Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, is an essential effector of B-cell receptor (BCR) signaling. Chronic activation of BTK-mediated BCR signaling is a hallmark of many hematological malignancies, which makes it an attractive therapeutic target. Pharmacological inhibition of BTK enzymatic function is now a well-proven strategy for the treatment of patients with these malignancies. We report the discovery and characterization of NX-2127, a BTK degrader with concomitant immunomodulatory activity. By design, NX-2127 mediates the degradation of transcription factors IKZF1 and IKZF3 through molecular glue interactions with the cereblon E3 ubiquitin ligase complex. NX-2127 degrades common BTK resistance mutants, including BTKC481S. NX-2127 is orally bioavailable, exhibits in vivo degradation across species, and demonstrates efficacy in preclinical oncology models. NX-2127 has advanced into first-in-human clinical trials and achieves deep and sustained degradation of BTK following daily oral dosing at 100 mg.

Background The E3 ubiquitin ligase Casitas B-lineage lymphoma B (CBL-B) is expressed in leukocytes and regulates signaling pathways in T and NK cells, significantly limiting their antitumor effector function. In T cells CBL-B attenuates activation initiated by TCR engagement, in part by mediating the requirement for CD28 co-stimulation, thus setting the threshold for T cell activation. In NK cells, CBL-B functions downstream of TAM receptors and negatively regulates cytokine production and cytotoxicity. Methods Here we describe the effects of NX-1607, an orally bioavailable intramolecular glue inhibitor of CBL-B, on primary human T and NK cells and assess NX-1607 in combination with Rituximab in a murine xenograft model of Non-Hodgkin's Lymphoma (NHL). Results Previously, we showed that NX-1607 enhances IL-2 and IFN-g secretion in human T cells following TCR stimulation. Regulatory T cells (Tregs) produce multiple cytokines in the tumor microenvironment (TME) that work to counteract the antitumor response by suppressing T-cell activation. Proliferation of CD4+ effector T cells activated by anti-CD3/CD28 was suppressed when cultured 1:1 with Tregs or TGF-b. Addition of NX-1607 recovered the proliferative capacity of CD4+ effector T cells to levels equivalent to that of anti-CD3/CD28 stimulation alone. Therefore, in addition to enhancing T-cell activation, NX-1607 renders T cells resistant to Treg and TGF-β-mediated suppression. In an in vitro ADCC assay, addition of NX-1607 significantly enhanced TNF-α and IFN-γ production in human primary NK cells. The efficacy of NX-1607 in combination with Rituximab was evaluated in a Raji NHL model where Raji cells were administered by IV to establish disseminated tumors followed by treatment with NX-1607 (30 mg/kg QD) and/or Rituximab (10 mg/kg). Both NX-1607 and Rituximab given as monotherapy provided a significant survival benefit. Combination of NX-1607 and Rituximab significantly enhanced tumor growth inhibition and stable rejections when compared to single agent activity. Importantly, the survival benefit provided by NX-1607 was abrogated by depletion of NK cells. Therefore, NX-1607 augments NK cell activity both in human NK cells and in mouse tumor models. Conclusions These studies provide insight into the antitumor activity of this novel, small molecule inhibitor of CBL-B, demonstrating that NX-1607 enhances both innate and adaptive immune responses, both of which are important for overcoming a suppressive TME. These studies also provide support for clinical development of NX-1607 as a monotherapy or in combination with antibody therapeutics to enhance ADCC antitumor effects. We have initiated a clinical trial with NX-1607 in patients with advanced solid tumors NX-1607-101 (NCT05107674).

