Michael DiDonato

Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors

EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harboring activating mutations in the EGFR kinase1,2, but resistance arises rapidly, most frequently due to the secondary T790M mutation within the ATP-site of the receptor.3,4 Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant5,6, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond7. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternate mechanisms of action. Here we describe rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild type receptor. A crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays, but as a single agent is not effective in blocking EGFR-driven proliferation in cells due to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state8. We observe dramatic synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization9,10, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by L858R/T790M EGFR and by L858R/T790M/C797S EGFR, a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.

Clinical stage EGFR inhibitors irreversibly alkylate Bmx Kinase

Irreversible HER/erbB inhibitors selectively inhibit HER-family kinases by targeting a unique cysteine residue located within the ATP-binding pocket. Sequence alignment reveals that this rare cysteine is also present in ten other protein kinases including all five Tec-family members. We demonstrate that the Tec-family kinase Bmx is potently inhibited by irreversible modification at Cys496 by clinical stage EGFR inhibitors such as CI-1033. This cross-reactivity may have significant clinical implications.

EGF816 Exerts Anticancer Effects in Non–Small Cell Lung Cancer by Irreversibly and Selectively Targeting Primary and Acquired Activating Mutations in the EGF Receptor

Non-small cell lung cancer patients carrying oncogenic EGFR mutations initially respond to EGFR-targeted therapy, but later elicit minimal response due to dose-limiting toxicities and acquired resistance. EGF816 is a novel, irreversible mutant-selective EGFR inhibitor that specifically targets EGFR-activating mutations arising de novo and upon resistance acquisition, while sparing wild-type (WT) EGFR. EGF816 potently inhibited the most common EGFR mutations L858R, Ex19del, and T790M in vitro, which translated into strong tumor regressions in vivo in several patient-derived xenograft models. Notably, EGF816 also demonstrated antitumor activity in an exon 20 insertion mutant model. At levels above efficacious doses, EGF816 treatment led to minimal inhibition of WT EGFR and was well tolerated. In single-dose studies, EGF816 provided sustained inhibition of EGFR phosphorylation, consistent with its ability for irreversible binding. Furthermore, combined treatment with EGF816 and INC280, a cMET inhibitor, resulted in durable antitumor efficacy in a xenograft model that initially developed resistance to first-generation EGFR inhibitors via cMET activation. Thus, we report the first preclinical characterization of EGF816 and provide the groundwork for its current evaluation in phase I/II clinical trials in patients harboring EGFR mutations, including T790M.

Crystal structure of the global regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a new fold

Chris Rife, Robert Schwarzenbacher, Daniel McMullan, Polat Abdubek, Eileen Ambing, Herbert Axelrod, Tanya Biorac, Jaume M. Canaves, Hsiu-Ju Chiu, Ashley M. Deacon, Michael DiDonato, Marc-André Elsliger, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Joanna Hale, Eric Hampton, Gye Won Han, Justin Haugen, Michael Hornsby, Lukasz Jaroszewski, Heath E. Klock, Eric Koesema, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Mitchell D. Miller, Kin Moy, Edward Nigoghossian, Jessica Paulsen, Kevin Quijano, Ron Reyes, Eric Sims, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Aprilfawn White, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson* The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The University of California, San Diego, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, California The Scripps Research Institute, La Jolla, California

A scaleable and integrated crystallization pipeline applied to mining the Thermotoga maritima proteome

The wealth of genomic data available for many organisms has set the stage for the next phase of structure—function analysis. High-throughput structural genomics is currently the method of choice for rapid analysis of protein structure—function relationships on a proteome-wide basis. The Joint Center for Structural Genomics (JCSG), established in 2000 under the NIH/NIGMS Protein Structure Initiative, has developed and implemented an integrated high-throughput structure pipeline and applied it in a 2-tiered approach to mining the proteome of the thermophilic bacterium Thermotoga maritima. In the first tier, the successful application of this integrated pipeline has resulted in the cloning and expression of 73% of the T. maritima proteome (1376 out of 1877 predicted genes), and has identified 465 proteins which produced crystal hits. These 465 proteins were compared with existing structural information and a subset of 269 targets were selected to process towards structure determination in a second tier effort. To date, the JCSG pipeline applied to the Thermotoga maritima proteome has resulted in 55 new structures and has identified 6 novel folds and continues to identify structures with novel features.

