Kenneth D. Westover

Quantitative Analysis of Hsp90-Client Interactions Reveals Principles of Substrate Recognition

HSP90 is a molecular chaperone that associates with numerous substrate proteins called clients. It plays many important roles in human biology and medicine, but determinants of client recognition by HSP90 have remained frustratingly elusive. We systematically and quantitatively surveyed most human kinases, transcription factors, and E3 ligases for interaction with HSP90 and its cochaperone CDC37. Unexpectedly, many more kinases than transcription factors bound HSP90. CDC37 interacted with kinases, but not with transcription factors or E3 ligases. HSP90::kinase interactions varied continuously over a 100-fold range and provided a platform to study client protein recognition. In wild-type clients, HSP90 did not bind particular sequence motifs, but rather associated with intrinsically unstable kinases. Stabilization of the kinase in either its active or inactive conformation with diverse small molecules decreased HSP90 association. Our results establish HSP90 client recognition as a combinatorial process: CDC37 provides recognition of the kinase family, whereas thermodynamic parameters determine client binding within the family.

Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center.

Binding of a ribonucleoside triphosphate to an RNA polymerase II transcribing complex, with base pairing to the template DNA, was revealed by X-ray crystallography. Binding of a mismatched nucleoside triphosphate was also detected, but in an adjacent site, inverted with respect to the correctly paired nucleotide. The results are consistent with a two-step mechanism of nucleotide selection, with initial binding to an entry (E) site beneath the active center in an inverted orientation, followed by rotation into the nucleotide addition (A) site for pairing with the template DNA. This mechanism is unrelated to that of single subunit RNA polymerases and so defines a new paradigm for the large, multisubunit enzymes. Additional findings from these studies include a third nucleotide binding site that may define the length of backtracked RNA; DNA double helix unwinding in advance of the polymerase active center; and extension of the diffraction limit of RNA polymerase II crystals to 2.3 A.

Structural basis of transcription: backtracked RNA polymerase II at 3.4 angstrom resolution.

Stepping Back to Go Forward Insight into the mechanism of transcription has come from crystal structures of actively transcribing RNA polymerase II complexes in the pre- and posttranslocation states. RNA polymerase also backtracks on the DNA template. Backtracking by only a few residues is reversible, but longer backtracking leads to arrest that is relieved by cleavage of the transcript by the transcription elongation factor SII (TFIIS). Now Wang et al. (p. 1203) report x-ray structures of backtracked ternary complexes and of a backtracked complex bound to a noncleaving mutant of TFIIS. The structures show a defined one-residue, backtracked state supporting the idea that RNA polymerase oscillates between backward and forward motion during active transcription. Mismatched residues disfavor forward translocation, increasing the lifetime of the backtracked state and facilitating cleavage by TFIIS. Thus, TFIIS-induced cleavage is likely to provide an important proofreading function during transcription. A backtracked RNA polymerase II reveals how the enzyme proofreads the RNA transcript. Transcribing RNA polymerases oscillate between three stable states, two of which, pre- and posttranslocated, were previously subjected to x-ray crystal structure determination. We report here the crystal structure of RNA polymerase II in the third state, the reverse translocated, or “backtracked” state. The defining feature of the backtracked structure is a binding site for the first backtracked nucleotide. This binding site is occupied in case of nucleotide misincorporation in the RNA or damage to the DNA, and is termed the “P” site because it supports proofreading. The predominant mechanism of proofreading is the excision of a dinucleotide in the presence of the elongation factor SII (TFIIS). Structure determination of a cocrystal with TFIIS reveals a rearrangement whereby cleavage of the RNA may take place.

Therapeutic Targeting of Oncogenic K‐Ras by a Covalent Catalytic Site Inhibitor

We report the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggest that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. These first-in-class covalent K-Ras inhibitors demonstrate that irreversible targeting of the K-Ras guanine-nucleotide binding site is potentially a viable therapeutic strategy for inhibition of Ras signaling.

Structural basis of eukaryotic gene transcription

An RNA polymerase II promoter has been isolated in transcriptionally activated and repressed states. Topological and nuclease digestion analyses have revealed a dynamic equilibrium between nucleosome removal and reassembly upon transcriptional activation, and have further shown that nucleosomes are removed by eviction of histone octamers rather than by sliding. The promoter, once exposed, assembles with RNA polymerase II, general transcription factors, and Mediator in a ∼3 MDa transcription initiation complex. X‐ray crystallography has revealed the structure of RNA polymerase II, in the act of transcription, at atomic resolution. Extension of this analysis has shown how nucleotides undergo selection, polymerization, and eventual release from the transcribing complex. X‐ray and electron crystallography have led to a picture of the entire transcription initiation complex, elucidating the mechanisms of promoter recognition, DNA unwinding, abortive initiation, and promoter escape.

In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C.