Background Adoptive cell transfer (ACT) involving engineered T cells (CAR-T) or autologous tumor-specific lymphocytes (TIL) induces effective antitumor response in advanced cancer patients. However, tumors frequently relapse after an initial response due to suboptimal T-cell activation and expansion within the tumor microenvironment. Moreover, current ACT treatment paradigms require application of high dose bolus infusions of IL-2 which are associated with acute toxicities restricting the use of ACT in the clinic. The E3 ubiquitin ligase Casitas B-lineage lymphoma B (CBL-B) is highly expressed in T cells, where it functions as an intracellular checkpoint that constrains T-cell activation following T cell receptor (TCR) engagement, therefore limiting T cell-mediated antitumor responses. Methods We have developed two highly potent small molecule inhibitors of CBL-B to increase T-cell antitumor function both in vitro (NX-0255) and in vivo (NX-1607). Results We previously reported that addition of NX-0255 during in vitro treatment of tumor-specific T cells increases the frequency and absolute numbers of less exhausted CD8+ memory T cells, profoundly improving their functionality and ability to control tumor growth following ACT in tumor-bearing mice. Here, we hypothesized that CBL-B inhibition could reduce the requirement for IL-2 bolus and utilized the Pmel-1 ACT/B16 melanoma tumor model to compare the antitumor effect of post infusion in vivo treatments with NX-1607 to high dose IL-2. C57BL/6 mice were implanted with B16-OVA and received Pmel-1 CD8+ T cells activated in vitro using anti-CD3 stimulation and NX-0255 combined with IL-2, followed by systemic treatment with either IL-2 (IP, 150000 IU for three days, BID) or oral NX-1607 (30 mg/kg, QD). We found that ACT supported by in vivo treatment with NX-1607 increased the antitumor activity of Pmel-1 cells when compared to ACT alone. Importantly, the increased antitumor activity of NX-1607-supported ACT was comparable to IL-2. Spectral cytometry analysis performed at 7 and 14 days after ACT showed that following NX-1607, a larger fraction of circulating Pmel-1 cells had a central-memory phenotype and expressed high levels of Granzyme B. Interestingly, both in vivo treatments induced increased 4-1BB/CD137 expression that significantly correlates with antitumor response. Conclusions These findings demonstrated that oral dosing of NX-1607 in combination with ACT can support the functionality of transferred cells providing a robust antitumor response in the aggressive B16-OVA model. Treatment with NX-1607 induces a more favorable T cell phenotype compared to IL-2 treatment and is well tolerated. The observed antitumor effects of NX-1607 support its potential use in combination with cell-based therapeutics.

BackgroundAdoptive cell transfer (ACT) of TIL effects durable responses in patients with melanoma and some epithelial tumors. It is thought that poor in vitro cell expansion and inefficient T-cell migration to the tumor limits the broader application of this approach. The E3 ubiquitin ligase, Casitas B-lineage lymphoma b (CBL-B) is expressed in T-cells where it functions as a regulator of immune cell activation, in part by requiring CD28 co-stimulation in addition to T-cell receptor activation. We have developed NX-0255, a highly potent small molecule inhibitor of CBL-B, demonstrating its ability to increase T-cell derived cytokine secretion and proliferation in the presence or absence of co-stimulation. Here, we investigated the effects of NX-0255 on the ex vivo growth and characteristics of human TIL to create drug-enhanced TIL (DeTIL-0255) as an ACT product for treating patients with cancer.MethodsTIL from ovary, colon, lung, head and neck, breast, and vulva carcinomas were cultured with IL-2 and compared in two experimental groups: NX-0255 without IL-2, or NX-0255 in combination with IL-2. Following 22 days of culture, cell number, and phenotype were assessed by flow cytometry and single-cell transcriptomics.ResultsCulturing of TIL in the presence of NX-0255 alone resulted in the expansion of cells, with numbers comparable to that of conventionally cultured TIL with IL-2. The addition of NX-0255 in combination with IL-2 significantly increased the number of cells expanded compared to TIL (n=16, p=0.004). Flow cytometric analysis demonstrated that DeTIL-0255 were significantly less exhausted compared to TIL, as shown by the significant reduction of CD8+ T-cells expressing PD-1 (p=0.02), and co-expressing PD-1+TIM-3+ (p=0.03) and PD-1+LAG-3+ (p=0.03).Furthermore, upon stimulation, the functional capacity of DeTIL-0255 was differentially enhanced, with significant increases in the absolute numbers of CD8+ T-cells expressing intracellular perforin (p=0.001), granzyme-B (p=0.005) and CD107a (p=0.01) when comparing DeTIL-0255 to TIL. An increase of CD8+ T-cells expressing CD137/4-1BB, a biomarker of CD8+ T-cell tumor reactivity was also observed (p=0.03). TCR repertoire and single-cell sequencing analysis demonstrated that DeTIL-0255 had increased TCR diversity and enhanced expression of genes associated with stemness (CD127+,CCR7+,CD62L+) and cytotoxicity (GNLY+,GZMB+,NKG7+).ConclusionsCollectively, these data suggest that DeTIL-0255 increases the proportion and absolute numbers of less exhausted CD8+ memory T-cells, enhancing cytolytic T-cell function. Adoptive transfer of DeTIL-0255 may increase persistence and exhibit broader functional activity than conventional TIL, potentially conferring improved anti-tumor activity. Taken together, these data support the clinical development of DeTIL-0255 for the treatment of patients with cancer.

