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LETTER REPLY TO TRAN ET AL.: Dimeric KRAS protein protein interaction stabilizers Dirk Kessler a , Andreas Gollner a , Michael Gmachl a , Andreas Mantoulidis a , Laetitia J. Martin a , Andreas Zoephel a , Moriz Mayer a , David Covini a , Silke Fischer a , Thomas Gerstberger a , Teresa Gmaschitz a , Craig Goodwin b , Peter Greb a , Daniela Häring a , Wolfgang Hela a , Johann Hoffmann a , Jale Karolyi-Oezguer a , Petr Knesl a , Stefan Kornigg a , Manfred Koegl a , Roland Kousek a , Lyne Lamarre a , Franziska Moser c , Silvia Munico-Martinez a , Christoph Peinsipp a , Jason Phan b , Jörg Rinnenthal a , Jiqing Sai b , Christian Salamon a , Yvonne Scherbantin a , Katharina Schipany a , Renate Schnitzer a , Andreas Schrenk a , Bernadette Sharps a , Gabriella Siszler a , Qi Sun b , Alex Waterson d,e , Bernhard Wolkerstorfer a , Markus Zeeb c , Mark Pearson a , Stephen W. Fesik b,d,e , and Darryl B. McConnell a,1 We thank Tran et al. (1) for their appreciation of our open innovation platform (https://opnme.com/) and interest in our manuscript (2) in which we demonstrate that the small-molecule KRAS inhibitor BI-2852 re- duces pERK (EC 50 = 6 μM) and inhibits proliferation (EC 50 = 7 μM) in NCI-H358 cells. Tran et al. (1) propose that BI-2852 exerts its cellular effects on the MAPK pathway at least in part through stabilization of a non- functional KRAS dimer. No cellular data for KRAS di- mer induction are provided and the hypothesis is primarily based on KRAS dimer formation observed with size exclusion chromatography using concentra- tions 1,000 times higher (3 mM) than those in which the cellular effects of BI-2852 are observed. Hence, inhibiting the proteinprotein interactions between KRAS and its GEFs, GAPs, and downstream effectors is a more plausible explanation than induction of inactive KRAS dimers for the cellular mode of action for BI-2852 (2). However, we concur that stabilization of nonfunc- tional KRAS dimers as a potential therapeutic approach to treat KRAS-driven cancers is intriguing. Accordingly, we have synthesized a range of dimeric switch I/II pocket binders in analogy to linkerology (3) in the PROTAC field. We discovered compound 2, which dimerizes KRAS with a K D of 3.8 μM according to isothermal calorimetry measurements (Fig. 1E). Using our high- throughput crystal-soaking system for active KRAS G12D (4) (Fig. 1C), we obtained a dimeric KRAS structure with 2 (Fig. 1D) at a resolution of 1.9 Å (Fig. 1G), which repre- sents the inactivating effector lobe dimer form 1 iden- tified by Jang et al. (5) using molecular dynamics calculations. Cocrystallization of 2 with GCP-KRAS G12D leads to the same KRAS dimer as observed for BI-2852 (Fig. 1 A and B), which is the second effector lobe form proposed by Jang et al. (5) at a resolution of 1.57 Å (Fig. 1F). In order to investigate the potential of small- molecule stabilization of nonfunctional KRAS dimers for the treatment of KRAS-driven cancers, more perme- able and potent compounds will be needed to enable cellular studies. a Discovery Research, Boehringer Ingelheim RCV GmbH & Co KG, 1120 Vienna, Austria; b Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37235; c Discovery Research, Boehringer Ingelheim Pharma GmbH & Co KG, D-88397 Biberach an der Riss, Germany; d Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37235; and e Department of Chemistry, Vanderbilt University, Nashville, TN 37235 Author contributions: D.K., A.G., M.G., A.M., L.J.M., A.Z., M.M., T. Gerstberger, M.K., J.P., J.R., R.S., A.W., M.Z., M.P., S.W.F., and D.B.M. designed research; D.K., A.G., M.G., A.M., L.J.M., A.Z., M.M., D.C., S.F., T. Gerstberger, T. Gmaschitz, C.G., P.G., D.H., W.H., J.H., J.K.-O., P.K., S.K., M.K., R.K., L.L., F.M., S.M.-M., C.P., J.P., J.R., J.S., C.S., Y.S., K.S., R.S., A.S., B.S., G.S., Q.S., A.W., B.W., and M.Z. performed research; D.C., S.F., T. Gmaschitz, C.G., P.G., D.H., W.H., J.H., J.K.-O., P.K., S.K., R.K., L.L., F.M., S.M.-M., C.P., J.S., C.S., Y.S., K.S., A.S., B.S., G.S., Q.S., and B.W. contributed new reagents/analytic tools; D.K., A.G., M.G., L.J.M., A.Z., M.M., T. Gerstberger, M.K., J.P., J.R., R.S., A.W., M.Z., M.P., S.W.F., and D.B.M. analyzed data; and D.K. and D.B.M. wrote the paper. Competing interest statement: D.K., A.G., M.G., A.M., L.J.M., A.Z., M.M., D.C., S.F., T. Gerstberger, T. Gmaschitz, P.G., D.H., W.H., J.H., J.K.-O., P.K., S.K., M.K., R.K., L.L., F.M., S.M.-M., C.P., J.R., C.S., Y.S., K.S., R.S., A.S., B.S., G.S., B.W., M.Z., M.P., and D.B.M. were employees of Boehringer Ingelheim at the time of the work. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). 1 To whom correspondence should be addressed. Email: [email protected]. First published February 11, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1921236117 PNAS | February 18, 2020 | vol. 117 | no. 7 | 33653367 LETTER Downloaded by guest on September 2, 2020

