druglike and the series is potentially patentable Compounds ADME/Tox ple e Mo Modul ule le Design and Synthesiss Information Compounds In vivo levo efficacy Nunez, S.; et al, DDT 202, 7, 0. and Johnstone, C. DDT 202, 7, 538. Harvey, A. L.; et al, The re-emergence of natural products for drug discovery in the genomics era NRDD 205, 4,. Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 2 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 22 Mo - The Pharmacophore and Molecular Recognition Change in Free Energy for: E + I E I Change in free energy erg (ΔG) from reactants products is a measure of the amount of work done - the more work done the more spontaneouss the reaction. G = G products - G reactants G = RT log(/k i ) For μm nm (i.e.,k i 000-fold) G - 4. kcal/mol Change in Free Energy for: E + I E I Gibbs Free Energy Equation: G = H T S Enthalpy change (i.e., amount of heat produced): H = HP - HR - Bond strength (e.g., optimized distance/geometry of polar groups) - Difficult to optimize (more to come) Entropy change (much easier to optimize): S = SP - SR - Desolvation entropy change (always favorable) Predominant force in hydrophobic interactions - Conformational entropy change (always unfavorable but penalty can be ) Bissantz, C.; Kuhn, B.; Stahl, M. JMC. 200, 53, 506. Ladbury, J. E., et al, NRDD 200, 9, 23 Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 23 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 24 Mo - The Pharmacophore and Molecular Recognition

7 Change in Free Energy for: E + I E I Gibbs Free e Energy Equation: G = H T S For Lead Op, G should be as negative as possible, but how should this be accomplished? By making H as negative as possible? By making S as positive as possible? Or both? Example mple le Mo Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 25 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 26 Mo - The Pharmacophore and Molecular Recognition Hydrogen Bonding of Water Examp Pauling electronegativities: O (3.44) N (3.04) C (2.55) H (2.22) X Hydrophobic Interactions - Entropic Considerations At the interface between a hydrophobic drug and water, stronger than normal H-bonds between the water molecules are formed to compensate for the weaker interactions between drug and water, therefore water is more ordered. E+ water release = S O N H HB strength = 4.3 kcal/mol (generic C-H ~ kcal/mol) Average of 3 HBs per water (out of total of 4) Each HB last about 0-2 second X O N H m e Drug Water release also stabilizes: CH 2 Phe Drug N H NH CH 2 Trp od Stacking Edge-to-face Favorable desolvation entropy is the predominant factor in hydrophobic interactions. The larger the surface area the greater the effect (~ 28 cal/mole/å 2 ) Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 27 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 28 Mo - The Pharmacophore and Molecular Recognition

8 Hydrophobic Collapse Change in conformation of a drug bought about by dissolution in H 2 O. Impact on drug binding: Enhanced binding affinity is observed if the hydrophobic-collapsed conformation is very similar to the bioactive conformation (i.e, the molecule is "preorganized ), or conversely; Energy in the form of decreaseded binding affinity may be required to adopt the bioactive conformation when that drug exists in a different, but stable conformation in water due to intramolecular hydrophobic interactions Drug-Protein Non-covalent Binding Forces: Hydrogen Bonding Neutral-neutral H-bond: X H ----:Y Where X is O or N and Y = O, N or F Optimal when X, H and lone pair of Y are linear and distance between X and Y is Å ΔG o ~ 0 to -.5 kcal/mol Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 29 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 30 Mo - The Pharmacophore and Molecular Recognition Abl-Tk/Gleevec H-Bond Interactions from IEP Drug-Protein Non-covalent Binding Forces: Hydrogen Bonding Example mple M thr35 glu286 Neutral-neutral H-bond: IC 50 = 38 nm e sc3d - Contribution of a neutral-neutral HB is unpredictable & contributes 0- to 5-fold in binding affinity - Benefit in establishing H-bond contacts with MT may be offset by unfavorable desolvation enthalpy (i.e., an uncompensatable desolvation penalty). Then what is a chemist to do? - Ask how strong the H-bond is with the protein relative to water and if a problem, optimize geometry and distance [difficult at best (e.g., lack of resolution in X-ray structures)] - Simultaneously optimize enthalpy and entropy - Think charge reinforced H-bond Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 3 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 32 Mo - The Pharmacophore and Molecular Recognition

9 r Hydrogen Bonding and other Polar Interactions Free Energy of Binding (E + I E I): Summary ΔG = Gproducts - Greactants ΔG = ΔH - TΔS Example e Mo Enthalpy change (i.e., amount of heat produced): H = HP - HR - Difficult to optimize due to two conflicting contributions: Optimization of polar group interactions always favorable but difficult Desolvation of polar groups (always unfavorable) Entropy change (much easier to optimize): S = SP - SR - Desolvation entropy change is the predominant force in hydrophobic interactions Always favorable, BUT... (solubility, lity, MW) - Conformational entropy change (always unfavorable but penalty can often be ) Unbound inhibitor (a reactant) S (translational tional and rotational energies) Bound inhibitor (product) S (fewer degrees of freedom) S is negative more positive G Optimize the thermodynamic profile: Balance enthalpy and entropy to optimize lead for both binding affinity and drug-like properties. Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 33 Mo - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 34 Mo - The Pharmacophore and Molecular Recognition Reducing the Conformational Entropy Penalty Overall goal: make S more positive more negative G (mem: ΔG = ΔH - TΔS) - Almost always accomplished by preorganization of the actual bioactive conformation: Hydrophobic collapse (mem: S = SP - SR) am Preparation of a conformationally Rigid analogue of a Floppy lead such that: SR >> SF Example: Inhibition of HIV Protease by Cyclic Ureas (Lam, P.Y.S.; et al. J. Med. Chem. 996, 39, 354) Modul ule le - Factors contributing to the poor binding of "floppy" analogue: Entropic penalty Preference for other conformations (e.g., hydrophoblic collapse) Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 35 Mo - The Pharmacophore and Molecular Recognition Goal: Balance enthalpy and entropy to optimize lead for both binding affinity and drug-like properties. kcal/mol (Ki ~ nm) Indinavir Saquinavir Thermodynamic Profiling Example mple Mo From: Ladbury, J. E., et al, Nature Rev. Drug Disc. 200, 9, 23: Nelfinavir ΔG = ΔH - TΔS Ritonavir Ampreavir Loprinavir Atazanavir Tipranavir dranavir For more info on Isothermal Titration Calorimetry (ITC) to assess the ΔH of binding, see: - Nunez, S.; et al, DDT 202, 7, 0. - Jean-Paul Renaud, J-P.; et al, NRDD 206, 5, 679 Geschwindner, S.; et al, Ligand Binding Thermodynamics in Drug Discovery: Still a Hot Tip? JMC 205, DOI:0.02/jm505f Klebe, G. Applying thermodynamic profiling in lead finding and optimization. NRDD 205, 4, 95. Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 36 Mo - The Pharmacophore and Molecular Recognition Darinavir