Crystallography as a Drug Design and Delivery Tool

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2016 Drug Design and Delivery Symposium “Crystallography as a Drug Design and Delivery Tool”

Vincent Stoll

Andrew Brunskill

Research Fellow and Associate Director of Structural Biology, AbbVie

Associate Principal Scientist, Merck

Robert Wenslow Vice President, Business Development, Crystal Pharmatech

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Drug Design Using Structural Biology Vincent S. Stoll, Ph.D. Associate Director Structural Biology R&D, AbbVie

No Pro Approval Code A4360572

The Drug Design Cycle Iterative structure-based drug design cycle Lead Generation

Preclinical Studies

Chemical Synthesis Medicinal Chemistry-based Cycle Biological Assays Analog Design

Structure-based Drug Design Cycle 3D Structure Determination Crystallography/NMR Homology Modeling

Protein Biochemistry

Molecular Biology


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Protein Production – Protein Biochemistry

Protein production pathway – an iterative process Production

Cloning and expression

Fermentation

Purification Characterization

Customers

Beneficiaries

Crystallography NMR

Project team Chemists Biologists

.

Cycle to improve expression, solubility, activity, stability Cycle to improve crystallizability, spectral quality, etc.


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How Do We Determine the Crystal Structure?

Solution of target protein

Add precipitant Crystal of protein in drop


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X-ray Generator

Soak ligand into crystal

Ligand soaks in over 2-24 hours

Crystal mounted at liquid nitrogen temperature (–196 °C) SBBD| Drew Medicinal Chemistry Course| 6.8.16

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Protein Crystal X-ray Diffraction

Intensities transformed to electron density

mounted crystal

X-ray beam

3D atomic model

Scatters X-rays in discrete directions Single wavelength (~0.1 nm) Single crystal (10~100 µm)

(image 0.2° wedge of 180° crystal rotation scan)

Integrate with AbbVie Projects


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Driving FBDD & SBDD on small Molecules and Biologics

Global X-ray Crystallography

Using State of the Art Technologies • Highly automated crystallization process

– UV Florescence and Absorbance crystal viewing – Lipidic cubic phase dispenser – Micro crystal mounting system

Crystallization Optimization

Crystal Plate Viewing Tower

– Currently manual, automation in development

– Image viewing software – Novel approach to compound solubility measurement (Critical Aggregation Concentration) – Helps characterize and prioritize compounds for crystallization

Crystal Identification Crystallization Screening

• Commercially purchased automation – Mosquito for nanoliter solution dispensing

• Protein supplied by centralized Protein Biochemistry Microcrystal Scooping SBBD| Drew Medicinal Chemistry Course| 6.8.16

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Enabling Challenging Experiments

• High Flux

• Mini-Beam 50, 20, 10, 5 m

>1013 ph/s @ 12.4 keV

• Micro-Focused Beam

30 m(v) x 70 m(h) FWHM

• Fast Detector

Weakly Diffracting Crystals

Small Crystals

auto positional feedback

• High-Precision Alio Goniometer reliable sample positioning

Pilatus 6M, no readout noise

• Collect-Along-Vector

• Stable Beam

Radiation SensitiveC rystals

Lipidic Cubic Phase Crystals

• Diffraction Rastering sample centering


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Crystallography Experiments @ IMCA-CAT


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INDUSTRY IMCA Members

EXPERIMENT Beamline 17-ID @ APS

CAPABILITIES

PRODUCTIVITY

• diffraction rastering

20,000+

• collect-along-vector

DISCOVER

structures annually

• auto collect & process

• micro crystals • membrane proteins • MAD / SAD • in situ • high-throughput • fast, encrypted data transfer

• proprietary • rapid & frequent access

IMCA-CAT Subscribers

• focused, intense beam

New subscriptions available

• mini beam 5-50 m

• real-time integration to company pipelines

• on-site, remote, mail-in

• pucks: Uni, ACTOR, ALS

www.imca-cat.org


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Structure-based Drug Design

Bcl Inhibitors, Fragment-based Drug Design, and Anti-targets


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Bcl-2 Family Inhibitor – a Protein-Protein Interaction

