Conformation-sensing antibodies stabilize the oxidized form of PTP1B and inhibit its phosphatase activity.

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and leptin signaling and is an established therapeutic target for diabetes and obesity. PTP1B is regulated by reactive oxygen species (ROS) produced in response to various stimuli, including insulin. The reversibly oxidized form of the enzyme (PTP1B-OX) is inactive and undergoes profound conformational changes at the active site. We generated conformation-sensor antibodies, in the form of single-chain variable fragments (scFvs), that stabilize PTP1B-OX and thereby inhibit its phosphatase function. Expression of conformation-sensor scFvs as intracellular antibodies (intrabodies) enhanced insulin-induced tyrosyl phosphorylation of the ß subunit of the insulin receptor and its substrate IRS-1 and increased insulin-induced phosphorylation of PKB/AKT. Our data suggest that stabilization of the oxidized, inactive form of PTP1B with appropriate therapeutic molecules may offer a paradigm for phosphatase drug development.

Design of BET Inhibitor Bottlebrush Prodrugs with Superior Efficacy and Devoid of Systemic Toxicities.

Prodrugs engineered for preferential activation in diseased versus normal tissues offer immense potential to improve the therapeutic indexes (TIs) of preclinical and clinical-stage active pharmaceutical ingredients that either cannot be developed otherwise or whose efficacy or tolerability it is highly desirable to improve. Such approaches, however, often suffer from trial-and-error design, precluding predictive synthesis and optimization. Here, using bromodomain and extra-terminal (BET) protein inhibitors (BETi)-a class of epigenetic regulators with proven anticancer potential but clinical development hindered in large part by narrow TIs-we introduce a macromolecular prodrug platform that overcomes these challenges. Through tuning of traceless linkers appended to a "bottlebrush prodrug" scaffold, we demonstrate correlation of in vitro prodrug activation kinetics with in vivo tumor pharmacokinetics, enabling the predictive design of novel BETi prodrugs with enhanced antitumor efficacies and devoid of dose-limiting toxicities in a syngeneic triple-negative breast cancer murine model. This work may have immediate clinical implications, introducing a platform for predictive prodrug design and potentially overcoming hurdles in drug development.

Cross-talk between chromatin acetylation and SUMOylation of tripartite motif-containing protein 24 (TRIM24) impacts cell adhesion.

Proteins with domains that recognize and bind post-translational modifications (PTMs) of histones are collectively termed epigenetic readers. Numerous interactions between specific reader protein domains and histone PTMs and their regulatory outcomes have been reported, but little is known about how reader proteins may in turn be modulated by these interactions. Tripartite motif-containing protein 24 (TRIM24) is a histone reader aberrantly expressed in multiple cancers. Here, our investigation revealed functional cross-talk between histone acetylation and TRIM24 SUMOylation. Binding of TRIM24 to chromatin via its tandem PHD-bromodomain, which recognizes unmethylated lysine 4 and acetylated lysine 23 of histone H3 (H3K4me0/K23ac), led to TRIM24 SUMOylation at lysine residues 723 and 741. Inactivation of the bromodomain, either by mutation or with a small-molecule inhibitor, IACS-9571, abolished TRIM24 SUMOylation. Conversely, inhibition of histone deacetylation markedly increased TRIM24's interaction with chromatin and its SUMOylation. Of note, gene expression profiling of MCF7 cells expressing WT versus SUMO-deficient TRIM24 identified cell adhesion as the major pathway regulated by the cross-talk between chromatin acetylation and TRIM24 SUMOylation. In conclusion, our findings establish a new link between histone H3 acetylation and SUMOylation of the reader protein TRIM24, a functional connection that may bear on TRIM24's oncogenic function and may inform future studies of PTM cross-talk between histones and epigenetic regulators.

Observed bromodomain flexibility reveals histone peptide- and small molecule ligand-compatible forms of ATAD2.

Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2 (ATPase family AAA domain-containing 2 isoform A) in cancer cells is associated with poor prognosis making the bromodomain of ATAD2 a promising epigenetic therapeutic target. In the development of an in vitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2 bromodomain. Structural studies on apo-, peptide-and small molecule-ATAD2 complexes (by co-crystallization) revealed that the bromodomain adopts a 'closed', histone-compatible conformation and a more 'open' ligand-compatible conformation of the binding site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2 binders which aided in the validation of the in vitro screen and in the analysis of these conformational studies.

