From Concept to Pharmacy

One of many modules taught in the course, Medicinal Chemistry (CHEM-4300 and CHEM-6300) at Rensselaer Polytechnic Institute by Mark P. Wentland

The Pharmacophore and Molecular Recognition

Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 1 Module - The Pharmacophore and Molecular Recognition

From Concept to Pharmacy

Molecular target discovery & validation; “Payer”

Screen to identify hit/lead

Cellular +/or functional activity

SAR-potency-selectivity

PatentIn vivo efficacy

Discovery ADME/Tox

Scale-up/cost

Drug deliveryClinical

FDA

Development ADME/Tox

Medicinal Chemistry:- What to make- How to make it

Lead Optimization

Molecular target discovery & validation; “Payer”

Screen to identify hit/lead

Scale-up/cost

Drug deliveryClinical

FDA

Development ADME/Tox


Protein Molecular Targets for Small Molecule Drug Discovery

Other protein molecular targets: Growth factor receptors, nuclear receptors, biogenic amine transporters, transcription factors, numerous protein-protein and DNA-protein interactions, etc.

Transcription

Nucleus

Cytoplasm

Translation

DNA

3'5'

5'3'

(protein synthesis)

mRNA

5'

3'

Druggability of MTs: • Link to disease • Amenable to:

- Screening (HTS)- Small molecule - Selectivity- Structure-based design

Enzyme

GPCR

Ion channel

S5'

3'


Molecular Targets of FDA-Approved DrugsSantos, R., et al, Nat. Rev. Drug Disc. 2017, 16, 19.

GPCRs33%

Ion Channels18%

3

Nuclearreceptors

16%

Other30%

GPCRs (7TM)ion channelsKinasesNuclear receptorsOther

Of the 1,348 US FDA-approved small molecule drugs for which the biological target is known:

999 target 549 human proteins

215 target 184 pathogen proteins

63 target 9 other human biopolymers

71 target 7 other pathogen biopolymers

Proportion of small-molecule drugs that target major families:


Screening Small Structure- Link to Molecular target (MT) (HTS) molecules Selectivity based design disease Enzymes + + + +

GPCRs + +

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

Druggability of Molecular Targets for Small Molecule Drug Discovery


Gleevec®bioactive conformation

Gleevec® bound to Abl-TK

The PharmacophoreThe functional groups (ionization considered) of a drug and the bioactiveconformation they must adopt to sustain high-affinity and specific non-covalentinteractions with the molecular target.

Binding forces responsible for this molecular recognition are the same that stabilize protein tertiary structure:

• Hydrophobic interactions• Hydrogen bonds• Numerous other polar interactions


Inhibitor design strategies: Mimic S, TS‡ or P(s)

Enzyme-Ligand Non-Covalent Interactions: Competitive Inhibition

E · I E + I NCC

koff

konKi = [E · I]

[E] [I] koff= kon

NCC (non-covalent complexes)E · S [E · S]‡ E · P(s)E + SH2O P(s) + E

X-H SubstrateX-H

SubstrateInhibitor

X-H Inhibitor

Orthostericsite


Lineweaver-Burk Plots for Determination of Ki of a Competitive Inhibitor

1Km

1Km

app1

Km (1 + [I]/Ki)

{

0 1 2 3 4 5 6 7 8 9 10

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

[I] = 0 μM

[I] = 3

X μM

[I] = 2X μM

[I] = 1X μM

1[S] (mM-1)

1[v] (min/mM)


IC50 Value and Dose Response Curves

IC50 = Ki 1 + [S]Km

Inhibitor concentration - μMEn

zym

e in

hibi

tion

(%)

102030405060

708090

100

1.0 100.10 0.30 3.0

IC50 = 1.0 μM IC50 = 10 μM

30 100

Both inhibitors are equally active; one is 10-fold more potent

IC50 = [Inhibitor] that reduces product formation by 50%


Medicinal

Chemistry

Sample

Module

Quantification of Biological ActivityIn vitro (outside the body)

• Acellular assays: ◦ Ki - inhibition constant

◦ Kd - dissociation constant

◦ 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) - 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 (http://www.iupac.org) Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 10 Module - The Pharmacophore and Molecular Recognition

Protein Ligand Binding: A Closer LookProtein Flexibility and Induced Fit


Protein Ligand Binding: A Closer LookProtein Flexibility and Induced Fit

Induced fit (Koshland, 1958):

Lock and key (Fisher, 1894):

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, 51, 6237.