DimericKRASprotein REPLY TO TRAN ET AL.: proteininteractionstabilizers LETTER · LETTER REPLY TO TRAN ET AL.: DimericKRASprotein–proteininteractionstabilizers Dirk Kessler a , Andreas

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Page 1: DimericKRASprotein REPLY TO TRAN ET AL.: proteininteractionstabilizers LETTER · LETTER REPLY TO TRAN ET AL.: DimericKRASprotein–proteininteractionstabilizers Dirk Kessler a , Andreas

LETTER

REPLY TO TRAN ET AL.:

Dimeric KRASprotein–protein interaction stabilizersDirk Kesslera, Andreas Gollnera, Michael Gmachla, Andreas Mantoulidisa, Laetitia J. Martina,Andreas Zoephela, Moriz Mayera, David Covinia, Silke Fischera, Thomas Gerstbergera, Teresa Gmaschitza,Craig Goodwinb, Peter Greba, Daniela Häringa, Wolfgang Helaa, Johann Hoffmanna, Jale Karolyi-Oezguera,Petr Knesla, Stefan Kornigga, Manfred Koegla, Roland Kouseka, Lyne Lamarrea, Franziska Moserc,Silvia Munico-Martineza, Christoph Peinsippa, Jason Phanb, Jörg Rinnenthala, Jiqing Saib, Christian Salamona,Yvonne Scherbantina, Katharina Schipanya, Renate Schnitzera, Andreas Schrenka, Bernadette Sharpsa,Gabriella Siszlera, Qi Sunb, Alex Watersond,e, Bernhard Wolkerstorfera, Markus Zeebc, Mark Pearsona,Stephen W. Fesikb,d,e, and Darryl B. McConnella,1

We thank Tran et al. (1) for their appreciation of ouropen innovation platform (https://opnme.com/) andinterest in our manuscript (2) in which we demonstratethat the small-molecule KRAS inhibitor BI-2852 re-duces pERK (EC50 = 6 μM) and inhibits proliferation(EC50 = 7 μM) in NCI-H358 cells. Tran et al. (1) proposethat BI-2852 exerts its cellular effects on the MAPKpathway at least in part through stabilization of a non-functional KRAS dimer. No cellular data for KRAS di-mer induction are provided and the hypothesis isprimarily based on KRAS dimer formation observedwith size exclusion chromatography using concentra-tions 1,000 times higher (3 mM) than those in whichthe cellular effects of BI-2852 are observed. Hence,inhibiting the protein–protein interactions between KRASand its GEFs, GAPs, and downstream effectors is a moreplausible explanation than induction of inactive KRASdimers for the cellular mode of action for BI-2852 (2).

However, we concur that stabilization of nonfunc-tional KRAS dimers as a potential therapeutic approach

to treat KRAS-driven cancers is intriguing. Accordingly,we have synthesized a range of dimeric switch I/II pocketbinders in analogy to linkerology (3) in the PROTACfield. We discovered compound 2, which dimerizesKRAS with a KD of 3.8 μM according to isothermalcalorimetry measurements (Fig. 1E). Using our high-throughput crystal-soaking system for active KRASG12D

(4) (Fig. 1C), we obtained a dimeric KRAS structure with 2(Fig. 1D) at a resolution of 1.9 Å (Fig. 1G), which repre-sents the inactivating effector lobe dimer form 1 iden-tified by Jang et al. (5) using molecular dynamicscalculations. Cocrystallization of 2 with GCP-KRASG12D

leads to the same KRAS dimer as observed for BI-2852(Fig. 1 A and B), which is the second effector lobe formproposed by Jang et al. (5) at a resolution of 1.57 Å (Fig.1F). In order to investigate the potential of small-molecule stabilization of nonfunctional KRAS dimersfor the treatment of KRAS-driven cancers, more perme-able and potent compounds will be needed to enablecellular studies.

