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Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity, and cell permeability to give BJP-06–005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.

Chronic inflammation is linked to diverse disease processes, but the intrinsic mechanisms that determine cellular sensitivity to inflammation are incompletely understood. Here, we show the contribution of glucose metabolism to inflammation-induced changes in the survival of pancreatic islet β-cells. Using metabolomics, biochemical and functional analyses, we investigate the protective versus non-protective effects of glucose in the presence of pro-inflammatory cytokines. When protective, glucose metabolism augments anaplerotic input into the TCA cycle via pyruvate carboxylase (PC) activity, leading to increased aspartate levels. This metabolic mechanism supports the argininosuccinate shunt, which fuels ureagenesis from arginine and conversely diminishes arginine utilization for production of nitric oxide (NO), a chief mediator of inflammatory cytotoxicity. Activation of the PC-urea cycle axis is sufficient to suppress NO synthesis and shield cells from death in the context of inflammation and other stress paradigms. Overall, these studies uncover a previously unappreciated link between glucose metabolism and arginine-utilizing pathways via PC-directed ureagenesis as a protective mechanism.

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide (Pom) bind to cereblon (CRBN) and trigger proteasomal degradation of neo-substrates IKZF1/3 leading to multiple myeloma (MM) cell apoptosis. Pomalidomide (Pom) also binds albeit weakly to p53-related protein kinase (PRPK, aka TP53RK), an understudied kinase reported to be associated with poor prognosis in MM patients. Here, we developed a series of IMiDs based on Pom and conducted a structure-activity relationship (SAR) study to identify a potent and selective PRPK binder. Structural analysis showed that IMiDs bind PRPK in a fundamentally different way from CRBN, and suggested specific derivatization to improve affinity. We employed a structure-guided strategy to develop compound TXM-02-118, which exhibited nanomolar affinityfor PRPK in binding assays, and showed high selectivity for PRPK over CRBN. Overall, the work represents an initial effort to develop tool compounds for studying PRPK. Moreover, it illustrates how a single class of molecules can use different recognition elements to bind diverse targets using fundamentally different binding poses. This has broad implications for chemical probe and lead compound selectivity profiling, and argues for more wide-spread use of global proteomics or similar methodologies.

SUMMARYDevelopment of affordable and effective vaccines that can also protect vulnerable populations such as the elderly from COVID-19-related morbidity and mortality is a public health priority. Here we took a systematic and iterative approach by testing several SARS-CoV-2 protein antigens and adjuvants to identify a combination that elicits neutralizing antibodies and protection in young and aged mice. In particular, SARS-CoV-2 receptorbinding domain (RBD) displayed as a protein nanoparticle (RBD-NP) was a highly effective antigen, and when formulated with an oil-in-water emulsion containing Carbohydrate fatty acid MonoSulphate derivative (CMS) induced the highest levels of cross-neutralizing antibodies compared to other oil-in-water emulsions or AS01B. Mechanistically, CMS induced antigen retention in the draining lymph node (dLN) and expression of cytokines, chemokines and type I interferon-stimulated genes at both injection site and dLN. Overall, CMS:RBD-NP is effective across multiple age groups and is an exemplar of a SARS-CoV-2 subunit vaccine tailored to the elderly.

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5BN642H, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5BN642H in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5BN642H-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5BN642H patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5BN642H crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5BN642H can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5BN642H, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5BN642H activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups (∼80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups.One Sentence SummaryAlum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.

Alterations in non-driver genes represent an emerging class of potential therapeutic targets in cancer. Hundreds to thousands of non-driver genes undergo loss of heterozygosity (LOH) events per tumor, generating discrete differences between tumor and normal cells. Here we interrogate LOH of polymorphisms in essential genes as a novel class of therapeutic targets. We hypothesized that monoallelic inactivation of the allele retained in tumors can selectively kill cancer cells but not somatic cells, which retain both alleles. We identified 5664 variants in 1278 essential genes that undergo LOH in cancer and evaluated the potential for each to be targeted using allele-specific gene-editing, RNAi, or small-molecule approaches. We further show that allele-specific inactivation of either of two essential genes (PRIM1 and EXOSC8) reduces growth of cells harboring that allele, while cells harboring the non-targeted allele remain intact. We conclude that LOH of essential genes represents a rich class of non-driver cancer vulnerabilities., In tumors, hundreds of genes can undergo loss of heterozygosity (LOH). Here, the authors investigate the potential for this LOH as a class of non-driver cancer vulnerabilities.