Une production excessive et inappropriée de cytokines pro-inflammatoire telles que l’interleukine IL-1, IL-6 et IL-18, est un élément central dans la physiopathologie de la maladie de Still de l’adulte (MSA) et de l’arthrite juvénile idiopathique. Au-delà des thérapies ciblant IL-1 ou IL-6, les inhibiteurs de Janus kinases (JAK) ont été proposés pour les patients avec une maladie de Still réfractaire ou présentant une intolérance aux biothérapies. Récemment, il a été suggéré que les inhibiteurs de JAK pourraient être efficaces dans la MSA réfractaire. Cette étude rétrospective s’appuie sur une enquête nationale des services de rhumatologie adulte et pédiatrique et médecine interne des hôpitaux français à partir d’un appel en ligne du « Club Rhumatismes et Inflammation ». Les données ont été collectées à l’aide d’un questionnaire standardisé et analysées à différents moments (initiation du traitement, M1, M3, M6 et à la fin du suivi). La réponse aux inhibiteurs de JAK a été classée en 3 catégories : rémission complète (résolution de l’ensemble des signes clinicobiologique), rémission partielle (amélioration clinique avec la persistance de quelques symptômes) ou échec (absence d’amélioration clinique ou biologique). Un total de 7 patients (5 adultes et 2 enfants) ont été recrutés. L’âge moyen à l’initiation du traitement était de 39,6 ± 9,6 ans pour les patients avec une MSA et de 11,0 ± 7,07 ans pour les enfants, et la durée moyenne de la maladie était de 6,7 ± 7,16 ans. L’expression clinique prédominante était la forme systémique pour 6 patients et la forme articulaire chronique pour un patient uniquement. La réponse aux corticostéroïdes, cDMARD ou bDMARD est considérée comme inadéquate chez l’ensemble des patients. Le baricitinib est utilisé pour 4 patients (dont 1 a ensuite été remplacé par upadacitinib au cours du suivi), ruxolitinib pour 2 patients et un patient sous tofacitinib. Les corticostéroïdes sont associés chez l’ensemble des patients, anakinra pour un patient, méthotrexate et anakinra pour un autre patient au début du suivi puis relayé par colchicine et anakinra. Pour une durée moyenne de suivi de 11,3 ± 9,3 mois : une réponse partielle est observée chez 4 patients (57 %) (patients traités par ruxolitinib, baricitinib ou tofacitinib) et un échec chez 3 patients (43 %) (patients sous baricitinib ou ruxolitinib). Aucun patient n’a atteint une rémission complète. Au cours de la dernière visite, les corticostéroïdes ont pu être diminué progressivement, cependant aucun patient n’a pu les arrêter définitivement. Les patients avec une réponse partielle présentaient une diminution moyenne de la corticothérapie de 63 % (40 % pour le tofacitinib et 80 % pour le ruxolitinib entre le début et la fin du suivi) et les patients en échec ont également eu une décroissance des corticoïdes évaluée à 65 %. La tolérance aux inhibiteurs de JAK a été excellente chez tous les patients sauf un qui a présenté une pneumopathie organisée ce qui a conduit à l’arrêt du traitement. Les inhibiteurs de JAK peuvent présenter un bénéfice pour certains patients présentant une MSA réfractaire, mais probablement pas en monothérapie. En effet, aucune rémission complète n’a été observée dans cette série de cas. Il existe potentiellement une différence de réponse entre les molécules, cependant le nombre de patients est trop faible pour conclure. Des informations additionnelles sont nécessaires afin d’évaluer plus précisément la balance bénéfice-risque de ce traitement ainsi que l’analyse d’une différence d’efficacité entre les différentes molécules du groupe des inhibiteurs de JAK.

