2015 Chemical Biology for Target Validation and Chemical Proteomics for Target Validation Banner

Chemical proteomics is emerging as a powerful pre-clinical component for the identification and validation of novel drug targets. A substantial challenge in probing biological systems remains the identification of targets being perturbed and causing favorable phenotypic response. Critical to the success of discovery programs is the challenging aspect of deconvoluting targets and pathways elucidating the mechanism of action of chemical compounds. Advances in chemical proteomics and quantitative mass spectrometry, introducing high-throughput workflows for target deconvolution, disease pathway analysis and understanding cellular protein dynamics, have recently improved pre-clinical target validation.

Cambridge Healthtech Institute’s Inaurugal Chemical Proteomics for Target Validation meeting will gather an interdisciplinary collection of leaders to discuss these emerging tools and strategies to de-risk novel discovery initiatives

Thursday, June 11

12:00 pm Registration

2:00 Chairperson's Opening Remarks

Iván Cornella-Taracido, Ph.D., Senior Principal Scientist, Discovery Chemistry, Merck



2:05: BioPlex 1.0: An Orfeome-Based, Mass Spectrometry-Driven, Human Protein Interaction Network

StevenGygiSteven P. Gygi, Ph.D., Professor, Cell Biology, Harvard Medical School

A protein’s membership in a complex imparts its molecular interactions, cellular localization, and biological function. We report a scalable affinity-purification mass spectrometry (AP-MS) platform and identify interacting partners for 2,594 proteins in HEK293T cells. The resulting network (BioPlex 1.0) contains 23,744 interactions, 86% previously unknown, among 7,668 proteins. This lecture will highlight network’s construction and its insights into human disease. Within two years, the platform described here will be used to determine interacting partners from a complete pass through the Orfeome collection (~13,000 human genes).

2:50 Mass Spectrometry-Based Proteomics in Preclinical Drug Discovery

BernhardKusterBernhard Kuster, Ph.D., Professor, Co-Founder and Chair, Proteomics and Bioanalytics, Technische Universität München, OmicScouts GmbH

Preclinical stages in the drug discovery process require a multitude of biochemical and genetic assays in order to characterize the effects of drug candidates on cellular systems and model organisms. Dramatic technological improvements in mass spectrometry-based proteomic and chemical proteomic strategies substantially facilitate decision-making throughout the drug discovery process. Here, we highlight proteomic approaches suitable for preclinical drug discovery and illustrate the potential of exciting recent developments.

Selected Poster Presentations: 

3:35 Development of a Novel Protein Affinity Mass Spectrometric Assay for Lead Generation

Elisabeth Walker, Ph.D., Postdoctoral Fellow, University of Illinois at Chicago

3:50 Drug Target Identification Using Energetics-Based Chemical Proteomics Approaches

Ryenne Ogburn, Research Scientist, Department of Chemistry, Duke University

4:05 Refreshment Break in the Exhibit Hall with Poster Viewing



4:45 Proteomics as a Contributing Technology in Drug Discovery

Kieran-GeogheganKieran Geoghegan, Ph.D., Research Fellow, Pfizer, Inc.

The relationship of proteomics to drug discovery continues to be explored and developed. Hopes for a bounty of new targets derived from comparisons of healthy and diseased tissues may have faded, but in their place have come opportunities to define the functional properties of lead and candidate molecules. Among the trends driving interest in such approaches are the targeting of proteins with deep controlling effects on cell metabolism, a revived interest in drugs that form covalent bonds with their targets, and the need to deconvolute the action of potent compounds for which no molecular target is known. All aspects of the continuum existing between classical proteomics and chemical biology offer potential to elucidate highly valued new information about drug action.