Summary The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) “killer domains” of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.

The peptidyl-prolyl cis-trans isomerase, Pin1, acts as a unified signaling hub that is exploited in cancer to activate oncogenes and inactivate tumor suppressors, in particular through up-regulation of c-Myc target genes. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to discover covalent inhibitors targeting Pin1’s active site nucleophile - Cys113, leading to the development of Sulfopin, a double-digit nanomolar Pin1 inhibitor. Sulfopin is highly selective for Pin1, as validated by two independent chemoproteomics methods, achieves potent cellular andin vivotarget engagement, and phenocopies genetic knockout of Pin1. Although Pin1 inhibition had a modest effect on viability in cancer cell cultures, Sulfopin induced downregulation of c-Myc target genes and reduced tumor initiation and tumor progression in murine and zebrafish models of MYCN-driven neuroblastoma. Our results suggest that Sulfopin is a suitable chemical probe for assessing Pin1-dependent pharmacology in cells andin vivo. Moreover, these studies indicate that Pin1 should be further investigated as a potential cancer target.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1’s active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long distance transport of non-membrane enclosed organelles such as RNA granules is not known. Here we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts directly with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that binding of SFPQ to KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie-Tooth (CMT) Disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations show that non-membrane enclosed organelles can move autonomously and that replacing axonally translated proteins provides a therapeutic approach to axonal degenerative disorders.

Arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) combination safely cures fatal acute promyelocytic leukemia, but their mechanisms of action and efficacy are not fully understood. ATRA inhibits leukemia, breast, and liver cancer by targeting isomerase Pin1, a master regulator of oncogenic signaling networks. Here we show that ATO targets Pin1 and cooperates with ATRA to exert potent anticancer activity. ATO inhibits and degrades Pin1, and suppresses its oncogenic function by noncovalent binding to Pin1’s active site. ATRA increases cellular ATO uptake through upregulating aquaporin-9. ATO and ATRA, at clinically safe doses, cooperatively ablate Pin1 to block numerous cancer-driving pathways and inhibit the growth of triple-negative breast cancer cells and tumor-initiating cells in cell and animal models including patient-derived orthotopic xenografts, like Pin1 knockout, which is substantiated by comprehensive protein and microRNA analyses. Thus, synergistic targeting of Pin1 by ATO and ATRA offers an attractive approach to combating breast and other cancers., Arsenic trioxide and all-trans retinoic acid combination safely cures human acute promyelocytic leukemia. Here, the authors show that this combination has potent anticancer activity in triple negative breast cancer by cooperatively targeting Pin1, a master regulator of oncogenic signaling networks, to eliminate cancer stem cells.

Purinergic receptor-7 (P2X7R) signaling controls Th17 and Th1 generation/differentiation, while NOD-like receptor P3 (NLRP3) acts as a Th2 transcriptional factor. Here, we demonstrated the existence of a P2X7R/NLRP3 pathway in T cells that is dysregulated by a P2X7R intracellular region loss-of-function mutation, leading to NLRP3 displacement and to excessive Th17 generation due to abrogation of the NLRP3-mediated Th2 program. This ultimately resulted in poor outcomes in cardiac-transplanted patients carrying the mutant allele, who showed abnormal Th17 generation. Transient NLRP3 silencing in nonmutant T cells or overexpression in mutant T cells normalized the Th profile. Interestingly, IL-17 blockade reduced Th17 skewing of human T cells in vitro and abrogated the severe allograft vasculopathy and abnormal Th17 generation observed in preclinical models in which P2X7R was genetically deleted. This P2X7R intracellular region mutation thus impaired the modulatory effects of P2X7R on NLRP3 expression and function in T cells and led to NLRP3 dysregulation and Th17 skewing, delineating a high-risk group of cardiac-transplanted patients who may benefit from personalized therapy.

Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.

Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.