Significance SML-8-73-1 (SML) is the first example, to our knowledge, of a GTP-competitive inhibitor of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras). A high-resolution structure of K-Ras G12C bound to SML shows K-Ras in an inactive conformation. In situ proteomic-based chemical profiling of SML demonstrates that SML is highly selective for K-Ras G12C over other small GTPases. A novel chemosensor-based assay allows measurement of covalent reaction rates between K-Ras G12C and SML and enables characterization of this reaction in the context of millimolar concentrations of GTP and GDP, well in exccss of what is found in living cells. These results demonstrate that even in the presence of high concentrations of GTP and GDP, SML is able to exchange into the GN site. Directly targeting oncogenic V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) with small-molecule inhibitors has historically been considered prohibitively challenging. Recent reports of compounds that bind directly to the K-Ras G12C mutant suggest avenues to overcome key obstacles that stand in the way of developing such compounds. We aim to target the guanine nucleotide (GN)-binding pocket because the natural contents of this pocket dictate the signaling state of K-Ras. Here, we characterize the irreversible inhibitor SML-8-73-1 (SML), which targets the GN-binding pocket of K-Ras G12C. We report a high-resolution X-ray crystal structure of G12C K-Ras bound to SML, revealing that the compound binds in a manner similar to GDP, forming a covalent linkage with Cys-12. The resulting conformation renders K-Ras in the open, inactive conformation, which is not predicted to associate productively with or activate downstream effectors. Conservation analysis of the Ras family GN-binding pocket reveals variability in the side chains surrounding the active site and adjacent regions, especially in the switch I region. This variability may enable building specificity into new iterations of Ras and other GTPase inhibitors. High-resolution in situ chemical proteomic profiling of SML confirms that SML effectively discriminates between K-Ras G12C and other cellular GTP-binding proteins. A biochemical assay provides additional evidence that SML is able to compete with millimolar concentrations of GTP and GDP for the GN-binding site.

Kinome-wide Selectivity Profiling of ATP-competitive Mammalian Target of Rapamycin (mTOR) Inhibitors and Characterization of Their Binding Kinetics

Background: Several new ATP-competitive mTOR inhibitors have been described, but their kinome-wide selectivity profiles have not been disclosed. Results: Four different profiling technologies revealed a different spectrum of targets for four recently described mTOR inhibitors. Conclusion: Diverse heterocyclic mTOR inhibitors have unique pharmacology. Significance: Profiling data guide choices of mTOR inhibitors for particular applications and provide new potential targets for medicinal chemistry efforts. An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC50 = 2 nm) relative to other inhibitors. In vitro biochemical profiling at 10 μm revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μm but did show that PP242 efficiently inhibited the RET receptor (EC50, 42 nm) and JAK1/2/3 kinases (EC50, 780 nm). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μm and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate

Serine is a both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical glucose-derived serine synthesis pathway, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic towards PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we use a quantitative high-throughput screen to identify small molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and suggest that one-carbon unit wasting may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

Pharmacological targeting of the pseudokinase Her3

Her3 (ErbB3) belongs to the epidermal growth factor receptor tyrosine kinases and is well credentialed as an anti-cancer target but is thought to be “undruggable” using ATP-competitive small molecules because it lacks significant kinase activity. Here we report the first selective Her3 ligand, TX1-85-1, that forms a covalent bond with Cys721 located in the ATP-binding site of Her3. We demonstrate that covalent modification of Her3 inhibits Her3 signaling but not proliferation in some Her3 dependent cancer cell lines. Subsequent derivatization with a hydrophobic adamantane moiety demonstrates that the resultant bivalent ligand (TX2-121-1) enhances inhibition of Her3 dependent signaling. Treatment of cells with TX2-121-1 results in partial degradation of Her3 and serendipitously interferes with productive heterodimerization between Her3 with either Her2 or c-Met. These results suggest that small molecules will be capable of perturbing the biological function of Her3 and the approximately 60 other pseudokinases found in human cells.

Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations.

KRAS mutations are the most common genetic abnormalities in cancer, but the distribution of specific mutations across cancers and the differential responses of patients with specific KRAS mutations in therapeutic clinical trials suggest that different KRAS mutations have unique biochemical behaviors. To further explain these high-level clinical differences and to explore potential therapeutic strategies for specific KRAS isoforms, we characterized the most common KRAS mutants biochemically for substrate binding kinetics, intrinsic and GTPase-activating protein (GAP)–stimulated GTPase activities, and interactions with the RAS effector, RAF kinase. Of note, KRAS G13D shows rapid nucleotide exchange kinetics compared with other mutants analyzed. This property can be explained by changes in the electrostatic charge distribution of the active site induced by the G13D mutation as shown by X-ray crystallography. High-resolution X-ray structures are also provided for the GDP-bound forms of KRAS G12V, G12R, and Q61L and reveal additional insight. Overall, the structural data and measurements, obtained herein, indicate that measurable biochemical properties provide clues for identifying KRAS-driven tumors that preferentially signal through RAF. Implications: Biochemical profiling and subclassification of KRAS-driven cancers will enable the rational selection of therapies targeting specific KRAS isoforms or specific RAS effectors. Mol Cancer Res; 13(9); 1325–35. ©2015 AACR.