Structure-guided development of a potent and selective non-covalent active-site inhibitor of USP7.
Lamberto ILiu XSeo HSSchauer NJIacob REHu WDas DMikhailova TWeisberg EL,
Engen JRAnderson KCChauhan DDhe-Paganon SBuhrlage SJ.

Cell Chemical Biology. 2017. Dec 21;24(12):1490-1500.e11.


Deubiquitinating enzymes (DUBs) have garnered significant attention as drug targets in the last 5-10 years. The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins, especially proteins that are challenging to directly target but which are stabilized by DUB family members. Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools. Most reported DUB inhibitors, however, are polypharmacological agents possessing weak (micromolar) potency toward their primary target, limiting their utility in target validation and mechanism studies. Due to a lack of high-resolution DUB:small-molecule ligand complex structures, no structure-guided optimization efforts have been reported for a mammalian DUB. Here, we report a small-molecule:ubiquitin-specific protease (USP) family DUB co-structure and rapid design of potent and selective inhibitors of USP7 guided by the structure. Interestingly, the compounds are non-covalent active-site inhibitors.

Preview by M. Desroses & M. Altun

Pubmed: 29056421

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Subzero Celsius separations in three-zone temperature controlled hydrogen deuterium exchange mass spectrometry.
Wales TE, Fadgen KE, Eggertson MJ, Engen JR.
J Chromatogr A. 2017. Nov 10;1523:275-282.

Hydrogen deuterium exchange mass spectrometry (HDX MS) reports on the conformational landscape of proteins by monitoring the exchange between backbone amide hydrogen atoms and deuterium in the solvent.  To maintain the label for analysis, quench conditions of low temperature and pH are required during the chromatography step performed after protease digestion but before mass spectrometry.  Separation at 0 °C is often chosen as this is the temperature where the most deuterium can be recovered without freezing of the typical water and acetonitrile mobile phases.  Several recent reports of separations at subzero Celsius emphasize the promise for retaining more deuterium and using a much longer chromatographic gradient or direct infusion time.  Here we present the construction and validation of a modified Waters nanoACQUITY HDX manager with a third temperature-controlled zone for peptide separations at subzero temperatures.  A new Peltier-cooled door replaces the door of a traditional main cooling chamber and the separations and trapping column are routed through the door housing.  To prevent freezing, 35% methanol is introduced post online digestion.  No new pumps are required and online digestion is performed as in the past.  Subzero separations, using conventional HPLC column geometry of 3 mm particles in a 1x50mm column, did not result in major changes to chromatographic efficiency when lowering the temperature from 0 to -20 °C.  There were significant increases in deuterium recovery for both model peptides and biologically relevant protein systems.  Given the higher levels of deuterium recovery, expanded gradient programs can be used to allow for higher chromatographic peak capacity and therefore the analysis of larger and more complex proteins and systems. 
Pubmed: 28596009

Achieving a graded immune response: BTK adopts a range of active/inactive conformations dictated by multiple interdomain contacts.
Joseph RE, Wales TE, Fulton DB, Engen JR, Andreotti AH.
Structure. 2017. Oct 3;25(10):1481-1494.e4.


Capturing the functionally relevant forms of dynamic, multidomain proteins is extremely challenging. Bruton's tyrosine kinase (BTK), a kinase essential for B and mast cell function, has stubbornly resisted crystallization in its full-length form. Here, nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry show that BTK adopts a closed conformation in dynamic equilibrium with open, active conformations. BTK lacks the phosphotyrosine regulatory tail of the SRC kinases, yet nevertheless achieves a phosphotyrosine-independent C-terminal latch. The unique proline-rich region is an internal “on” switch pushing the autoinhibited kinase toward its active state. Newly identified autoinhibitory contacts in the BTK pleckstrin homology domain are sensitive to phospholipid binding, which induces large-scale allosteric changes. The multiplicity of these regulatory contacts suggests a clear mechanism for gradual or “analog” kinase activation as opposed to a binary “on/off” switch. The findings illustrate how previously modeled information for recalcitrant full-length proteins can be expanded and validated with a convergent multidisciplinary experimental approach.