SummaryThe PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here we present the discovery and characterization of a novel pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K’s reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

[Image: see text] Recent reports have highlighted the dual bromodomains of TAF1 (TAF1(1,2)) as synergistic with BET inhibition in cellular cancer models, engendering interest in TAF/BET polypharmacology. Here, we examine structure activity relationships within the BI-2536 PLK1 kinase inhibitor scaffold, previously reported to bind BRD4. We examine binding by this ligand to TAF1(2) and apply structure guided design strategies to discriminate binding to both the PLK1 kinase and BRD4(1) bromodomain while retaining activity on TAF1(2). Through this effort we discover potent dual inhibitors of TAF1(2)/BRD4(1), as well as biased derivatives showing marked TAF1 selectivity. We resolve X-ray crystallographic data sets to examine the mechanisms of the observed TAF1 selectivity and to provide a resource for further development of this scaffold.

The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As interest in covalent drugs has increased, so too has the need for analytical platforms that can leverage their mechanism-of-action to characterize modified protein targets. Here we describe novel gas phase dissociation pathways which yield predictable fragment ions during MS/MS of inhibitor-modified peptides. We find that these dissociation pathways are common to numerous cysteine-directed probes as well as the covalent drugs, Ibrutinib and Neratinib. We leverage the predictable nature of these fragment ions to improve the confidence of peptide sequence assignment in proteomic analyses and explore their potential use in selective mass spectrometry-based assays.

A Correction to this paper has been published: https://doi.org/10.1038/s41594-020-00540-y.

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.

p53 is a critical tumor suppressor protein that guards the human genome against mutations by inducing cell cycle arrest or apoptosis. Cancer cells subvert p53 by deletion, mutation, or overexpression of the negative regulators HDM2 and HDMX. For tumors that retain wild-type p53, its reactivation by pharmacologic targeting of HDM2 and/or HDMX represents a promising strategy, with a series of selective small-molecule HDM2 inhibitors and a dual HDM2/HDMX stapled-peptide inhibitor being evaluated in clinical trials. Because selective HDM2 targeting can cause hematologic toxicity, selective HDMX inhibitors could provide an alternative p53-reactivation strategy, but clinical candidates remain elusive. Here, we applied a mutation-scanning approach to uncover p53-based stapled peptides that are selective for HDMX. Crystal structures of stapled-peptide/HDMX complexes revealed a molecular mechanism for the observed specificity, which was validated by HDMX mutagenesis. Thus, we provide a blueprint for the development of HDMX-selective inhibitors to dissect and target the p53/HDMX interaction.

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

Extracellular ATP (eATP) activates T cells by engaging the P2X7R receptor. We identified two loss-of-function P2X7R mutations that are protective against type 1 diabetes (T1D) and thus hypothesized that eATP/P2X7R signaling may represent an early step in T1D onset. Specifically, we observed that in patients with newly diagnosed T1D, P2X7R is upregulated on CD8+ effector T cells in comparison with healthy control subjects. eATP is released at high levels by human/murine islets in vitro in high-glucose/inflammatory conditions, thus upregulating P2X7R on CD8+ T cells in vitro. P2X7R blockade with oxidized ATP reduces the CD8+ T cell–mediated autoimmune response in vitro and delays diabetes onset in NOD mice. Autoreactive CD8+ T-cell activation is highly dependent upon eATP/P2X7R-mediated priming, while a novel sP2X7R recombinant protein abrogates changes in metabolism and the autoimmune response associated with CD8+ T cells. eATP/P2X7R signaling facilitates the onset of autoimmune T1D by fueling autoreactive CD8+ cells and therefore represents a novel targeted therapeutic for the disorder.