+ L1

L1

L2+L2

L2


Saquinavir Ritonavir Indinavir Nelfinavir(1HXB) (1HXW) (1HSG) (1OHR)

X-Ray Crystal Structures of HIV-PR/Inhibitor Complexes


Allosteric Enzyme Modulation

Abood, M. E. JMC 2016, 59, 42.

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

Positive Allosteric Modulator: A drug that binds to an enzyme at an allosteric site and stabilizes a conformation having good substrate recognition Activation

X-H X-H X-H

NAM

NAM

Allosteric site

Orthostericsite

X-H X-H

PAM

PAM

X-H


Irreversible Enzyme Inhibition

- Singh, J.; et al. “The resurgence of covalent drugs.” Nat. Rev. Drug Disc. 2011, 10, 307.- Wilson, A. J. et al. “Keep Calm, and Carry on Covalently.” J. Med. Chem. 2013, 56, 7463.

SH

+SH S Inh*

BrInh* irrev.reversible

Br

Inh*

Irreversible inhibition: Covalent bonding of inhibitor to enzyme

E · I* E − I*


sc18i

Non-Superimposable Mirror Images


Drugs Marketed as Racemates over Time

SER

A

1983-1987

Freq

uenc

y (%

)

RacematesSingle EnantiomersAchiral

Nat. Rev. Drug Disc. 2003, 2, 424.

15

30

45

60

SE

R

A

1998-2002

Update: Agranat, I.; et al. “The predicated demise of racemic new molecular entities is an exaggeration.” Nat. Rev. Drug Disc. 2012, 11, 972.

Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 17 Module - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 18 Module - The Pharmacophore and Molecular Recognition

Medicinal

Chemistry

Sample

Module

Chirality Effects on Drug BindingHigh ER's:• 3/4 Points of complementarity (See: Crossley, R. Tetrahedron, 1992, 48, 8155)

• Both enantiomers have similar contacts with MT, however, the distomer has an unfavorable conformation (See: Huai, Q.; et al, JMC 2006, 49, 1867)

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

Low affinity ligandHigh affinity ligand

BS 2

BS 3

BS 4

BS 1

R1

R2R4

R3 BS 2

BS 3

BS 4

BS 1

R1

R4R2

R3


From one Extreme to Another


Lead OptimizationOverall goal: Identify and correct the deficiencies of a lead molecule in order to advance a candidate to the clinic.

In general, leads come from high-throughput screening. i.e., HTS Hits Leads

*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

HTL*

Nunez, S.; et al, DDT 2012, 17, 10. and Johnstone, C. DDT 2012, 17, 538.Harvey, A. L.; et al, “The re-emergence of natural products for drug discovery in the genomics era” NRDD 2015, 14, 111.

Molecular target-based orPhenotypic-based screening


Information

Compounds

ADME/Tox In vivoefficacy

Design/Synthesis-Biological Evaluation Iterations:

Cellularactivity

Design and

Synthesis

Potencyselectivity

Information

Compounds

Lead OptimizationOverall goal: Identify and correct the deficiencies of a lead molecule in order to advance a candidate to the clinic.


Change in Free Energy for: E + I E ∙ I

G = Gproducts - Greactants

Change in free energy (ΔG) from reactants → products is ameasure of the amount of work done - the more work donethe more spontaneous the reaction.