Ubiquitinated PCNA Drives USP1 Synthetic Lethality in Cancer.

CRISPR Cas9-based screening is a powerful approach for identifying and characterizing novel drug targets. Here, we elucidate the synthetic lethal mechanism of deubiquitinating enzyme USP1 in cancers with underlying DNA damage vulnerabilities, specifically BRCA1/2 mutant tumors and a subset of BRCA1/2 wild-type (WT) tumors. In sensitive cells, pharmacologic inhibition of USP1 leads to decreased DNA synthesis concomitant with S-phase-specific DNA damage. Genome-wide CRISPR-Cas9 screens identify RAD18 and UBE2K, which promote PCNA mono- and polyubiquitination respectively, as mediators of USP1 dependency. The accumulation of mono- and polyubiquitinated PCNA following USP1 inhibition is associated with reduced PCNA protein levels. Ectopic expression of WT or ubiquitin-dead K164R PCNA reverses USP1 inhibitor sensitivity. Our results show, for the first time, that USP1 dependency hinges on the aberrant processing of mono- and polyubiquitinated PCNA. Moreover, this mechanism of USP1 dependency extends beyond BRCA1/2 mutant tumors to selected BRCA1/2 WT cancer cell lines enriched in ovarian and lung lineages. We further show PARP and USP1 inhibition are strongly synergistic in BRCA1/2 mutant tumors. We postulate USP1 dependency unveils a previously uncharacterized vulnerability linked to posttranslational modifications of PCNA. Taken together, USP1 inhibition may represent a novel therapeutic strategy for BRCA1/2 mutant tumors and a subset of BRCA1/2 WT tumors.

Genetic and pharmacological inhibition of PDK1 in cancer cells: characterization of a selective allosteric kinase inhibitor.

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.

VRK1 Is a Synthetic-Lethal Target in VRK2-Deficient Glioblastoma.

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic-lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2-M arrest and DNA damage. The VRK1-VRK2 synthetic-lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line-derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM. SIGNIFICANCE: A paralog synthetic-lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

Discovery of PDK1 kinase inhibitors with a novel mechanism of action by ultrahigh throughput screening.

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K(m) values were determined as 5.6 mum for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1x and 10x K(m) concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.

Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs.

At present there are no drugs for the treatment of chronic liver fibrosis that have been approved by the Food and Drug administration of the United States. Telmisartan, a small-molecule antihypertensive drug, displays antifibrotic activity, but its clinical use is limited because it causes systemic hypotension. Here, we report the scalable and convergent synthesis of macromolecular telmisartan prodrugs optimized for preferential release in diseased liver tissue. We optimized the release of active telmisartan in fibrotic liver to be depot-like (that is, a constant therapeutic concentration) through the molecular design of telmisartan brush-arm star polymers, and show that these lead to improved efficacy and to the avoidance of dose-limiting hypotension in both metabolically and chemically induced mouse models of hepatic fibrosis, as determined by histopathology, enzyme levels in the liver, intact-tissue protein markers, hepatocyte necrosis protection, and gene-expression analyses. In rats and dogs, the prodrugs are retained long-term in liver tissue and have a well-tolerated safety profile. Our findings support the further development of telmisartan prodrugs that enable infrequent dosing in the treatment of liver fibrosis.

Publisher Correction: Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs.

In the version of this Article originally published, the author Peter Blume-Jensen was not denoted as a corresponding author; this has now been amended and the author's email address has been added. The 'Correspondence and requests for materials' statement was similarly affected and has now been updated with the author's initials 'P.B-J.'

TRIM28 multi-domain protein regulates cancer stem cell population in breast tumor development.