Crystal structure of acireductone dioxygenase (ARD) from Mus musculus at 2.06 Å resolution

Qingping Xu, Robert Schwarzenbacher, S. Sri Krishna, Daniel McMullan, Sanjay Agarwalla, Kevin Quijano, Polat Abdubek, Eileen Ambing, Herbert Axelrod, Tanya Biorac, Jaume M. Canaves, Hsiu-Ju Chiu, Marc-André Elsliger, Carina Grittini, Slawomir K. Grzechnik, Michael DiDonato, Joanna Hale, Eric Hampton, Gye Won Han, Justin Haugen, MichaelHornsby, Lukasz Jaroszewski, Heath E. Klock, Mark W. Knuth, Eric Koesema, Andreas Kreusch, Peter Kuhn, Mitchell D. Miller, Kin Moy, Edward Nigoghossian, Jessica Paulsen, Ron Reyes, Chris Rife, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Aprilfawn White, Guenter Wolf, Keith O. Hodgson, John Wooley, Ashley M. Deacon, Adam Godzik, Scott A. Lesley, and Ian A. Wilson* The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The University of California San Diego, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, California The Scripps Research Institute, La Jolla, California

Discovery of (R,E)-N-(7-Chloro-1-(1-[4-(dimethylamino)but-2-enoyl]azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (EGF816), a Novel, Potent, and WT Sparing Covalent Inhibitor of Oncogenic (L858R, ex19del) and Resistant (T790M) EGFR Mutants for the Treatment of EGFR Mutant Non-Small-Cell Lung Cancers.

Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.

Crystal structure of an indigoidine synthase A (IndA)‐like protein (TM1464) from Thermotoga maritima at 1.90 Å resolution reveals a new fold

Inna Levin, Mitchell D. Miller, Robert Schwarzenbacher, Daniel McMullan, Polat Abdubek, Eileen Ambing, Tanya Biorac, Jamison Cambell, Jaume M. Canaves, Hsiu-Ju Chiu, Ashley M. Deacon, Michael DiDonato, Marc-André Elsliger, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Joanna Hale, Eric Hampton, Gye Won Han, Justin Haugen, Michael Hornsby, Lukasz Jaroszewski, Cathy Karlak, Heath E. Klock, Eric Koesema, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Andrew Morse, Kin Moy, Edward Nigoghossian, Jie Ouyang, Rebecca Page, Kevin Quijano, Ron Reyes, Alyssa Robb, Eric Sims, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Xianhong Wang, Bill West, Guenter Wolf, Qingping Xu, Olga Zagnitko, Keith O. Hodgson, John Wooley, and Ian A. Wilson* Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California San Diego Supercomputer Center, La Jolla, California Genomics Institute of the Novartis Research Foundation, San Diego, California University of California, San Diego, La Jolla, California Scripps Research Institute, La Jolla, California

Crystal structure of a single‐stranded DNA‐binding protein (TM0604) from Thermotoga maritima at 2.60 Å resolution

Michael DiDonato, S. Sri Krishna, Robert Schwarzenbacher, Daniel McMullan, Lukasz Jaroszewski, Mitchell D. Miller, Polat Abdubek, Sanjay Agarwalla, Eileen Ambing, Herbert Axelrod, Tanya Biorac, Hsiu-Ju Chiu, Ashley M. Deacon, Marc-André Elsliger, Julie Feuerhelm, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Joanna Hale, Eric Hampton, Justin Haugen, Michael Hornsby, Heath E. Klock, Mark W. Knuth, Eric Koesema, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Kin Moy, Edward Nigoghossian, Linda Okach, Jessica Paulsen, Kevin Quijano, Ron Reyes, Chris Rife, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Aprilfawn White, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California San Diego Supercomputer Center, La Jolla, California Genomics Institute of the Novartis Research Foundation, San Diego, California University of California, San Diego, La Jolla, California The Scripps Research Institute, La Jolla, California

Crystal structure of Hsp33 chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution

Lukasz Jaroszewski, Robert Schwarzenbacher, Daniel McMullan, Polat Abdubek, Sanjay Agarwalla, Eileen Ambing, Herbert Axelrod, Tanya Biorac, Jaume M. Canaves, Hsiu-Ju Chiu, Ashley M. Deacon, Michael DiDonato, Marc-André Elsliger, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Joanna Hale, Eric Hampton, Gye Won Han, Justin Haugen, Michael Hornsby, Heath E. Klock, Eric Koesema, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Mitchell D. Miller, Kin Moy, Edward Nigoghossian, Jessica Paulsen, Kevin Quijano, Ron Reyes, Chris Rife, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Aprilfawn White, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson* The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The University of California, San Diego, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, California The Scripps Research Institute, La Jolla, California