Human ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that prevents protein degradation by removing polyubiquitin chains from its substrates. It regulates the stability of a number of human transcription factors and tumor suppressors and plays a critical role in the development of several types of cancer, including prostate and small cell lung cancer. In addition, human USP7 is targeted by several viruses of the Herpesviridae family and is required for effective herpesvirus infection. The USP7 C-terminal region (C-USP7) contains five ubiquitin-like domains (UBL1-5) that interact with several USP7 substrates. Although structures of the USP7 C terminus bound to its substrates could provide vital information for understanding USP7 substrate specificity, no such data has been available to date. In this work we have demonstrated that the USP7 ubiquitin-like domains can be studied in isolation by solution NMR spectroscopy, and we have determined the structure of the UBL1 domain. Furthermore, we have employed NMR and viral plaque assays to probe the interaction between the C-USP7 and HSV-1 immediate-early protein ICP0 (infected cell protein 0), which is essential for efficient lytic infection and virus reactivation from latency. We have shown that depletion of the USP7 in HFF-1 cells negatively affects the efficiency of HSV-1 lytic infection. We have also found that USP7 directly binds ICP0 via its C-terminal UBL1-2 domains and mapped the USP7-binding site for ICP0. Therefore, this study represents a first step toward understanding the molecular mechanism of C-USP7 specificity toward its substrates and may provide the basis for future development of novel antiviral and anticancer therapies.

Proline-directed phosphorylation at serine or threonine residues (pSer/Thr-Pro) regulates numerous cellular processes, including the cell cycle, transcription, and differentiation. Deregulation of such signaling networks is a hallmark of transformation and oncogenesis. Pin1, a peptidyl-prolyl isomerase, regulates the function and stability of phosphoproteins by catalyzing the cis/trans isomerization of pSer/Thr-Pro motifs. Pin1 is frequently overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC), and Pin1 is required for activated Ras to induce tumorigenesis. While mutations in KRAS are observed in 90-95% of human PDAC cases, it has historically proven very challenging to develop small molecules that inhibit mutant Ras function. Consequently, drug discovery efforts have turned to targets required for Ras-mediated transformation, such as Pin1. However, existing Pin1 inhibitors lack the potency, selectivity, and/or cell permeability to serve as informative cellular probes. We report a highly potent, cell-permeable Pin1 inhibitor that covalently targets Cys113, a conserved cysteine residue in the Pin1 active site. Through iterative rounds of synthesis and characterization, we developed inhibitor 1b. With a Ki of 15 nM as measured in biochemical binding and isomerase inhibition assays, 1bis currently the most potent Pin1 inhibitor available. Furthermore, in a chemoproteomic study using Covalent Inhibitor Target Site Identification (CITe-Id) to quantify the dose-dependent covalent labeling of 1b to individual cysteines across the proteome, Pin1 Cys113 was the only identified target, highlighting the pronounced selectivity of 1b for Pin1. We show that treatment with 1b diminishes viability of human PDAC cell lines, which can be fully rescued in corresponding Pin1 knockout cells generated using CRISPR/Cas9, showing that this phenotype is on-target. In parallel to inhibitor development, we used CRISPR/Cas9 GFP-dropout screens to further validate the dependence of these cell lines on Pin1. Genetic disruption of Pin1 led to antiproliferative effects, confirming the results of 1b treatment. We also employed the degradation tag (dTAG) approach to assess the effects of rapid and selective targeted Pin1 degradation through generation of FKBP12F36V-Pin1, Pin1-/-human PDAC cell lines. Treatment with a small molecule FKBP12F36V-degrader led to rapid ubiquitination and degradation of FKBP12F36V-Pin1, enabling comparisons of targeted inhibition and Pin1 degradation. Through the development of a selective Pin1 inhibitor coupled with genetic approaches and the chemical-genetic dTAG strategy, we demonstrate that Pin1 inhibition represents a tractable strategy in PDAC. Citation Format: Benika Pinch, Zainab Doctor, Christopher M. Browne, Hyuk-Soo Seo, Behnam Nabet, Shingo Kozono, Xiaolan Lian, Daniel Zaidman, Dina Daitchman, Nir London, Lu Gong, Theresa Manz, Yujin Chun, Li Tan, Jarrod Marto, Stephen Buratowski, Sirano Dhe-Paganon, Xiao Zhou, Kun Ping Lu, Nathanael S. Gray. Discovery and characterization of covalent Pin1 inhibitors targeted to an active site cysteine [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 2757.