Abstract The E3 ubiquitin ligase Casitas B-lineage lymphoma b (CBL-B) is expressed in T cells where it functions as an important negative regulator of immune activation. CBL-B d attenuates T-cell activation initiated by TCR engagement in part by mediating the requirement for CD28 co-stimulation, thus setting the threshold for T cell activation. CD4+ and CD8+ T cells from mice deficient in cbl-b have 5 to 10-fold enhanced secretion of IL-2 and IFN-γ when stimulated ex vivo with anti-CD3. Cbl-b deficient mice also demonstrate enhanced NK cell function. Here we describe NX-1607, an investigational, orally bioavailable, small molecule inhibitor of CBL-B. NX-1607 demonstrates potent biochemical inhibition of CBL-B leading to stimulatory effects on human immune cells at low nanomolar concentrations. NX-1607 induction of IL-2 and IFN-γ secretion occurs in primary human T cells stimulated with anti-CD3 antibodies, in both the presence and absence of CD28 co-stimulation, although to a lesser degree in the absence of co-stimulation. In vivo, oral administration of NX-1607 in mice demonstrated significant tumor growth inhibition in two colon carcinoma tumor models, CT26 and MC38, as well as a triple negative breast tumor model, 4T1. The change in tumor microenvironment caused by NX-1607 treatment leads to rapid NK cell infiltration followed by infiltration of activated CD8+ T cells. Depletion of CD8+ T cells or NK cells completely abrogated NX-1607 antitumor activity. Importantly, the combination of NX-1607 and anti-PD-1 can substantially increase the median overall survival and the frequency of complete tumor rejections in all three tumor models. These studies provide significant insights into the antitumor activity of this novel, small molecule E3 ligase inhibitor and deliver experimental support for clinical development of NX-1607 given as monotherapy or in combination with PD-1 blockade. Citation Format: Ryan Rountree, Frederick Cohen, Austin Tenn-McClellan, Alexandra Borodovsky, Marilena Gallotta, Jennifer Stokes, Jose Gomez Romo, Chris Karim, Gwenn M. Hansen, Cristiana Guiducci, Arthur Sands, Jennifa Gosling. Small molecule inhibition of the ubiquitin ligase CBL-B results in potent T and NK cell mediated anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1595.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a Zn2+ deacetylase that is essential for the survival of most pathogenic Gram-negative bacteria. ACHN-975 (N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(((1R,2R)-2-(hydroxymethyl)cyclopropyl)buta-1,3-diyn-1-yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose-limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN-975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC-516, (S)-N-(2-(hydroxyamino)-1-(3-methoxy-1,1-dioxidothietan-3-yl)-2-oxoethyl)-4-(6-hydroxyhexa-1,3-diyn-1-yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30 mg mL-1 for parenteral administration and conversion into the active drug with a t1/2 of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development.

