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Optimizing Binding Kinetics in Medicinal Chemistry: facts or fantasy?

‡ -Gon /RT kon  e -G‡ /RT ‡ off ΔG on koff  e -ΔGd/RT Kd  e  koff /kon

P + L ‡ ΔG off

ΔGd

PL Gerhard Müller Ex-Head of Med Chem Mercachem, Nijmegen, NL Binding coordinate

11 The topic is hot, complex, and I am just an end-user & controversial

“You can’t optimize koff, and you don’t need to optimize koff, you simply need to optimize Kd ! ”

Head Med Chem, (top-5 Pharma), West Coast, US, Jan 2016 Optimizing Binding Kinetics in Medicinal Chemistry: facts or fantasy?

‡ -Gon /RT kon  e -G‡ /RT ‡ off ΔG on koff  e -ΔGd/RT Kd  e  koff /kon

P + L ‡ ΔG off

ΔGd

PL Gerhard Müller Chief Scientific Officer Gotham Therapeutics, New YorkBinding coordinate

2 confidential Streetlight effect in medicinal chemistry Top-heavy distributions, rich-get-richer mechanisms

5% / 75% J. Med. Chem., 54 ,6405–6416 (2011) , US J. Org. Chem. 73, 4443-4451 (2008)

MW clogP

shape clogP flatness

Fsp3 flatland J. Med. Chem., 58, 2390−2405 (2015)

para meta ortho

J. . Med. Chem., 59, 4443–4458 (2016) 3 Cheminformatics Analysis – Kinase Family Ligand and target promiscuity (ChEMBL21) Hu, Kunimoto, Bajorath Chemical Biology & Drug Design, 89, 834-845 (2017) quantitative kinome coverage 270 kinases with high-confidence data Top-10 kinases (45% of human kinome still unexplored) VEGFR2 TK 2239 erbB1 TK 1814 22.537 distinct IC50 values p38a CMGC 1509 9191 distinct scaffolds HGFR TK 1411 31.873 kinase-inhibitor combinations PI3a lipid 844 erbB-2 TK 768 GSK3b CMGC 743 SRC TK 709 Chk1 CAMK 693 AKT AGC 621 S 11351 PI3g lipid 567 Aurora-A OTHER 558 LCK TK 489 c-Jun CMGC 478 Aurora-B OTHER 445 FLT3 TK 444

4 35 Approved Kinase Inhibitors - I Wave of low-molecular weight compounds F O O Cl O O S N N O O HN Cl HN O N NH H N O O N N N N N N O N H H F3C N N N N O H H N O N O N FASUDIL (Eril) GLEEVEC () IRESSA () TARCEVA () NEXAVAR () Ischemia CML, GIST NSCLC NSCLC Renal tumors RHO-kinase (AGC) Bcr-Abl, c-Kit, PDGFR EGFR EGFR RAF, KDR, PDGF, Flt3, c-Kit Asahi Kasei (JP) 1995 Novartis (CH) 2001 F AstraZeneca (US) 2003 OSI//Roche (US) 2004 Onyx/Bayer (US) 2005 HO Cl N N H O O H N N N S S F N N N H O H N N N N NH N H N N N HN O HN O NH O O O Cl N TASIGNA () CF SPRYCEL () 3 SUTENT () TYKERB () resistant CML resistant CML RCC, GIST N Bcr-Abl, c-Kit, PDGFR Bcr-Abl, Src, Lck, c-Kit, PDGFR Flt3, c-Kit, KDR, PDGFR … EGFR Novartis (CH) 2007 BMS (US) 2006 Sugen/ (US) 2006 GSK (UK) 2007 F Br O N HN S O N *ALS N F H N O H N N N N N N N 2 N N H H S N N N H N O H O O N O N H N VOTRIENT () (SU-11654) Vandetinib (ZD-6474) Mast cell tumors - DOGS Renal tumors Mast cell tumors - DOGS Medullary thyroid cancer FGFRs, PDGFRs,cKit VEGF’s, PDGF’s, c-Kit, … FGFRs, PDGFRs,cKit VEGFRs, EGFRs, others AB Science (F) 2008 GSK (UK) 2009 Pfizer (US) 2009 AstraZeneca (US) 2011