106 Business Week | December 12, 2005

• Potential as a therapeutic option in a range of cancers (ABT-737is an investigational Compound. Safety and Efficacy have not been established) SBBD| Drew Medicinal Chemistry Course| 6.8.16

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Bcl-2 Family Inhibitor Helps Turns on Death Switch In cancer cells Overproduction of Bcl-2 protein binds to death proteins and shuts off the death switch allowing cancers to stay alive BH3only

+

Death Protein

Bcl-2

BH3only

like

Bcl-2 like

How Bcl-xL binds to death proteins

Cancer cells live

Bcl-xL

Bcl-2 family inhibitor binds to Bcl-2 and helps blocks death protein interaction, allowing death protein to kill cancer +

Bcl-2

Bcl-2

like

like

BH3only

Cancer cells die Muchmore et al., Nature 381, 335 (1996) Sattler et al., Science, 275, 983 (1997)


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Bcl-xL Structure of Binding Site Bcl-xL complexed to Bak peptide • Goal is to design a compound to mimic Bak peptide binding • Hydrophobic residues interact with Bcl-xL: – Leu 78, Ile 85

D83

• Hydrophilic residues point out into solvent:

R139

– Arg 76, Asp 83, Asp 84

R76

• Take advantage of groups to design compound

I85

L78

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HN

10,000 fragment library

O Cl

H3CO

NH2

13C

COOH

(ppm)

Fragment Screening and Linking – SAR by NMR

NH N

CH3 H3C

N

CH3

HN

HN

N O

O

N

1H

(ppm)

N

Screen for fragment #1


O OH

O


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Fragment Screening and Linking – SAR by NMR • Perform 2nd site screen

COOH

HN

Cl

H3CO

10,000 fragment library

O

NH2

• Determine ternary structure

NH N

CH3 H3C

N HN

N O

• Design linkers and synthesize

CH3

HN O

N

• Confirm design with structure

N



O OH O

O

Link fragments


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Overall Fragment Screening Paradigm Fragment Collection

Ro3 and Ro3.5 Collections

HTS

Fragment Screening

Fragment Optimization

Biochemical assays XRC

SBDD Design (XRC and Props)

Triage BP Collection

Purification and sample logistics

BP Similars BioPhys (BP)

Synthesis

Screening

BPS

SPR NMR Screening and confirmation

Lead SBBD| Drew Medicinal Chemistry Course| 6.8.16 32

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Screen for First-Site Ligands using NMR Monitor Binding with 15N-HSQC spectrum 105.0

• 10,000 compound library • ~ 215 • [Compound] = 1 mM

15

107.0 108.0 109.0

O

G94

G196

111.0

Kd = 300 M

110.0

OH

F

N ppm

106.0

G138

9.0

8.5

8.0

1

H ppm


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NMR Structure of Bound Fragment

Binds to peptide “hot spot” • Two key interactions maintained (Leu78 and Asp83)

R139

Second site accessible • Ile85 pocket of Bak peptide

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Screen for Second-Site Ligand • Screen in excess of biaryl acid • 3,500 compound library • ~ 150 R139

• [Compound] = 5 mM

OH

Kd = 2000 M

• Binds to second “hot spot” ─ Ile of Bak peptide


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Linking Strategy OH

O

linker

OH

Kd = 2000 – 6000 M

6.1 Å

Kd = 300 M

F97

F

O O O


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Linking Strategy O

OH

F

FPA IC50 = 1.4 µM

• Accesses hydrophobic second site • 200-fold gain in potency – Expected >150-fold

• Still room for improvement


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Acylsulfonamide Linking Strategy Acidic Hydrogen OH

O

H N

O

O

S

R O

F97

F

F

Kd = 300 µM

Kd = 320 µM R = Me

O O O

New trajectory: avoids F97 Maintains acidic nature SBBD| Drew Medicinal Chemistry Course| 6.8.16

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Diversity Approach to 2nd Site Binders Parallel synthesis O

OH

React with 120 diverse sulfonamides

NO2 H N

O

O

Kd = 300,000 nM

NO2 S

N

React with 125 diverse amines

H N

O

O

O

S

H N S

O

HNRR'

R-SO2NH2 F

S

H N

Kd = 245 nM

Kd = 36 nM Kd = >10 µM

F

F

in 10% serum!