5:15 Scoring Target Selectivity of Drugs in Live Cells by Thermal Proteome Profiling

Marcus-BantscheffMarcus Bantscheff, Ph.D., Head, Technology, Cellzome GmbH, a GSK Company

The thermal stability of proteins can be used to assess ligand binding in living cells. We have generalized this concept by determining the thermal profiles of more than 7,000 proteins in human cells by means of mass spectrometry. Monitoring the effects of small-molecule ligands on thermal profiles enables discovery of new inhibitor targets and a quantitative assessment of target and off-target occupancy. For example, we identified the heme biosynthesis enzyme ferrochelatase as a target of kinase inhibitors and suggest that its inhibition causes the phototoxicity observed with vemurafenib and alectinib. Thermal shifts can also be observed for downstream effectors of drug treatment, e.g., the Abl inhibitor dasatinib induced shifts in BCR-ABL pathway proteins, including CRK/CRKL. This presentation will provide an introduction to thermal proteome profiling as an unbiased measure of drug-target engagement and highlight complementarity to alternative approaches such as affinity-based chemoproteomics.

5:45 Proteomics-Based Methods for In-Depth Analysis of Key Molecular Events in Tumorogenesis

Jarrod-MartoJarrod Marto, Ph.D., Associate Professor, Department of Biological Chemistry and Molecular Pharmacology, Dana-Farber

Proteomics-based methods provide a highly parallel readout of multiple biologically relevant events in a single experiment. Collectively these data provide a detailed view of key molecular mechanisms in cancer initiation and progression and can also facilitate drug target discovery and improved characterization of small molecule-based therapeutics.

6:15 Close of Day

Friday, June 12

7:30 am Interactive Breakout Discussion Groups with Continental Breakfast

Each discussion group in this session is led by a moderator/s who ensures focused conversations around key issues. Attendees join a specific group and the small, informal setting facilitates sharing of ideas and active networking.

TABLE 16: Uncovering Current Challenges and Technical Limitations in Chemical Proteomics


Marcus Bantscheff, Ph.D., Head, Technology, Cellzome GmbH, a GSK Company

  • What is the accessible target space, what can be covered with these techniques and what are we missing?
  • Target and off-target engagement in live primary cells and tissues, aren’t we there yet?
  • Impact of chemical proteomics on toxicology, and safety predictions
  • The challenge of linking the binding of a drug to a target or off-target to a clinical, toxicological, cellular phenotype

TABLE 17: Assessing the Chemoproteomic Landscape in Drug Discovery


Markus Schirle, Ph.D., Senior Investigator, Developmental and Molecular Pathways/Chemogenetics, Novartis Institutes for BioMedical Research

  • In what areas do scientists see chemoproteomics having the most impact?
  • De novo target deconvolution vs. off-/tox-target ID vs. target engagement/PD vs. screening applications?
  • Is it a stand-alone solution or is Chemoproteomics used within a larger, multi-pronged efforts?
  • What are gaps and blind spots for the various variants of Chemoproteomics and how do people approach these?




8:35 Chairperson’s Remarks

Alexander Statsyuk, Ph.D., Assistant Professor, Chemistry, Northwestern University


8:45 Chemical Proteomic Strategies to Investigate Reactive Cysteines

Eranthie-WeerapanaEranthie Weerapana, Ph.D., Assistant Professor, Chemistry, Boston College

Cysteine residues play diverse functional roles in proteins, including catalysis, metal-binding, structural stabilization and redox regulation. These functional cysteines are highly reactive and can be targeted by irreversible inhibitors. We aim to identify new functional cysteines within the human proteome and develop chemical probes to covalently modify these residues. To achieve this, we have developed a chemical-proteomic platform to identify cysteines that are highly sensitive to S-nitrosation, which is a posttranslational modification known to regulate protein activity and localization. We identified several previously unannotated cysteines and demonstrate that they allosterically regulate protein activity through S-nitrosation. In order to develop chemical probes to modify these and other functional cysteines in the proteome, a library of cysteine-reactive small-molecules was generated. Our initial library is based on a trifunctionalized 1,3,5-triazine scaffold, from which a potent and selective cysteine-reactive inhibitor for protein disulfide isomerase was identified. Our studies illustrate the potential of irreversible cysteine-targeted inhibitors as pharmacological agents for a large subsection of the proteome.