The expression of Tripartite motif-containing protein 28 (TRIM28)/Krüppel-associated box (KRAB)-associated protein 1 (KAP1), is elevated in at least 14 tumor types, including solid and hematopoietic tumors. High level of TRIM28 is associated with triple-negative subtype of breast cancer (TNBC), which shows higher aggressiveness and lower survival rates. Interestingly, TRIM28 is essential for maintaining the pluripotent phenotype in embryonic stem cells. Following on that finding, we evaluated the role of TRIM28 protein in the regulation of breast cancer stem cells (CSC) populations and tumorigenesis in vitro and in vivo. Downregulation of TRIM28 expression in xenografts led to deceased expression of pluripotency and mesenchymal markers, as well as inhibition of signaling pathways involved in the complex mechanism of CSC maintenance. Moreover, TRIM28 depletion reduced the ability of cancer cells to induce tumor growth when subcutaneously injected in limiting dilutions. Our data demonstrate that the downregulation of TRIM28 gene expression reduced the ability of CSCs to self-renew that resulted in significant reduction of tumor growth. Loss of function of TRIM28 leads to dysregulation of cell cycle, cellular response to stress, cancer cell metabolism, and inhibition of oxidative phosphorylation. All these mechanisms directly regulate maintenance of CSC population. Our original results revealed the role of the TRIM28 in regulating the CSC population in breast cancer. These findings may pave the way to novel and more effective therapies targeting cancer stem cells in breast tumors.

Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context.

The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway.

Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor.

The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and ITC Kd = 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.

A genomic perspective on protein tyrosine phosphatases: gene structure, pseudogenes, and genetic disease linkage.

The protein tyrosine phosphatases (PTPs) are now recognized as critical regulators of signal transduction under normal and pathophysiological conditions. In this analysis we have explored the sequence of the human genome to define the composition of the PTP family. Using public and proprietary sequence databases, we discovered one novel human PTP gene and defined chromosomal loci and exon structure of the additional 37 genes encoding known PTP transcripts. Direct orthologs were present in the mouse genome for all 38 human PTP genes. In addition, we identified 12 PTP pseudogenes unique to humans that have probably contaminated previous bioinformatics analysis of this gene family. PCR amplification and transcript sequencing indicate that some PTP pseudogenes are expressed, but their function (if any) is unknown. Furthermore, we analyzed the enhanced diversity generated by alternative splicing and provide predicted amino acid sequences for four human PTPs that are currently defined by fragments only. Finally, we correlated each PTP locus with genetic disease markers and identified 4 PTPs that map to known susceptibility loci for type 2 diabetes and 19 PTPs that map to regions frequently deleted in human cancers. We have made our analysis available at http://ptp.cshl.edu or http://science.novonordisk.com/ptp and we hope this resource will facilitate the functional characterization of these key enzymes.

Development of novel cellular histone-binding and chromatin-displacement assays for bromodomain drug discovery.

BACKGROUND: Proteins that 'read' the histone code are central elements in epigenetic control and bromodomains, which bind acetyl-lysine motifs, are increasingly recognized as potential mediators of disease states. Notably, the first BET bromodomain-based therapies have entered clinical trials and there is a broad interest in dissecting the therapeutic relevance of other bromodomain-containing proteins in human disease. Typically, drug development is facilitated and expedited by high-throughput screening, where assays need to be sensitive, robust, cost-effective and scalable. However, for bromodomains, which lack catalytic activity that otherwise can be monitored (using classical enzymology), the development of cell-based, drug-target engagement assays has been challenging. Consequently, cell biochemical assays have lagged behind compared to other protein families (e.g., histone deacetylases and methyltransferases). RESULTS: Here, we present a suite of novel chromatin and histone-binding assays using AlphaLISA, in situ cell extraction and fluorescence-based, high-content imaging. First, using TRIM24 as an example, the homogenous, bead-based AlphaScreen technology was modified from a biochemical peptide-competition assay to measure binding of the TRIM24 bromodomain to endogenous histone H3 in cells (AlphaLISA). Second, a target agnostic, high-throughput imaging platform was developed to quantify the ability of chemical probes to dissociate endogenous proteins from chromatin/nuclear structures. While overall nuclear morphology is maintained, the procedure extracts soluble, non-chromatin-bound proteins from cells with drug-target displacement visualized by immunofluorescence (IF) or microscopy of fluorescent proteins. Pharmacological evaluation of these assays cross-validated their utility, sensitivity and robustness. Finally, using genetic and pharmacological approaches, we dissect domain contribution of TRIM24, BRD4, ATAD2 and SMARCA2 to chromatin binding illustrating the versatility/utility of the in situ cell extraction platform. CONCLUSIONS: In summary, we have developed two novel complementary and cell-based drug-target engagement assays, expanding the repertoire of pharmacodynamic assays for bromodomain tool compound development. These assays have been validated through a successful TRIM24 bromodomain inhibitor program, where a micromolar lead molecule (IACS-6558) was optimized using cell-based assays to yield the first single-digit nanomolar TRIM24 inhibitor (IACS-9571). Altogether, the assay platforms described herein are poised to accelerate the discovery and development of novel chemical probes to deliver on the promise of epigenetic-based therapies.