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Structure-Based Optimization of Bcl-xL Inhibitors NO2 H N

O

O

NO2

H N O

S

S

H N

O

O

H N

H

N

S

S

Structure-based reduction in protein-binding

O

Tail collapses increases potency N N

Polar isostere for phenyl ring

F

Accessing a “third” pocket In the groove Cl

Kd = 36 nM

ABT-737 KD < 1 nM Nature 435, 677-681 (2005) SBBD| Drew Medicinal Chemistry Course| 6.8.16

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ABT-737 – The Clinical Candidate Fits the structure-based design rubric NO2 H N

O

O

NO2

H N O

S

S

H N

O

O

H N

H

S

S

N

O

N N F

Cl

ABT-737 Clinical candidate Kd < 1 nM


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Audience Survey Question ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT

What are some of the advantages of using fragments in drug discovery? • They identify and bind key hotspots in a binding site. • Linking two fragments together is easy. • They often lead to more efficient drugs with better physical properties. • Fragments are easy to optimize in chemistry without protein structural information. 42

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ABT-263: Making an Oral Drug NO2

H N

H N

O

S O O

N S

Main liability of ABT-737 is its poor oral bioavailability

• Must be dosed IV This is due to limited absorption

N N

• Very basic dimethyl amine • Rigid chloro-biphenyl

Cl

Nitro group potentially toxic

ABT-737 Oral Bioavailability ~ 5 %


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H N

H N

O

S O O

N S

Remove potential toxicity

O F3C NO S O 2

N N

H N

O

S O O

H N

N S

Cl

N

ABT-737

N

Oral Bioavailability ~ 5 %

Cl


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H N

H N

O

S O O

Decrease basicity by one log unit

N S


O F3C S O

N N

O

O

H N

H N

S O O

N S

Cl

N

ABT-737

N


Cl


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H N

H N

O

S O O


N S


O F3C S O

N N

H N

O

S O O

Decrease rigidity, improve absorption

O

H N

N S

Cl

N

ABT-737

N


Cl


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H N

H N

O

S O O


N S


O F3C S O

N N

H N

O

S O O

Decrease rigidity, improve absorption

O

H N

N S

Cl

N

ABT-737

N


Cl

ABT-263 Oral Bioavailability ~ 30 %


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ABT-263 – The Current Clinical Candidate Also fits the structure-based design rubric O F3C S O

H N

O

S O O

O

H N

N S

N N

Cl

ABT-263 Clinical candidate Kd < 1 nM

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Navitoclax Induces Thrombocytopenia Bcl-xL driven toxicity Platelet count vs. dose % of Baseline Platelets

% of Baseline Platelets

Platelet count vs. drug exposure 90 80 70 60 50

R2 = 0.9466

40 30 20 10 0 1

AUC0-inf (ug·h/mL)

10

100

1000

Dose (mg)

• Platelet survival is dependent on Bcl-xL – Mason, et. al., Cell 128, 1173, (2007).