5 35 Approved Kinase Inhibitors - II Wave of low-molecular weight compounds F Cl Cl chiral N NH 2 Cl N N Cl O O N F N N O HN OMe N O S N N chiral N MeO CN O chiral F H N N H N Cl N N N N H N O N N H (PF-02341066) (INCB-018424) (PLX-4032) N Bosulif () NSCLC Myelofibrosis, bone marrow cancer Metastatic melanoma Xeljanz (Tofacitinib) N CML H Alk, cMet, RON Jak1, Jak2 B-Raf, SRMS, ACK1, FGR, … RA, proriasis, iBD Src, Bcr-Abl Pfizer (US) 2011 Incyte /Novartis (US) 2011 Plexxikon/Daiichi/Roche (US) 2011 JAK3 /Pfizer (US) 2012 Pfizer (US) 2012 N HH O NN Cl O O N ONO H H H O Cl N N N N F C N N MeO N 3 H H S H F O O O F (AG013736) MeO NTivopath () HN O MeO Renal cell carcinoma Cometriq () renal cancer VEGFR1,2,3, PDGFR,cKit Stivarga () metastatic medullary thyroid cancer VEGFRs Pfizer (US) 2012 anti-angiogenic MeO N Ret, Met, Flt1, KDR, Kit, Axl, Tie2,... AVEO Pharm (US) 2012 VEGFR2, Tie2 Exelixis (US) 2012 Bayer (D) 2012 N F F F N covalent O N H S N N O N O O N S F N HN Cl N N CF O O H H 3 F N N N N HN H N O NH2 O N N I O N O Iclusig (, AP24534) Tafinlar () Mekinist () Gilotrif () CML, ALL metastatic melanoma metastatic melanoma NSCLC, breast cancer multi-target TK inhibitor b-RAF (V600E) mutant MEK EGFRs ARIAD Pharma. (US) 2012 GSK (UK) 2013 GSK (UK) 2013 Boehringer Ingelheim (D) 2013

6 35 Approved Kinase Inhibitors - III Wave of low-molecular weight compounds Cl N N F O N H covalent O N 2 HN N NH O O N N N O S N N O HN N Zydelig () Imbruvica () Pulmonary fibrosis Zykadia () CLL mantle cell lymphoma HN N PDGFR, VEGFR ALK-positive NSCLC PI3Kd BTK N Boehringer Ingelheim 2014 ALK, IGF-1R, InsR, ROS1 Gilead (US), 2014 Celera, Pharmacyclis, J&J (US) 2013 NH Novartis (CH), 2014

covalent O Cl HN O HN F 2 H I N O N N H O N O H N N N N N O O N H F N N Lenvima () H O Ibrance () H Tagrisso () Thyroid cancer F Breast cancer Astra Zeneca Multi kinase inhibitor, VEGF‘s Cotellic () CDK4,6 inhibitor Eisai (JP) 2015 TK (EGFR) Pfizer (US) 2015 Breast cancer Lung cancer MEK FDA-approved in November 2015 Exelixix, Roche (US) 2015 HN N O N

NNNNN H

Kisqali () Alecensa (alecitinib) Aliqopa () Verzenio (abemaciclib, LY2835219) Hoffmann-La Roche Novartis Bayer CDK4/6 inhibitor Eli Lilly Multi-kinase inhibitor (ALK, RET) Follicular Lymphoma adv metastatic breast cancer ALK-positive non-small lung cancer combi with – breast PI3K-inhibitor approved March 2017 CDK4/6-inhibitor FDA-approved in December 2015 Approved Sep 2017 Approved Sep 2017

7 Dominating Inhibitor Design Paradigm  type I inhibitors Long tradition: Traxler scheme Bioorg. Med. Chem. Lett. 23, 1238–1244 (2013)

N-terminal domain E470 O H H HN N Cl L471 N O hinge N N H N L472 N O IC50: 0.6 µM

OH

ATP-binding cleft H H N

N N

N O H IC : 40 nM modified from: MeO 50 P. Furet et al. J. Comp.-Aid. Mol Des. 1995, 9, 465-472 P. Traxler, Exp.Opin. Ther. Patents 1998, 8, 1599-1625

C-terminal domain

NF-kB-inducing kinase (NIK) inhibitors – , US 8 DFG-in, -out, -undefined: spine functional connection between C- and N-lobe: dynamics

Cl PD173955 : c-Abl N O Gleevec : c-Abl N N N N N N Cl H H S N N N O H N

competitive inhibition of active state stabilizing inactive kinase conformation type I type II DFG-in DFG-out