9:15 Serendipitous Discovery of the Selective Inhibitor of the Ubiquitin System Using Chemoproteomic Approaches

Alexander-SatsyukAlexander Statsyuk, Ph.D., Assistant Professor, Chemistry, Northwestern University

While trying to design chemoproteomic probes for UBL proteins based on covalent Nedd8 E1 enzyme inhibitor MLN4924, we discovered a molecule ABP3 that potently and covalently labeled ubiquitin and Nedd8 proteins inside A549 cells. The key to this discovery was the use of click chemistry that allowed us to visualize and identify protein targets of ABP3, due to the presence of an alkyne tag in the molecule. Subsequent follow up experiments showed that ABP3 is a potent inhibitor of Nedd8 ubiquitin conjugation in cells, but not SUMO, ISG15, and Ufm1 conjugation. We have found that ABP3 leads to a complete inhibition of the ubiquitin system in cells, including CLR E3s enzymes that require Nedd8 conjugation for their activation, and stimulates apoptosis. We have shown that in contrast to proteasome inhibitors inhibitors of the ubiquitin system do not induce the formation of aggressomes which are believed to be a critical factor limiting clinical efficacy of proteasome inhibitors. We believe that the developed inhibitor of the ubiquitin system will serve as a useful tool compound to study the function of the ubiquitin system.

Selected Poster Presentations: 

9:45 Identification of Novel Targets of CDK4/6 Inhibitors in Squamous Cell Lung Cancer by Chemical Proteomics

Natalia Sumi, Research Scientist, H Lee Moffitt Cancer Center & Research Institute

10:00 Functional Proteomics of Matrix Metalloproteases in a Model of Osteoarthritis

Ravindra Kodihalli, Ph.D., Research Scientist, Biological Engineering, MIT

10:15 Coffee Break in the Exhibit Hall with Poster Viewing



11:00 Towards Comprehensive Coverage of Drug Target Space in Chemical Proteomics

Markus-SchirleMarkus Schirle, Ph.D., Senior Investigator, Developmental and Molecular Pathways/Chemogenetics, Novartis Institutes for BioMedical Research

Quantitative chemical proteomics has emerged as a powerful affinity-based approach for the elucidation of cellular targets of bioactive compounds, e.g. hits from phenotypic screens, by generation of protein interaction profiles for the compound under investigation in a disease-relevant cellular background. Standard lysate-based, non-covalent approaches have been highly successful for certain target classes, including kinases and other enzyme families and in particular soluble members. However, they have a significantly lower success rate for important target classes such as ion channels and G-protein coupled receptors that require the intact cellular environment for compound-binding competence. In these cases, covalent strategies such as photocrosslinking-based experiments using live cell treatment have proven to be successful but require careful experimental design and optimization. Our efforts towards a comprehensive chemical proteomics strategy for de-novo target deconvolution that includes covalent and non-covalent approaches will be presented.

11:30 Case Studies in Target Identification and Mechanism of Action in Drug Discovery

Monica-SchenoneMonica Schenone, Ph.D., Technical and Scientific Leader, Biochemical Target ID, Proteomics Platform, Broad Institute

12:00 pm Small Molecule Profiling by Protein Stability-Based Interaction Proteomics (ProSIP)

Kilian-HuberKilian Huber, Ph.D., Senior Fellow, Giulio Superti-Furga Laboratory, CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences

Phenotypic screens provide an exciting and orthogonal approach to target-based drug discovery. However, target deconvolution remains a major challenge in this area and has precluded both academia and the pharmaceutical industry from conducting such screening campaigns. Our novel approach not only overcomes many limitations of classical target identification approaches such as extensive chemical derivatization but also allows for a proteome-wide survey of small molecule targets in intact living cells considering physiological context and thus important parameters such as drug uptake, metabolism and drug access to selected cellular compartments. We report an unbiased systems-level approach to monitor small-molecule target engagement in live cells based on protein thermal stability and quantitative mass spectrometry. The procedure does not require chemical modification of the compound of interest and constitutes a powerful means to assess target binding across different protein classes in a physiological context. Protein Stability-based Interaction Proteomics (ProSIP) should allow for the systematic mapping of chemical agents, including metabolites, to their natural partners.