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Pubmed: 28867612

Chemically Induced Degradation of the Oncogenic Transcription Factor BCL6.
Kerres NSteurer SSchlager SBader GBerger HCaligiuri MDank CEngen JREttmayer P,
Fischerauer BFlotzinger GGerlach DGerstberger TGmaschitz TGreb PHan BHeyes E,
Iacob REKessler DKölle HLamarre LLancia DRLucas SMayer MMayr KMischerikow N,
Mück KPeinsipp CPetermann OReiser URudolph DRumpel KSalomon CScharn DSchnitzer R,
Schrenk ASchweifer NThompson DTraxler EVarecka RVoss TWeiss-Puxbaum AWinkler S,
Zheng XZoephel AKraut NMcConnell DPearson MKoegl M.

Cell Rep. 2017. Sept 19;20(12):2860-2875.


The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.

Pubmed: 28930682

Mechanism of enzyme repair by the AAA+ chaperone rubisco activase.
Bhat JY, Miličić G, Thieulin-Pardo G, Bracher A, Maxwell A, Ciniawsky S, Mueller-Cajar O, Engen JR,
Hartl FU, Wendler P, Hayer-Hartl M.

Mol. Cell. 2017. Sept 7;67(5):744-756.e6.


How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair.

Pubmed: 28803776

KRAS G12C drug development: Discrimination between switch II pocket configurations using Hydrogen/Deuterium-Exchange Mass Spectrometry.
Lu J, Harrison RA, Li L, Zeng M, Gondi S, Scott D, Gray NS, Engen JR, Westover KD.
Structure. 2017. Sept 5;25(9):1442-1448.


KRAS G12C, the most common RAS mutation found in non-small-cell lung cancer, has been the subject of multiple recent covalent small-molecule inhibitor campaigns including efforts directed at the guanine nucleotide pocket and separate work focused on an inducible pocket adjacent to the switch motifs. Multiple conformations of switch II have been observed, suggesting that switch II pocket (SIIP) binders may be capable of engaging a range of KRAS conformations. Here we report the use of hydrogen/deuterium-exchange mass spectrometry (HDX MS) to discriminate between conformations of switch II induced by two chemical classes of SIIP binders. We investigated the structural basis for differences in HDX MS using X-ray crystallography and discovered a new SIIP configuration in response to binding of a quinazoline chemotype. These results have implications for structure-guided drug design targeting the RAS SIIP.

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Pubmed: 28781083

Allosteric sensitization of proapoptotic BAX.
Pritz JR, Wachter F, Lee S, Luccarelli J, Wales TE, Cohen DT, Coote P, Heffron GJ, Engen JR, Massefski W, Walensky LD.
Nat Chem Biol. 2017. Sep;13(9):961-967.


BCL-2-associated X protein (BAX) is a critical apoptotic regulator that can be transformed from a cytosolic monomer into a lethal mitochondrial oligomer, yet drug strategies to modulate it are underdeveloped due to longstanding difficulties in conducting screens on this aggregation-prone protein. Here, we overcame prior challenges and performed an NMR-based fragment screen of full-length human BAX. We identified a compound that sensitizes BAX activation by binding to a pocket formed by the junction of the α3-α4 and α5-α6 hairpins. Biochemical and structural analyses revealed that the molecule sensitizes BAX by allosterically mobilizing the α1-α2 loop and BAX BH3 helix, two motifs implicated in the activation and oligomerization of BAX, respectively. By engaging a region of core hydrophobic interactions that otherwise preserve the BAX inactive state, the identified compound reveals fundamental mechanisms for conformational regulation of BAX and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.

Pubmed: 28692068

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Engineering Aglycosylated IgG Variants with Wild-type or Improved Binding Affinity to Human Fc gamma RIIA and Fc gamma RIIIAs.
Chen TF, Sazinsky SL, Houde D, DiLillo DJ, Bird J, Li KK, Cheng GT, Qiu H, Engen JR, Ravetch JV,
Wittrup KD.
J Mol Biol. 2017. Aug 4;429(16):2528-2541.