1m52.pdb 1iep.pdb

DFGin DFGout

A.P. Kornev, N. M. Haste, S. S. Taylor, L. F. Ten Eyck, Proc. Natl. Acad. Sci., 103, 17783 (2006) • mutations/inhibitors change dynamic infrastructure of kinases A.P. Kornev, S. S. Taylor, L. F. Ten Eyck, Proc. Natl. Acad. Sci., 105, 14377 (2008) • structure and function are mediated by dynamics, even when A.P. Kornev, S. S. Taylor, Biochim. Biophys. Acta, 1804, 440-444 (2010) major structural changes are not apparent H.S. Meharena, et al, PLOS Biology, 11 (10), 1-11 (2013)

9 Sorafenib:CDK8 vs. Lapatinib:EGFR Various mechanisms to disturb intact spine arrangement type II (DMG-out) C-helix shift – type ?

Cl O O S F O C F 3 H N O O N H O HN Lapatinib: off-rate: 0.0023 min-1 N O N N Cl N -1 H H (koff) = RT = 435 min N Sorafenib IC50 (CDK8): 74 nM (k )-1 = RT = 576 min off J.Mol.Biol. 412, 251-266 (2011) Cancer Research, 64, 6652-59 (2004)

10 CDK8 – Transcription Regulation CDK8 deregulated in solid tumors: Colon // Gastric // Melanoma // Breast // AML

PharmacologyHN & Therapeutics, 2017 inO press N N

N N N N O CMGC: H CDK, MAPK, Palbociclib GSK, CLK families) approved 2015 (Pfizer)

HN N O N

NNNNN H CDK8:Cyclin C transiently associated with the Ribociclib 1.2 MDa Mediator complex (regulates transcription through RNA polymerase approved 2017 (Novartis) II interaction) combi with letrozole (aromatase inh)

Two major groups of CDKs: • regulating CDKs 1-6, 11, 14-18 Abemaciclib • transcription regulators Approved Sept 2017 (Eli Lilly) 7-13, 19, 20

11 Sorafenib inhibits CDK8 (in 2011) Retro-Design: targeting conformational states by deep pocket-directed scaffolds

traditional type I CDK inhibitors novel type II CDK inhibitors Retro-Design Approach: new artifacts that refer to ➢ ATP competitive inhibitors ➢ acting through induced fit mechanism Sets out with type III inhibitors particular modes, motifs, models, ➢ accomodated in ATP binding site ➢ accomodated in deep pocket ▪ Disrupt hydrophobic spine techniques, and materials of the ➢ no binding kinetic signature ➢ hydrophobic R spine disrupted ▪ Long residence time on target; slow k past. off ➢ hydrophobic R spine intact -1 ▪ Option for exploration of novel IP space (koff) = RT = 576 min

deep pocket

4acm.pdb Elisabeth Schneider 3rgf.pdb J.Mol.Biol. 412, 251-266 (2011)

12 Examples: “Retro-Design“ Approach Fragment-based lead generation – seed fragment positioned in deep pocket ACS Med. Chem. Lett. 3, 342 (2012), Takeda Bioorg. Med. Chem. Lett. 23, 2962 (2013) ACS Med. Chem. Lett. 6, 798-803 (2015) fragment screen against Bankog University, Scripps Res. Inst. Astex non-phosphorylated VEGFR2 retro-design – FBLG – DDR2 VEGFR2: back-to-front Cl counter-screen: phosphorylated form H H N N N O N 3 X P-o-C study: CF 3 Y Cl O N N A R O 4.4 mM (non-P) H H 32% inh @ 100 mM O N N > 100 mM (P) H H N N N H H N Cl H broader fragment screen: N O O IC50: 3 nM F O O N OH H Ki: 25 mM (non-P) N N N CF H 3 N O IC50: 6 nM; 55 %F

O H N N O O N H Cl N N N N H H N N N N ~ 0.5 mM N IC50: 6 nM; 97 %F F H 3vhe.pdb

13 Urea/Amide Replacements Application of traditional medicinal chemistry guidelines

R R R XX XN N X X N() O N X X X R X N()X don N N X H H

X X X

N()R N N X N prox dist X R X R X X X X N() don

X N R N R magci N R magic (N) m a gic X don O X X X X X X N H R goal: novelty = no prior art

XR NX N N magci R magci N() close magci R XN() X don contract rings X expand X X

14 Back-to-Front Approach From deep pocket-directed fragment to early lead structure • 9 novel scaffolds  200 analogs prepared