The lipid A biosynthesis pathway is essential in Pseudomonas aeruginosa. LpxA and LpxD are the first and third enzymes in this pathway respectively, and are regarded as promising antibiotic targets. The unique structural similarities between these two enzymes make them suitable targets for dual-binding inhibitors, a characteristic that would decrease the likelihood of mutational resistance and increase cell-based activity. We report the discovery of multiple small molecule ligands that bind to P. aeruginosa LpxA and LpxD, including dual-binding ligands. Binding poses were determined for select compounds by X-ray crystallography. The new structures reveal a previously uncharacterized magnesium ion residing at the core of the LpxD trimer. In addition, ligand binding in the LpxD active site resulted in conformational changes in the distal C-terminal helix-bundle, which forms extensive contacts with acyl carrier protein (ACP) during catalysis. These ligand-dependent conformational changes suggest a potential allosteric influence of reaction intermediates on ACP binding, and vice versa. Taken together, the novel small molecule ligands and their crystal structures provide new chemical scaffolds for ligand discovery targeting lipid A biosynthesis, while revealing structural features of interest for future investigation of LpxD function.

New drugs with novel mechanisms of resistance are desperately needed to address both community and nosocomial infections due to Gram-negative bacteria. One such potential target is LpxC, an essential enzyme that catalyzes the first committed step of lipid A biosynthesis. Achaogen conducted an extensive research campaign to discover novel LpxC inhibitors with activity against Pseudomonas aeruginosa. We report here the in vitro antibacterial activity and pharmacodynamics of ACHN-975, the only molecule from these efforts and the first ever LpxC inhibitor to be evaluated in phase 1 clinical trials. In addition, we describe the profiles of three additional LpxC inhibitors that were identified as potential lead molecules. These efforts did not produce an additional development candidate with a sufficiently large therapeutic window and the program was subsequently terminated.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a Zn2+ deacetylase that is essential for the survival of most pathogenic Gram-negative bacteria. ACHN-975 (N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(((1R,2R)-2-(hydroxymethyl)cyclopropyl)buta-1,3-diyn-1-yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose-limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN-975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC-516, (S)-N-(2-(hydroxyamino)-1-(3-methoxy-1,1-dioxidothietan-3-yl)-2-oxoethyl)-4-(6-hydroxyhexa-1,3-diyn-1-yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30 mg mL-1 for parenteral administration and conversion into the active drug with a t1/2 of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development.

A major challenge for new antibiotic discovery is predicting the physicochemical properties that enable small molecules to permeate Gram-negative bacterial membranes. We have applied physicochemical lessons from previous work to redesign and improve the antibacterial potency of pyridopyrimidine inhibitors of biotin carboxylase (BC) by up to 64-fold and 16-fold against Escherichia coli and Pseudomonas aeruginosa, respectively. Antibacterial and enzyme potency assessments in the presence of an outer membrane-permeabilizing agent or in efflux-compromised strains indicate that penetration and efflux properties of many redesigned BC inhibitors could be improved to various extents. Spontaneous resistance to the improved pyridopyrimidine inhibitors in P. aeruginosa occurs at very low frequencies between 10(-8) and 10(-9). However, resistant isolates had alarmingly high minimum inhibitory concentration shifts (16- to >128-fold) compared to the parent strain. Whole-genome sequencing of resistant isolates revealed that either BC target mutations or efflux pump overexpression can lead to the development of high-level resistance.