The SMARCA2/4 ATPase Domain Surpasses the Bromodomain as a Drug Target in SWI/SNF-Mutant Cancers: Insights from cDNA Rescue and PFI-3 Inhibitor Studies.

The SWI/SNF multisubunit complex modulates chromatin structure through the activity of two mutually exclusive catalytic subunits, SMARCA2 and SMARCA4, which both contain a bromodomain and an ATPase domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF-mutant tumors, including SMARCA4-deficient lung cancer; however, the contribution of conserved, druggable protein domains to this anticancer phenotype is unknown. Here, we functionally deconstruct the SMARCA2/4 paralog dependence of cancer cells using bioinformatics, genetic, and pharmacologic tools. We evaluate a selective SMARCA2/4 bromodomain inhibitor (PFI-3) and characterize its activity in chromatin-binding and cell-functional assays focusing on cells with altered SWI/SNF complex (e.g., lung, synovial sarcoma, leukemia, and rhabdoid tumors). We demonstrate that PFI-3 is a potent, cell-permeable probe capable of displacing ectopically expressed, GFP-tagged SMARCA2-bromodomain from chromatin, yet contrary to target knockdown, the inhibitor fails to display an antiproliferative phenotype. Mechanistically, the lack of pharmacologic efficacy is reconciled by the failure of bromodomain inhibition to displace endogenous, full-length SMARCA2 from chromatin as determined by in situ cell extraction, chromatin immunoprecipitation, and target gene expression studies. Furthermore, using inducible RNAi and cDNA complementation (bromodomain- and ATPase-dead constructs), we unequivocally identify the ATPase domain, and not the bromodomain of SMARCA2, as the relevant therapeutic target with the catalytic activity suppressing defined transcriptional programs. Taken together, our complementary genetic and pharmacologic studies exemplify a general strategy for multidomain protein drug-target validation and in case of SMARCA2/4 highlight the potential for drugging the more challenging helicase/ATPase domain to deliver on the promise of synthetic-lethality therapy.

Enzyme kinetic characterization of protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta, PTPepsilon, CD45, LAR, PTP1B and SHP-1), using pNPP as substrate. Most noticeable is the increase in the turnover number for PTPbeta with increasing pH and the weak pH-dependence of the turnover number of CD45. The kinetic data for PTPalpha-D1 and PTPalpha-D1D2 suggest that D2 affects the catalysis of pNPP. PTPepsilon and the closely homologous PTPalpha behave differently. The K(m) data were lower for PTPepsilon than those for PTPalpha, while the inverse was observed for the catalytic efficiencies.

Cancer genomics: from discovery science to personalized medicine.

Recent advances in genome technologies and the ensuing outpouring of genomic information related to cancer have accelerated the convergence of discovery science and clinical medicine. Successful examples of translating cancer genomics into therapeutics and diagnostics reinforce its potential to make possible personalized cancer medicine. However, the bottlenecks along the path of converting a genome discovery into a tangible clinical endpoint are numerous and formidable. In this Perspective, we emphasize the importance of establishing the biological relevance of a cancer genomic discovery in realizing its clinical potential and discuss some of the major obstacles to moving from the bench to the bedside.

Identification of direct target engagement biomarkers for kinase-targeted therapeutics.

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3'-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1(Ser410) and p-PDK1(Thr513) are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1(Ser241)), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.