aDiscovery Research, Boehringer Ingelheim RCV GmbH & Co KG, 1120 Vienna, Austria; bDepartment of Biochemistry, Vanderbilt University Schoolof Medicine, Nashville, TN 37235; cDiscovery Research, Boehringer Ingelheim Pharma GmbH & Co KG, D-88397 Biberach an der Riss, Germany;dDepartment of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37235; and eDepartment of Chemistry, VanderbiltUniversity, Nashville, TN 37235Author contributions: D.K., A.G., M.G., A.M., L.J.M., A.Z., M.M., T. Gerstberger, M.K., J.P., J.R., R.S., A.W., M.Z., M.P., S.W.F., and D.B.M. designedresearch; D.K., A.G., M.G., A.M., L.J.M., A.Z., M.M., D.C., S.F., T. Gerstberger, T. Gmaschitz, C.G., P.G., D.H., W.H., J.H., J.K.-O., P.K., S.K., M.K.,R.K., L.L., F.M., S.M.-M., C.P., J.P., J.R., J.S., C.S., Y.S., K.S., R.S., A.S., B.S., G.S., Q.S., A.W., B.W., and M.Z. performed research; D.C., S.F.,T. Gmaschitz, C.G., P.G., D.H., W.H., J.H., J.K.-O., P.K., S.K., R.K., L.L., F.M., S.M.-M., C.P., J.S., C.S., Y.S., K.S., A.S., B.S., G.S., Q.S., and B.W.contributed new reagents/analytic tools; D.K., A.G., M.G., L.J.M., A.Z., M.M., T. Gerstberger, M.K., J.P., J.R., R.S., A.W., M.Z., M.P., S.W.F.,and D.B.M. analyzed data; and D.K. and D.B.M. wrote the paper.Competing interest statement: D.K., A.G., M.G., A.M., L.J.M., A.Z., M.M., D.C., S.F., T. Gerstberger, T. Gmaschitz, P.G., D.H., W.H., J.H., J.K.-O.,P.K., S.K., M.K., R.K., L.L., F.M., S.M.-M., C.P., J.R., C.S., Y.S., K.S., R.S., A.S., B.S., G.S., B.W., M.Z., M.P., and D.B.M. were employees of BoehringerIngelheim at the time of the work.This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).1To whom correspondence should be addressed. Email: [email protected] published February 11, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.1921236117 PNAS | February 18, 2020 | vol. 117 | no. 7 | 3365–3367

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Fig. 1. KRAS dimers and biophysical validation. (A) KRAS G12D modeled dimer with BI-2852 (Protein Data Bank [PDB] ID code 6GJ8). The dimerinterface was generated with symmetry operations from the published structure, showing a β-sandwich interface. (B) Cocrystal structure of KRASG12D with compound 2 showing a similar β-sandwich interface as with BI-2852. (C) Published dimeric KRAS-G12D apo system (PDB ID code6QUU) with an extended β-sheet interface. (D) Structure of compound 2 soaked in published KRAS-G12D soaking system. The compound needsto adapt to the extended β-sheet interface. (E) Isothermal titration calorimetry experiments with compound 2 showing binding to KRAS-G12D.(F) 2FoFc density around cocrystallized compound 2 at 1.57-Å resolution showing unambiguous binding. The interacting amino acids of bothKRAS monomers are labeled. (G) 2FoFc density around compound 2 soaked in KRAS G12D at 1.9-Å resolution showing unambiguous binding ofthe ligand. The interacting amino acids of both KRAS monomers are labeled, respectively.

3366 | www.pnas.org/cgi/doi/10.1073/pnas.1921236117 Kessler et al.

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1 T. H. Tran et al., The small molecule BI-2852 induces a nonfunctional dimer of KRAS. Proc. Natl. Acad. Sci. U.S.A. 117, 3363–3364 (2020).2 D. Kessler et al., Drugging an undruggable pocket on KRAS. Proc. Natl. Acad. Sci. U.S.A. 116, 15823–15829 (2019).3 D. P. Bondeson, C. M. Crews, Targeted protein degradation by small molecules. Annu. Rev. Pharmacol. Toxicol. 57, 107–123 (2017).4 A. Bergner et al., KRAS binders hidden in nature. Chemistry 25, 12037–12041 (2019).5 H. Jang, S. Muratcioglu, A. Gursoy, O. Keskin, R. Nussinov, Membrane-associated Ras dimers are isoform-specific: K-Ras dimers differ from H-Ras dimers. Biochem.J. 473, 1719–1732 (2016).

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