The repair of DNA double strand breaks by homologous recombination relies on the unique topology of the chains formed by Lys-63 ubiquitylation of chromatin to recruit repair factors such as breast cancer 1 (BRCA1) to sites of DNA damage. The human RING finger (RNF) E3 ubiquitin ligases, RNF8 and RNF168, with the E2 ubiquitin-conjugating complex Ubc13/Mms2, perform the majority of Lys-63 ubiquitylation in homologous recombination. Here, we show that RNF8 dimerizes and binds to Ubc13/Mms2, thereby stimulating formation of Lys-63 ubiquitin chains, whereas the related RNF168 RING domain is a monomer and does not catalyze Lys-63 polyubiquitylation. The crystal structure of the RNF8/Ubc13/Mms2 ternary complex reveals the structural basis for the interaction between Ubc13 and the RNF8 RING and that an extended RNF8 coiled-coil is responsible for its dimerization. Mutations that disrupt the RNF8/Ubc13 binding surfaces, or that truncate the RNF8 coiled-coil, reduce RNF8-catalyzed ubiquitylation. These findings support the hypothesis that RNF8 is responsible for the initiation of Lys-63-linked ubiquitylation in the DNA damage response, which is subsequently amplified by RNF168. Background: RNF8 and RNF168 are essential RING-E3 ubiquitin ligases that catalyze the formation of Lys-63 ubiquitin chains in the DNA damage response (DDR). Results: We solved the crystal structures and probed the activity of RNF8 and RNF168 in vitro. Conclusion: RNF168 likely acts downstream of RNF8 given its deficient activity. Significance: Our data provide structural and catalytic insight into the relative activities of RNF8 and RNF168.

Ube3A (also referred to as E6AP for E6 Associated Protein) is a E3 ubiquitin-protein ligase implicated in the development of Angelman syndrome by controlling degradation of synaptic protein Arc and oncogenic papilloma virus infection by controlling degradation of p53. This article describe the solution NMR structure of the conserved N-terminal domain of human Ube3A (residues 24-87) that contains two residues (Cys44 and Arg62) found to be mutated in patients with Angelman syndrome. The structure of this domain adopts a novel Zn-binding fold we called AZUL (Amino-terminal Zn-finger of Ube3a Ligase). The AZUL domain has a helix-loop-helix architecture with a Zn ion coordinated by four Cys residues arranged in Cys-X(4)-Cys-X(4)-Cys-X(28)-Cys motif. Three of the Zn-bound residues are located in a 23-residue long and well structured loop that connects two α-helicies.

MLN4924 is a selective inhibitor of the NEDD8-activating enzyme (NAE) and has advanced into clinical trials for the treatment of both solid and hematological malignancies. In contrast, the structurally similar compound 1 (developed by Millennium: The Takeda Oncology Company) is a pan inhibitor of the E1 enzymes NAE, ubiquitin activating enzyme (UAE), and SUMO-activating enzyme (SAE) and is currently viewed as unsuitable for clinical use given its broad spectrum of E1 inhibition. Here, we sought to understand the determinants of NAE selectivity. A series of compound 1 analogues were synthesized through iterative functionalization of the purine C6 position and evaluated for NAE specificity. Optimal NAE specificity was achieved through substitution with primary N-alkyl groups, while bulky or secondary N-alkyl substituents were poorly tolerated. When assessed in vitro, inhibitors reduced the growth and viability of malignant K562 leukemia cells. Through this study, we have successfully identified a series of sub-10 nM NAE-specific inhibitors and thereby highlighted the functionalities that promote NAE selectivity.

BCL-2 family proteins are master regulators of the mitochondrial apoptotic pathway and dictate whether a cell will live or die in response to stress. Cancer cells hijack the anti-apoptotic arm of this signaling network, overexpressing BCL-2, MCL-1, and BFL-1, to enforce cellular immortality. Whereas BCL-2 has been drugged by the selective small-molecule inhibitor, venetoclax, and new MCL-1 inhibitor molecules have just entered phase I clinical testing, anti-apoptotic BFL-1 remains undrugged. Importantly, the expression of BFL-1 can cause resistance to these molecules and has been independently linked to the pathogenesis and chemoresistance of human cancers, including melanomas and hematologic malignancies. Given the emergence of BCL-2 family proteins as high-priority cancer targets, solving their structures, alone and in complex with their natural BH3 domain ligands, yields important blueprints for drug design. Whereas the structures of numerous “BH3-in-groove” complexes have been solved, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69 Å resolution, revealing similarities and key differences among unliganded anti-apoptotic targets. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by BH3-based stapled peptide inhibitors. The crystal structure of a complex between BFL-1 and a cysteine-reactive and stapled NOXA BH3 α-helix demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for selective targeting of BFL-1, and inform a new therapeutic strategy for overcoming BFL-1-dependent apoptotic blockades in human cancer. Citation Format: Edward P. Harvey, Hyuk-Soo Seo, Rachel M. Guerra, Gregory H. Bird, Sirano Dhe-Paganon, Loren D. Walensky. Structural basis for selective therapeutic targeting of BFL-1 in cancer by a covalent stapled peptide inhibitor [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 1901.