HH ONNCF 3 Retro-Fragments bind ! O Cl (presumably by disrupting the R-spine) • Incorporate hinge-binding functionality: proof of design concept MC85 O IC50: 15 nM HN MW: 463 H R clogP: 3.9 O #HBA: 8 I N H N R #HBD: 2 G O TPSA: 100 E HN #rbs: 5 Different hinge binders: steep SAR R

15 Rich SAR Findings in the Deep Pocket High sensitivity to sterics and electronics – steep SAR

loss of cellular efficacy difluoro restores Efflux? efficacy increased cell. efficacy (factor 8-9)

increased increased cell. efficacy cell. efficacy (factor 3) (factor 25)

smaller rings unfavorable? difluoro restores efficacy

16 In-vivo proof-of-concept with lead candidate Project ready for Lead Optimization – Mercachem seeking a partner CDK-panel MC116

Kinase IC50 (µM) Assay Result Kd kon koff residence time CDK1/CycA2 >10 [µM] [1/s 1/M] [1/s] [min] Biochem. IC50 6 nM CDK1/CycB1 >10 9.55E-04 2.54E+04 2.43E-05 687 CDK1/CycE1 1.182 Cell. (HCT116) 1300 nM CDK2/CycA2 >10 CDK2/CycD1 0.043 Cell. (DLD1) 1400 nM Inhibition of STAT1-phosphorylation on Ser727* CDK2/CycE1 >10 Cell. (SW620) 2600 nM (in HCT116 colon cancer cell lines) CDK3/CycC 0.232 CDK3/CycE1 0.740 CDK4/CycD1 0.864 CDK4/CycD2 2.045 IV (2 mg/kg) PO (10 mg/kg) CDK4/CycD3 2.210 Comp. AUC Total CL AUC C (%) inf t (h) inf max t (h) CDK5/p25NCK >10 (µg·hr/mL) 1/2 (mL/min/kg) (µg·hr/mL) (µg/mL) max CDK5/p35NCK >10 CDK6/CycD1 3.236 MC116 0.93 1.36 36.19 2.01 0.88 0.52 31.8 CDK6/CycD2 0.957 CDK6/CycD3 0.657 CDK7/CycH/MAT1 >10 600 Vehicle control

CDK8/CycC 0.018 )

3 ]

HCT116: 3 SW620: Competitor reference (70mg/kg bid) CDK9/CycK 4.728 600 CDK9/CycT1 5.794 400 MC116.TsOH (150mg/kg bid) CDK12 R722C/CycK >10 400 HCT116 SW620 CDK12wt/CycK 7.692 MC116 CDK13/CycK 7.047 200 TGI% TGI% CDK16/CycY 0.406 200

tumor volume [mm volume tumor volume 67 45 CDK17/p35NCK 0.438 (mm tumor volume CDK19/CycC >10 0 0 weight 59 45 CDK20/CycH >10 0 10 20 30 0 10 20 30 day CDK20/CycT1 >10 day

17 Structure-Kinetic Relationships Residence times from 2 hours to 2 days – kinetic selectivity

10000 2613 3852 1000 687

335 248 489 )

off 126

k 100 -1 -1

(1/ 2.75E+04 s M 2.54E+04 10

2.62E+04 Residence time[min] Residence 1 soraf MC085 MC147 MC111 MC103 MC116 MC100

2.5 days BIRB-796 p38

IC50: 8 nM 12 nM 6 nM

18 Structure-Kinetic Relationships Residence times against a kinase panel

• off-target effect of weakly active compound underestimated if residence time is long • off-target effect of highly active compound overestimated if residence time is short

19 Multi-step processes Escape Trajectory: retrograde induced-fit mechanims of dissociation

Target:Ligand‡

Target*:Ligand‡

G binding G 

Target

Target:Ligand

Target*:Ligand

with appreciation to Bob Copeland Escape Trajectory

20 Slow k-off inhibitors Not limited to distortion of hydrophobic R-spine

O OH O H N N N N N biochemical NH NH NH 2 Akt 1 - IC : 2 nM N 2 N 50 F N N Akt 2 - IC : 13 nM IC50 IGF-1R: 0.6 nM 50 O N O N N N Akt 3 - IC50: 9 nM N EC50 3T3/huIGF-1R: 4 nM O N N t IGR-1R: 133 h (!) ½ half-life: 38 min in Akt1 30 min in Akt2 F HO NH ACS Med. Chem. Lett. 4, 627-631 (2013) Caner R. 68, 2366-2374 (2008) AstraZeneca N N O O O S IC50 Jak2: < 3 nM N N IC50 AurB/INCENP: 15 nM N t½ Jak2: 6,6 h H after 1 hour @ [ATP] ~ 50 x Km: 1.7 nM