• Bcl-xL inhibition with navitoclax results in concentration and dose dependent thrombocytopenia • Thrombocytopenia is the clinical dose limiting toxicity of navitoclax (Navitoclax is an investigational Compound. Safety and Efficacy have not been established)

Roberts et al., J Clin Oncol 27:15s, abstr 3505 (2009) Wilson et al., Lancet Oncol 11, 1149, (2010) 49

Navitoclax Inhibition Profile Dictates Efficacy/Toxicity • Bcl-2 is an important survival factor in lymphoid malignancies – CLL, NHL • Bcl-xL is required for survival of circulating platelets – Bcl-xL inhibition leads to dose-limiting thrombocytopenia navitoclax

Bcl-2

Target efficacy in leukemia and lymphoma

Bcl-xL

Dose-limiting thrombocytopenia

(Navitoclax is an investigational Compound. Safety and Efficacy have not been established) SBBD| Drew Medicinal Chemistry Course| 6.8.16

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Bcl-2 Selective Inhibitors: Key Hypotheses • Bcl-2 selective inhibitors will maintain efficacy in lymphoid malignancies • Bcl-2 selective (Bcl-xL sparing) inhibitors will not induce thrombocytopenia • Will result in improved therapeutic window, allowing higher exposures and greater efficacy in Bcl-2 dependent malignancies Bcl-2 selective

navitoclax

Bcl-2

Target efficacy in leukemia and lymphoma

Bcl-xL

Dose-limiting thrombocytopenia

(Navitoclax is an investigational Compound. Safety and Efficacy have not been established) SBBD| Drew Medicinal Chemistry Course| 6.8.16

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Bcl-2 & Bcl-xL Binding Grooves Show High Similarity

• No naturally occurring, Bcl-2 selective BH3-only protein • Only 4 residues differ within binding groove Bcl-2

Bcl-xL

D100

E96

D108 R126

M112

A104 S122

L108


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Reverse Engineering Leads to Bcl-2 Selectivity • • • •

Modification of P4 binding region Loss of Bcl-xL affinity Reduced Bcl-2 affinity No cellular potency O H N S O O

O

O

O N

H N S O O

O

S

ON+ H N

O

O

N

-P4 binder

N

1

O

N

2

Cl

ON+ H N

N

-amine

N

H N S O O

O

N

N

Cl

ON+ H N

3

Cl

TR FRET, Ki, [nM] Bcl-2 Bcl-xL 0.20 1.3 29 >660 18 >660

1 2 3


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Design Cues From X-ray Crystal Artifact • Overall ligand binding conformation maintained • P4 pocket occupied by Trp28 in N-terminal loop of neighboring protein in crystal • Trp side chain forms H-bond with Asp100 of Bcl-2 – Corresponds to Glu96 of Bcl-xL • Suggests alternate approach to Bcl-2 P4 hot spot Bcl-2 X-ray

Bcl-2 X-ray

Asp 100 Asp 100

P4

Intercalating Trp


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Indole Substituent Fills P4 & Captures H-Bond • ~100-fold increase in Bcl-2 affinity • Selectivity vs. Bcl-xL maintained • Affinity & cellular efficacy still ~10-fold lower than navitoclax • Target second hydrogen bond and improve cellular activity NO2 H N

NO2 H N O O S O NH

O O S O NH

O

O

O

+ P4 indole

N N

Cl

N H

N N

3

R104

4

Cl

D100

2.8

ASP 100

TR FRET, Ki, [nM] Bcl-2 Bcl-xL 0.04 0.05 18 >660 0.30 >660

navitoclax 3 4


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ABT-199: a Selective Bcl-2 Inhibitor NO2 H N

• High Bcl-2 affinity

O O S O NH

• Lower affinity for Bcl-xL, Mcl-1

NO2 H N O O S O NH

O

O

• Ineffective in Bcl-xL-dependent human tumor cell lines (H146) Cl

Affinity

O

O

N H

N

N N

N

N H

N

4

Cl

ABT-199

Cellular Efficacy, EC50, nM

TR FRET, Ki, nM

Human Tumor Cell Lines, 10%HS

Agents

Bcl-2

Bcl-xL

Bcl-w

Mcl-1

RS4;11 (Bcl-2)

navitoclax

0.04

0.05

7

>440

110

75

4

0.30

>660

NT

>440

1180

>10,000

ABT-199

< 0.01

48

21

>440

8

3600

(ABBV-199 is an investigational Compound. Safety and Efficacy have not been established)

H146 (Bcl-xL)