We have identified two new human genes that encode proteins with tandem pleckstrin homology-phosphotyrosine binding (PH-PTB) domains at their amino termini. Because the other known PH-PTB proteins (insulin receptor substrates: IRS-1, IRS-2, IRS-3, and IRS-4, and the downstream of kinases: DOK-1, DOK-2, and DOK-3) are substrates of insulin and insulin-like growth factor (IGF)-1 receptors, we asked whether these new proteins, termed IRS5/DOK4 and IRS6/DOK5, might also have roles in insulin and IGF-1 signaling. Northern analyses indicate that IRS5/DOK4 is ubiquitously expressed but most abundant in kidney and liver. IRS6/DOK5 expression is highest in skeletal muscle. Both proteins are tyrosine-phosphorylated in response to insulin and IGF-1 in transfected cells, although the kinetics differ. Insulin receptor-phosphorylated IRS5/DOK4 associates with RasGAP, Crk, Src, and Fyn, but not phosphatidylinositol 3-kinase p85, Grb2, SHP-2, Nck, or phospholipase Cgamma Src homology 2 domains, and activates MAPK in cells. IRS6/DOK5 neither associates with these Src homology 2 domains nor activates MAPK. IRS5/DOK4 and IRS6/DOK5 represent two new signaling proteins with potential roles in insulin and IGF-1 action.

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.

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

Mevalonate pyrophosphate decarboxylase catalyzes the decarboxylation of mevalonate pyrophosphate to isopentyl pyrophosphate. The mechanism of action of this enzyme was investigated to elucidate the mechanism of inhibition by 3-hydroxy-3-(fluoromethyl)-5-pyrophosphopentanoic acid (II). It was previously found that II is a competitive inhibitor (Ki = 0.01 microM) of the enzyme reaction [Reardon, J.E., & Abeles, R.H. (1987) Biochemistry 26, 4717-4722; Nave, J.F., d'Orchymont, H., Ducep, J.B., Piriou, F., & Jung, M.J. (1985) Biochem. J. 227, 247-254]. We have now observed that II is decarboxylated 2500-fold more slowly than mevalonate pyrophosphate (3-hydroxy-3-methyl-5-pyrophosphopentanoic acid, I). The enzyme was exposed to saturating concentrations of II and ATP and then passed through a Penefsky column to remove excess substrate. The enzyme was denatured immediately upon emerging from the Penefsky column. Nearly 1 equiv of both 3-phospho-3-(fluoromethyl)-5-pyrophosphopentanoic acid and ADP was bound to the enzyme. 3-Hydroxy-5-pyrophosphopentanoic acid (III) is phosphorylated at the secondary hydroxyl group and released from the enzyme without decarboxylation. This reaction is 30-fold slower than the rate of decarboxylation of I. The introduction of the 3-fluoromethyl group as well as the removal of the 3-methyl group results in low rates of decarboxylation. These substrate analogs have decreased electron density relative to the tertiary carbon of the substrate. Therefore, the transition state of the decarboxylation step has considerable carbocationic character. Further support for the carbocationic transition state is provided by the finding that N-methyl-N-carboxymethyl-2-pyrophosphoethanolamine (IV) inhibits the enzyme reaction with Ki = 0.75 microM. IV is probably a transition-state analog in which the positively charged nitrogen atom is analogous to the carbocation.