N N H H N -1 N AurB/INCENP: koff < 0.0014 min N O k 5.6 x 105 M-1s-1 t > 480 min = 8 h N on ½ H -5 -1 koff 1.3 x 10 s -1 AurC/INCENP: koff 0.0026 min → Mol. Cancer Ther. 12, 460-470 (2013) t½ = 920 min = 15.3 h t½ = 270 min = 4.5 h Biochem. J. 420, 259-265 (2009) 21 Histone-Deacetylases – HDAC’s – epigenetic target class HDAC family, mechanism, and first inhibitors

III class I: HDAC1, 2, 3, 8 class IIa: HDAC4, 5, 7, 9 IV class IIb: HDAC6, 10 2+ class IV: HDAC11 Zn dependent I

IIb class III:sirtuins SIRT1-7 NAD+ dependent IIa O O OH first inhibitor (Yoshida et al. 1990): N H Trichostatin A - anti-fungal antibiotic activity N O H N

HN O

22 HDAC inhibitor classes Predominant pharmacophoric feature: hydroxamate as Zinc2+ chelating group O OPharm. Pat. Analyst (2012) 1(1), 75–90 OH O N O O H H S OH O N N N N H H H O N NH N 2 H Etinostat – phase II HN – phase II – FDA-approved (Novartis) oncology, HDAC1, 2 oncology, HDAC1, 2, 3, 6 oncology, HDAC1, 2, 3, 6 O N O

N OH O N N H OH Br NH N H O O MeO OH O N OH H H N HO N H N N H N O H O O N OH O O N H H H N OH N N O H O

23 Change in kinetics based on structure modifications Hydroxamate-to-amino benzamide exchange J. Biol. Chem. 51, 35402 (2008)

Cellular efficacy after wash-out

H2N H N

O Induced fit H N 106 O

O OH N H

106: conformational switch H N SAHA (no conformational selection) Recovered HDAC3 O ()  Bindnig kinetic signature  cellular activity

24 Exploration of “Foot Pocket” Time-dependency in biochemical inhibition – opportunities for design Takeda, San Diego: J. C. Bressi et al., Bioorg. Med. Chem. Lett. 20, 3142-3145 (2010) Genentech: B. E. L. Lauffer et al., J. Biol. Chem. 288, 26926-26943 (2013)

HN O HN O HN O HN O

NH2 NH2 NH2 NH2 HN O

O Br F HDAC2 IC HDAC2 IC (24h) HN O R 50 50 NH (1h) [µM] [µM] 2 S phenyl 0.9 0.027 2-furanyl 1.8 0.043 4-Br-phenyl 24 0.6 4-F-phenyl 1.4 0.032 HDAC1 IC50: 10 nM

SAHA 0.076 0.072 HDAC2 IC50: 56 nM

1/koff (HDAC1): 1207 min

1/koff (HDAC2): 1259 min

25 Summary

➢ Protein kinases remain a true hot topic in medicinal chemistry we just start to understand … Learn to treasure structural insights but don’t be misled/seduced too much – Xray  lacks a time axis

➢ Detailed understanding/optimization of binding kinetics is key still a widely underappreciated parameter (design guidelines poorly understood) if we, as Medicinal Chemists are honest: “our understanding is pretty limited!”

➢ Targeting dynamic equilibria instead of a static entity (kinases and phosphatases and …) capture biomolecular targets in inactive states study molecular dynamics

➢ There are so many other drug targerts that display dynamics, transient pockets !!!

26 Summary

Necessary: Sufficient:

Bob Copeland (CSO Accent Therapeutics) 10th Swiss med chem course, Leysin, 2012

„any chemist reporting IC50‘s should be boiled in oil!“ E. Fischer‘s lock & key hypothesis consider dynamic effects optimize stereochemical and physicochemical fit optimize residence time for multiple reasons

27 ProQinase: Christoph Schächtele Michael Kubbutat Mercachem: Koen Hekking

28 9 February 2016