12:30 A GLP-1R Positive Allosteric Modulator Acts through Covalent Modification

Whitney NolteWhitney Nolte, Ph.D., Senior Scientist, Medicinal Chemistry, Pfizer

The glucagon-like peptide-1 receptor (GLP-1R) is an important target for the treatment of type 2 diabetes. GLP-1 binds the GLP-1R and promotes glucose-dependent insulin secretion. Recent studies have identified several small molecule potentiators of the GLP-1R. The focus of these studies is to investigate the mechanism of action of one of the most studied GLP-1R potentiators, 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), which potentiates the signaling of weak agonists at the receptor. BETP is a reactive electrophile, making covalent modification of the receptor a potential mechanism for its action. Studies using mass spectrometry-based proteomics identified Cys347 and Cys438 as sites at which BETP modifies the GLP-1R. The functional relevance of the modification at Cys347 and not Cys438 was established through site-directed mutagenesis. Full agonists retained activity at the Cys347Ala mutant of GLP-1R; however, BETP no longer potentiated the function of the weak agonist, GLP-1(9-36)NH2. These results illustrate that BETP covalently modifies Cys347 in intracellular loop 3 (ICL3) of GLP-1R and that this modification is necessary for its potentiation of GLP-1R activity. We further show that introduction of a cysteine residue in ICL3 of the glucagon receptor sensitizes it to ligand potentiation by BETP. These results highlight the potential relevance of this site in ICL3 to enhancing the activity of class B GPCRs and may facilitate the discovery of new small molecule modulators.

1:00 Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own

1:30 Session Break



2:00 Chairperson’s Remarks

Andrea M. Zuhl, Ph.D., Fellow, Neuroscience, Pfizer; Former Research Associate, Cravatt Lab, The Scripps Research Institute


2:05 Tandem Photoaffinity Labeling - Bioorthogonal Conjugation in Medicinal Chemistry

David-LapinskyDavid Lapinsky, Ph.D., Associate Professor, Medicinal Chemistry, Division of Pharmaceutical Sciences, Duquesne University

Photoaffinity labeling has a longstanding history as a powerful biochemical technique. However, photoaffinity labeling has significantly evolved over the past decade principally due to its coupling with bioorthogonal/click chemistry reactions. This talk will aim to highlight recent applications of tandem photoaffinity labeling–bioorthogonal conjugation as a powerful and versatile chemical approach in medicinal chemistry and chemical biology. In particular, recent applications of this strategy towards affinity-based protein profiling (AfBPP), drug target identification, binding ensemble profiling, studying endogenous biological molecules, and imaging applications will be presented. Additionally, recent advances in the development of ‘all-in-one’ compact moieties possessing a photoreactive group and clickable handle will be presented.

2:35 A Modular and Traceless Chemical Method to Locate and Track Endogenous Protein Targets in Live Cells

James-ChambersJames Chambers, Ph.D., Assistant Professor, Chemistry, University of Massachusetts, Amherst

I will describe out rationale for designing a traceless, chemistry-based probe that allows for tagging endogenous receptors on neurons. The probe combines elements of medicinal chemistry, bio-conjugation, chemical biology, and neurobiology. I will provide a detailed discussion of our design and implementation for our first probe that was targeted to glutamate-gated AMPA receptors. I will then discuss our present efforts to modularize the system.


3:05 Chemoproteomic Profiling Reveals Cathepsin D Off-Target Activity Drives Ocular Toxicity of β-Secretase Inhibitors

Andrea M. Zuhl, Ph.D., Fellow, Neuroscience, Pfizer; Former Research Associate, Cravatt Lab, The Scripps Research Institute

Inhibition of β-secretase BACE1 is considered one of the most promising approaches for treating Alzheimer’s disease. Several structurally distinct BACE1 inhibitors have been withdrawn from development after inducing ocular toxicity in animal models, but the target mediating this toxicity has not been identified. Here we use a clickable photoaffinity probe to identify cathepsin D (CatD) as a principal off-target of BACE1 inhibitors in human cells. We found that several BACE1 inhibitors blocked CatD activity in cells with much greater potency than that displayed in cell-free assays with purified protein. Through a series of exploratory toxicology studies, we show that quantifying CatD target engagement in cells with the probe is predictive of ocular toxicity in vivo. Taken together, our findings designate CatD as a principal driver of ocular toxicity for BACE1 inhibitors and more generally underscore the power of chemical proteomics for discerning mechanisms of drug action.

3:35 Close of Conference