E3 ubiquitin ligases play critical roles in directing cellular protein fate by controlling the specificity of ubiquitin conjugation to substrate proteins and targeting them for cellular relocalization or degradation by the ubiquitin proteasome system. The E3 ubiquitin ligase CBL-B is expressed in immune cell lineages and negatively regulates activity of the T-cell receptor (TCR) by imposing a requirement for a costimulatory signal to mount a productive immune response upon TCR engagement. Mice deficient in Cbl-b, and more specifically in the RING Zn-finger ligase domain of Cbl-b, demonstrate a tumor rejection phenotype mediated by CD8+ T cells (Paolino et al., JI, 2011). We have reproduced these results and demonstrate that Cbl-b deficient mice show enhanced anti-tumor activity. In addition, we show that CD4+ and CD8+ T cells from mice deficient in Cbl-b have 5 to 10-fold enhanced secretion of IL-2 and IFN γ when stimulated ex vivo. These data provide a genetic rationale for the development of a small molecule inhibitor of CBL-B ligase activity for use in patients with tumor-mediated immune suppression of effector T cells. We have identified a series of small molecule inhibitors of CBL-B activity with biochemical potency at low nanomolar concentrations. CBL-B inhibitors increased cytokine secretion in vitro at low nanomolar concentrations, as measured by IL-2 and IFN γ secretion, in primary human and mouse T cells stimulated with CD3/CD28 or CD3 alone. The compounds also stimulated proliferation and elevated levels of the T cell surface activation markers CD25 and CD69. CBL-B inhibitors enhanced an antigen recall response in human PBMCs ex vivo, as measured by approximately 5-fold higher secretion of GM-CSF, TNF-α and RANTES, and demonstrated effects in an ex vivo model of exhausted T cell function. Oral dosing of an optimized CBL-B inhibitor enhanced anti-CD3 stimulated T cell activation in mouse CD4+ and CD8+ T cells, demonstrating a dose proportional pharmacodynamic effect. Oral administration over 28 days in the syngeneic CT-26 tumor model was well tolerated and resulted in single agent tumor growth inhibition. These data support the continued advancement of small molecule oral CBL-B inhibitors for future development in immuno-oncology. Citation Format: Jennifa Gosling, Ryan Rountree, Asad Taherbhoy, Chenbo Wang, Thomas Cummins, Frederick Cohen, Hiroko Tanaka, Dahlia Weiss, Mario Cardozo, Christopher Karim, May Tan, Joseph Juan, Austin Tenn-McClellan, Szerenke Kiss von Soly, Julie Sheung, Kathleen Boyle, Ketki Dhamnaskar, Katherine Kurylo, Jilliane Bruffey, Jennifer McKinnell, Dane Karr, Andria Christianson, Anne-Renee Van Der Vuurst de Vries, Pallavur Sivakumar, Mark Gallop, Paul A. Barsanti, Anjanabha Saha, Neil F. Bence, Christoph W. Zapf. Genetic and pharmacologic evaluation of the ubiquitin ligase CBL-B as a small-molecule, tumor immunotherapy target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2696.

The ubiquitin system regulates the majority of cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains, and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates, including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumor suppressor and other proteins critical for tumor cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance (NMR)-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumor cell death and enhance cytotoxicity with chemotherapeutics and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 noncovalently target USP7 12A distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate H-bond interactions with the ubiquitin Lys-48 side-chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties having free Lys-48 side-chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding via NMR, a study that substantiated our hypothesis. This preferential binding significantly protracted the depolymerization kinetics of Lys-48-linked ubiquitin chains relative to Lys-63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity. [LK, PDL, and LR contributed equally to this work.] Citation Format: Ingrid Wertz, Lorna Kategaya, Paola Di Lello, Lionel Rouge, Richard Pastor, Kevin R. Clark, Jason Drummond, Tracy Kleinheinz, Eva Lin, John-Paul Upton, Sumit Prakash, Johanna Heideker, Mark McCleland, Maria Stella Ritorto, Dario R. Alessi, Matthias Trost, Travis W. Bainbridge, Michael C. Kwok, Taylur P. Ma, Zachary Stiffler, Bradley Brasher, Yinyan Tang, Priya Jaishanker, Brian Hearn, Adam R. Renslo, Michelle R. Arkin, Frederick Cohen, Kebing Yu, Frank Peale, Florian Gnad, Matthew T. Chang, Christiaan Klijn, Elizabeth Blackwood, Scott E. Martin, William F. Forrest, James A. Ernst, Chudi Ndubaku, Xiaojing Wang, Maureen H. Beresini, Vickie Tsui, Carsten Schwerdtfeger, Robert A. Blake, Jeremy Murray, Till Maurer. Development and mechanistic characterization of USP7 deubiquitinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY23-03.