G = - 2.303RT log(1/Ki)

Bissantz, C.; Kuhn, B.; Stahl, M. JMC. 2010, 53, 5061.Ladbury, J. E., et al, NRDD 2010, 9, 23

For 1 μM → 1 nM (i.e., Ki 1000-fold) G - 4.1 kcal/mol



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 )



Gibbs Free Energy Equation: G = H – T S

For Lead Op, G should be as negative as possible, but howshould this be accomplished?

• By making H as negative as possible?

• By making S as positive as possible?

• Or both?

Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 25 Module - The Pharmacophore and Molecular Recognition Medicinal Chemistry (CHEM-4300/6300) Rensselaer Polytechnic Institute 26 Module - The Pharmacophore and Molecular Recognition

Hydrogen Bonding of Water

• Pauling electronegativities: O (3.44) N (3.04) C (2.55) H (2.22)

• HB strength = 4.3 kcal/mol (generic C-H ~ 88-95 kcal/mol)

• Average of 3 HBs per water (out of total of 4)

• Each HB last about 10-12 second


Medicinal

Chemistry

Sample

Module

O

NH

X

O

NH

X

+

Hydrophobic Interactions - Entropic ConsiderationsAt 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.

water release = S

• Favorable desolvation entropy is the predominant factor in hydrophobic interactions.• The larger the surface area the greater the effect (~ 28 cal/mole/Å2)

Water release also stabilizes:

Edge-to-face

N HNH

CH2

TrpDrug

Stacking

CH2

PheDrug


• Change in conformation of a drug bought about by dissolution in H2O.

• 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 decreased 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

Hydrophobic Collapse


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 2.4 - 3.0 Å• ΔGo ~ 0 to -1.5 kcal/mol

Drug-Protein Non-covalent Binding Forces: Hydrogen Bonding


sc13d

IC50 = 38 nM

Abl-Tk/Gleevec® H-Bond Interactions from 1IEP

thr315

glu286


Drug-Protein Non-covalent Binding Forces: Hydrogen Bonding

• Neutral-neutral H-bond:

- Contribution of a neutral-neutral HB is unpredictable & contributes 0- to 15-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 32 Module - The Pharmacophore and Molecular Recognition

Hydrogen Bonding and other Polar Interactions


Free Energy of Binding (E + I E ∙ I): Summary

ΔG = Gproducts - Greactants ΔG = ΔH - TΔS• 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, MW)

- Conformational entropy change (always unfavorable but penalty can often be )

• Unbound inhibitor (a reactant) S (translational 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.


• 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)

• 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. 1996, 39, 3514)

- Factors contributing to the poor binding of "floppy" analogue: • Entropic penalty • Preference for other conformations (e.g., hydrophoblic collapse)

Reducing the Conformational Entropy Penalty


-14.2

-12.4

+1.8

-13.0 -12.8 -13.7 -13.2 -15.1

-2.3

-12.7

-15.0-14.3 -14.3

kcal

/mol

Indi

navi

r

Saqu

inav

ir

Nel

finav

ir

Rito

navi

r

Ampr

eavi

r

Lopr

inav

ir

Ataz

anav

ir

Tipr

anav

ir

Dar

inav

ir

ΔG ΔH - TΔS=

(Ki ~ 1nM)

Goal: Balance enthalpy and entropy to optimize lead for both binding affinity and drug-like properties.

Thermodynamic Profiling

• From: Ladbury, J. E., et al, Nature Rev. Drug Disc. 2010, 9, 23:

• Geschwindner, S.; et al, “Ligand Binding Thermodynamics in Drug Discovery: Still a Hot Tip?” JMC 2015, DOI:10.1021/jm501511f• Klebe, G. “Applying thermodynamic profiling in lead finding and optimization.” NRDD 2015, 14, 95.

• For more info on Isothermal Titration Calorimetry (ITC) to assess the ΔH of binding, see: - Nunez, S.; et al, DDT 2012, 17, 10.- Jean-Paul Renaud, J-P.; et al, NRDD 2016, 15, 679


Medicinal

Chemistry

Sample

Module

Documents

From Concept to Pharmacy