Summary In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF Skp2 substrate p27 Kip1 . CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

The proteasomal pathway of protein degradation involves 2 discrete steps: ubiquitination and degradation. Here, we evaluated the effects of inhibiting the ubiquitination pathway at the level of the ubiquitin-activating enzyme UBA1 (E1). By immunoblotting, leukemia cell lines and primary patient samples had increased protein ubiquitination. Therefore, we examined the effects of genetic and chemical inhibition of the E1 enzyme. Knockdown of E1 decreased the abundance of ubiquitinated proteins in leukemia and myeloma cells and induced cell death. To further investigate effects of E1 inhibition in malignancy, we discovered a novel small molecule inhibitor, 3,5-dioxopyrazolidine compound, 1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furyl)methylene]-3,5-pyrazolidinedione (PYZD-4409). PYZD-4409 induced cell death in malignant cells and preferentially inhibited the clonogenic growth of primary acute myeloid leukemia cells compared with normal hematopoietic cells. Mechanistically, genetic or chemical inhibition of E1 increased expression of E1 stress markers. Moreover, BI-1 overexpression blocked cell death after E1 inhibition, suggesting ER stress is functionally important for cell death after E1 inhibition. Finally, in a mouse model of leukemia, intraperitoneal administration of PYZD-4409 decreased tumor weight and volume compared with control without untoward toxicity. Thus, our work highlights the E1 enzyme as a novel target for the treatment of hematologic malignancies.

Epigenetics is the study of the transmission of cell memory through mitosis or meiosis that is not based on the DNA sequence. At the molecular level the epigenetic memory of a cell is embedded in DNA methylation, histone post-translational modifications, RNA interference and histone isoform variation. There is a tight link between histone post-translational modifications (the histone code) and DNA methylation, as modifications of histones contribute to the establishment of DNA methylation patterns and vice versa. Interestingly, proteins have recently been identified that can simultaneously read both methylated DNA and the histone code. UHRF1 fulfills these requirements by having unique structural domains that allow concurrent recognition of histone modifications and methylated DNA. Herein, we review our current knowledge of UHRF1 and discuss how this protein ensures the link between histone marks and DNA methylation. Understanding the molecular functions of this protein may reveal the physiological relevance of the linkage between these layers of epigenetic marks.

Calpains are calcium activated cysteine proteases found throughout the animal, plant, and fungi kingdoms; 14 isoforms have been described in the human genome. Calpains have been implicated in multiple models of human disease; for instance, calpain 1 is activated in the brains of individuals with Alzheimer's disease, and the digestive tract specific calpain 9 is down-regulated in gastric cancer cell lines. We have solved the structures of human calpain 1 and calpain 9 protease cores using crystallographic methods; both structures have clear implications for the function of non-catalytic domains of full-length calpains in the calcium-mediated activation of the enzyme. The structure of minicalpain 1 is similar to previously solved structures of the protease core. Auto-inhibition in this system is most likely through rearrangements of a central helical/loop region near the active site cysteine, which occlude the substrate binding site. However, the structure of minicalpain 9 indicates that auto-inhibition in this enzyme is mediated through large intra-domain movements that misalign the catalytic triad. This disruption is reminiscent of the full-length inactive calpain conformation. The structures of the highly conserved, ubiquitously expressed human calpain 1 and the more tissue specific human calpain 9 indicate that although there are high levels of sequence conservation throughout the calpain family, isolated structures of family members are insufficient to explain the molecular mechanism of activation for this group of proteins.

Friedreich's ataxia, an autosomal recessive neurodegenerative disorder characterized by progressive gait and limb ataxia, cardiomyopathy, and diabetes mellitus, is caused by decreased frataxin production or function. The structure of human frataxin, which we have determined at 1.8-A resolution, reveals a novel protein fold. A five-stranded, antiparallel beta sheet provides a flat platform, which supports a pair of parallel alpha helices, to form a compact alphabeta sandwich. A cluster of 12 acidic residues from the first helix and the first strand of the large sheet form a contiguous anionic surface on the protein. The overall protein structure and the anionic patch are conserved in eukaryotes, including animals, plants, and yeast, and in prokaryotes. Additional conserved residues create an extended 1008-A(2) patch on a distinct surface of the protein. Side chains of disease-associated mutations either contribute to the anionic patch, help create the second conserved surface, or point toward frataxin's hydrophobic core. These structural findings predict potential modes of protein-protein and protein-iron binding.