Souers et al, Nature Medicine 2013 56

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Physicochemical Properties O

O

Property

ABT-199

Mol Wt (g/mol)

868.44

cLogP

8.1

PSA

183

N

Experimental logD

5.4

N

Aqueous solubility pH 1

O

O– N+ H N

O

O S NH O N H

N

2.3 ± 0.2

25 °C, (g/mL) Cl

Aqueous solubility pH 7.4 , 80% with improved bioavailability (Newman A. et al J Pharm Sci. 2012) Overcome Solubility of late stage polymorph

Amorphous coated pellets – Co-crystal also works

Dispersion co-crystal also works

85

Therapeutic Delivery, February 2015 ,Vol. 6, No. 2 , Pages 247-261

Kaletra Law et al., J. Pharm. Sci. 93 (2004) 563

Douslin et al. JACS 1946, 68, 173

A higher-energy form that offers enhanced solubility, dissolution rate and oral bioavailability

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Co-crystals – potential mechanism ccx = cocrystal

Extraction of coformer from lattice of ccx

concentration

BA

Exposure to water/SGF

Meta-stable

acid

time

concentration

free base

Solid ccx

+

Free base

No BA change

time

= “parachute” prolongs supersaturation (excipient(s) or crystal form by itself)

87

Audience Survey Question ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT

What year was the first recorded co-crystal discovered? • 1776 • 1942 • 1840 • 1996

Kobell FV, Prakt JF. D-glucose: Sodium chloride monohydrate. Chemie. 1843;28:489 88

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Itraconazole • Weak base, extremely low aqueous solubility – ~nanograms / mL RT solubility in water at pH 3 – 8 – 0.6 mg/mL at pH 1.2 • High dose drug, significant first-pass metabolism • Inconsistent oral bioavailability was a major challenge • Salt forms were intrinsically acidic and hygroscopic N N N

CH3

O

O

H3C N

N

N

N

O

O

N

H

Cl

Cl

89

Co-crystal vs. marketed product

[1] (M)

8x10

-4

Sporanox® beads  l-malic acid co-crystal l-tartaric acid co-crystal  succinic acid co-crystal cis-itraconazole

6 4 2 0 0

100

200 300 Time (min)

400

• Co-crystals display improved dissolution compared to the crystalline free base • Comparable dissolution to the amorphous drug coated on beads – SPORANOX® capsule formulation Remenar et al. JACS, 125, 8456 (2003)

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Regulatory IDENTIFY all relevant Crystals (or amorphous) for your compound

91

ICH Q6A, Federal Register, 2000, 65(251), 83041-83063

Regulatory Understand the IMPACT of all relevant Crystals (or amorphous) for your product

92

ICH Q6A, Federal Register, 2000, 65(251), 83041-83063

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Lessons Learned

 Crystals (or lack thereof) have significant impact on product quality and performance  Choosing the right Crystal is not a unit operation  Staged decision points  Constant Vigilance

 Crystals make your “molecule” a “medicine”

93

Special Thanks!

 Ann Newman – Seventh Street Development Group and Crystal Pharmatech

 Jun Huang – Crystal Pharmatech  Elizabeth Vadas – InSciTech Inc.  Örn Almarsson – Moderna Therapeutics  Carlos Sanrame – Crystal Pharmatech 94

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Vincent Stoll

Andrew Brunskill






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2016 Drug Design and Delivery Symposium

http://bit.ly/2016ddds

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Chemistry of Longevity: Rapamycin's Secret Past and Potential for a Longer Life Matt Kaeberlein, Professor of Pathology, University of Washington Bethany Halford, Senior Editor, Chemical & Engineering News 97



Vincent Stoll

Andrew Brunskill






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Chemistry of Longevity: Rapamycin's Secret Past and Potential for a Longer Life Matt Kaeberlein, Professor of Pathology, University of Washington Bethany Halford, Senior Editor, Chemical & Engineering News


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Crystallography as a Drug Design and Delivery Tool

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