The ubiquitin system regulates the majority of cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains, and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates, including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumor suppressor and other proteins critical for tumor cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance (NMR)-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumor cell death and enhance cytotoxicity with chemotherapeutics and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 noncovalently target USP7 12A distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate H-bond interactions with the ubiquitin Lys-48 side-chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties having free Lys-48 side-chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding via NMR, a study that substantiated our hypothesis. This preferential binding significantly protracted the depolymerization kinetics of Lys-48-linked ubiquitin chains relative to Lys-63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity. [LK, PDL, and LR contributed equally to this work.] Citation Format: Ingrid Wertz, Lorna Kategaya, Paola Di Lello, Lionel Rouge, Richard Pastor, Kevin R. Clark, Jason Drummond, Tracy Kleinheinz, Eva Lin, John-Paul Upton, Sumit Prakash, Johanna Heideker, Mark McCleland, Maria Stella Ritorto, Dario R. Alessi, Matthias Trost, Travis W. Bainbridge, Michael C. Kwok, Taylur P. Ma, Zachary Stiffler, Bradley Brasher, Yinyan Tang, Priya Jaishanker, Brian Hearn, Adam R. Renslo, Michelle R. Arkin, Frederick Cohen, Kebing Yu, Frank Peale, Florian Gnad, Matthew T. Chang, Christiaan Klijn, Elizabeth Blackwood, Scott E. Martin, William F. Forrest, James A. Ernst, Chudi Ndubaku, Xiaojing Wang, Maureen H. Beresini, Vickie Tsui, Carsten Schwerdtfeger, Robert A. Blake, Jeremy Murray, Till Maurer. Development and mechanistic characterization of USP7 deubiquitinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY23-03.

The development of selective ubiquitin-specific protease-7 (USP7) inhibitors GNE-6640 and GNE-6776, which induce tumour cell death and reveal differential kinetics of Lys-48 and Lys-63-linked ubiquitin chain depolymerization by USP7. Deubiquitinating enzymes remove the small modifier protein ubiquitin from target substrates regulating their stability. One such enzyme, USP7, is a potential target for anti-cancer therapy, as its inhibition would result in the degradation of the ubiquitinated oncoprotein MDM2, leading to reactivation of the tumour suppressor protein p53. However, selective inhibitors of USP7 have remained elusive. Here, Ingrid Wertz and team develop two USP7 inhibitors, providing structural insights into the mode of action of these compounds and demonstrating their toxicity towards tumour cells. Elsewhere in this issue, David Komander and colleagues independently report the identification of two small molecules that inhibit USP7 with high affinity and specificity both in vitro and within cells, also demonstrating their ability to inhibit tumour growth. The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation1. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease2; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival3. However, developing selective deubiquitinase inhibitors has been challenging4 and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain5, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

USP7 is a deubiquitinase implicated in destabilizing the tumor suppressor p53, and for this reason it has gained increasing attention as a potential oncology target for small molecule inhibitors. Herein we describe the biophysical, biochemical, and computational approaches that led to the identification of 4-(2-aminopyridin-3-yl)phenol compounds described by Kategaya ( Nature 2017, 550, 534-538) as specific inhibitors of USP7. Fragment based lead discovery (FBLD) by NMR combined with virtual screening and re-mining of biochemical high-throughput screening (HTS) hits led to the discovery of a series of ligands that bind in the palm region of the catalytic domain of USP7 and inhibit its catalytic activity. These ligands were then optimized by structure-based design to yield cell-active molecules with reasonable physical properties. This discovery process not only involved multiple techniques working in concert but also illustrated a unique way in which hits from orthogonal screening approaches complemented each other for lead identification.