The binding of human IgG1 to human Fc gamma receptors (hFcγR) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs DTT-IYG and DAT-IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFcγRI and hFcγRIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT-IYG to be the best at restoring WT-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT-IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function.
Pubmed: 28694069

Structures of PGAM5 provide insight into active site plasticity & multimeric assembly.
Chaikuad A, Filippakopoulos P, Marcsisin SR, Picaud S, Schröder M, Sekine S, Ichijo H, Engen JR,
Takeda K, Knapp S.
Structure. 2017. Jul 5;25(7):1089-1099.

PGAM5 is a mitochondrial membrane protein that functions as an atypical Ser/Thr phosphatase and is a regulator of oxidative stress response, necroptosis, and autophagy. Here we present several crystal structures of PGAM5 including the activating N-terminal regulatory sequences, providing a model for structural plasticity, dimerization of the catalytic domain, and the assembly into an enzymatically active dodecameric form. Oligomeric states observed in structures were supported by hydrogen exchange mass spectrometry, size-exclusion chromatography, and analytical ultracentrifugation experiments in solution. We report that the catalytically important N-terminal WDPNWD motif acts as a structural integrator assembling PGAM5 into a dodecamer, allosterically activating the phosphatase by promoting an ordering of the catalytic loop. Additionally the observed active site plasticity enabled visualization of essential conformational rearrangements of catalytic elements. The comprehensive biophysical characterization offers detailed structural models of this key mitochondrial phosphatase that has been associated with the development of diverse diseases. 
Pubmed: 28648608

Applicability of Hydrogen-Deuterium exchange in comparing conformations of innovator to biosimilar biopharmaceutical products.
Iacob RE, Engen JR, Krull IS.
LC-GC. 2017. Jun 1;35(6):382-390.

As the biopharmaceutical industry has grown, more and more drugs are coming off patent, leading to an increase on worldwide markets of biosimilars that compete with the innovator drugs.  All regulatory agencies are carefully monitoring just how closely a biosimilar company can mimic the innovator product, with similar or identical safety, efficacy, and therapeutic properties.  Here we discuss the potential applications of hydrogen–deuterium exchange mass spectrometry (HDX-MS) to determine similarities in the higher order structures of a biosimilar and its innovator drug.

Characterization of aggregation propensity of a Fc-fusion protein therapeutic by Hydrogen/Deuterium exchange mass spectrometry.

Huang RY-C, Iacob RE, Krystek SR, Jin M, Wei H, Tao L, Das TK, Tymiak AA, Engen JR, Chen G.
J Am Soc Mass Spectrom. 2017. May;28(5):795-802.

Cover Feature

Aggregation of protein therapeutics has long been a concern across different stages of manufacturing processes in biopharmaceutical industry. It is often indicative of aberrant protein therapeutic higher order structure. In this study, the aggregation propensity of a human Fc-fusion protein therapeutic was characterized. Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) was applied to examine the conformational dynamics of dimers collected from a bioreactor. HDX-MS data combined with spatial aggregation propensity (SAP) calculations revealed potential aggregation interface in the Fc domain. This study provides a general strategy for the characterization of the aggregation propensity of Fc fusion proteins at the molecular level. 
Pubmed: 27527097

Structural stability and local dynamics in disease-causing mutants of human apolipoprotein A-I: what makes the protein amyloidogenic?
Das M, Wilson CJ, Mei X, Wales TE, Engen JR, Gursky O.
Amyloid. 2017. Mar;24(sup1):11-12.

Pubmed: 28042708

Force interacts with macromolecular structure in activation of TGF-β.
Dong X, Zhao B, Iacob RE, Zhu J, Koksal AC, Lu C, Engen JR, Springer TA.
Nature. 2017. Feb 2;542:55-59.


Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-β1 precursor (pro-TGF-β1), integrins bind to the prodomain, apply force, and release the TGF-β growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVβ6 binds pro-TGF-β1 in an orientation biologically relevant for force-dependent release of TGF-β from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-β orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin β-subunit. Simulations of force-dependent activation of TGF-β demonstrate evolutionary specializations for force application through the TGF-β prodomain and through the β- and not α-subunit of the integrin.   

News & Views by T. Ha

Pubmed: 28117447