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational conjugation of NEDD8 onto target proteins. Cullin proteins are major substrates of the neddylation pathway, and are involved in diverse cellular processes. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924 in leukemic cells. MLN4924 shows potent activity in K562 human leukemia cells (EC50 = 100 nM). In contrast, by exposing K562 cells to increasing concentrations of MLN4924 over 6 months, we selected a population of cells resistant to MLN4924 (R-K562MLN; EC50 > 2 µM). R-K562MLN cells proliferated similarly to parental K562 cells, and remained resistant to MLN4924 even after culturing in the absence of MLN4924 for 5 weeks. Compared to parental cells, R-K562MLN cells were similarly sensitive to a broad spectrum of chemotherapeutic drugs, including daunorubicin, a classical substrate of the multidrug efflux transporter P-glycoprotein. Thus, the mechanism of resistance to MLN4924 did not appear to be related to generalized multidrug efflux. While basal levels of NEDD8-conjugated cullin proteins were equivalent in parental and resistant cells, NEDD8-cullins were diminished in parental cells treated with 25 nM MLN4924. In contrast, no appreciable reduction in NEDD8-cullins was observed in R-K562MLN cells after treatment with up to 250 nM MLN4924. However, knockdown of NEDD8 by shRNA was cytotoxic to R-K562MLNcells, suggesting that NEDD8 conjugation remains essential for the survival of resistant cells. To further probe mechanisms of MLN4924 resistance in these cells, we sequenced the coding region of the NAE catalytic subunit UBA3 from K562 and R-K562MLN cells, and identified a mutation in codon 310 [ATT (Isoleucine, I) > AAT (Asparagine, N)] in R-K562MLN cells. Biochemical analyses using recombinant wild-type and I310N NAE complexes indicated that the I310N mutation alters biochemical properties of the NAE, increasing the enzyme’s affinity for ATP while decreasing its affinity for NEDD8. The I310N NAE complex was ∼7-fold less sensitive to MLN4924 (IC50 = 225 nM) compared to wild-type NAE (IC50 = 32 nM), providing a mechanistic basis for MLN4924 resistance in R-K562MLNcells through the UBA3 I310N mutation. To investigate whether another malignant leukemic cell line employs a similar on-target resistance mechanism, we similarly selected a population of U937 cells resistant to MLN4924 (R-U937MLN). Parental U937 cells were sensitive to MLN4924 (EC50 = 25 nM), while R-U937MLN cells were more than 40-fold more resistant (EC50 > 1 µM). Sequencing of the coding region of UBA3 in U937 and R-U937MLN cells revealed a point mutation in codon 352 [TAT (Tyrosine, Y) > CAT (Histidine, H)] only in R-U937MLN cells. As for the UBA3 I301N mutation, the Y352H mutation conferred a ∼10-fold decrease in the sensitivity of the NAE to MLN4924 (IC50= 138.5 nM and 13 nM for Y352H and wild-type, respectively) through decreased NEDD8 and increased ATP affinities. These findings suggest that the Y352H mutation, like I310N, is sufficient to provide MLN4924 resistance in leukemia cells while sustaining adequate NAE function for leukemic cell survival. We next found that R-K562MLN cells were cross-resistant to other NAE-selective inhibitors derived from Compound 1, a pan-E1 inhibitor. Nevertheless, compared to parental cells, R-K562MLN cells were not refractory to Compound 1 (EC50 = 27 nM and 81 nM, respectively). The cytotoxicity of Compound 1 towards MLN4924-resistant cells might be explained by its inhibition of other E1 enzymes. Indeed, Compound 1 diminished the abundance of ubiquitinated proteins in R-K562MLNcells similarly to its effects in K562 cells, while NEDD8-cullins in the MLN4924-resistant cells were unaffected. Overall, as MLN4924 continues to be evaluated clinically, our work provides insight into the mechanisms of MLN4924 resistance, which may facilitate the development of more effective second-generation NAE inhibitors. Disclosures: Petroski: Allostere, Inc.: Consultancy, Equity Ownership.