Cryo-EM structure of SKP1-SKP2-CKS1 in complex with CDK2-cyclin A-p27KIP1.

p27KIP1 (cyclin-dependent kinase inhibitor 1B, p27) is a member of the CIP/KIP family of CDK (cyclin dependent kinase) regulators that inhibit cell cycle CDKs. p27 phosphorylation by CDK1/2, signals its recruitment to the SCFSKP2 (S-phase kinase associated protein 1 (SKP1)-cullin-SKP2) E3 ubiquitin ligase complex for proteasomal degradation. The nature of p27 binding to SKP2 and CKS1 was revealed by the SKP1-SKP2-CKS1-p27 phosphopeptide crystal structure. Subsequently, a model for the hexameric CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex was proposed by overlaying an independently determined CDK2-cyclin A-p27 structure. Here we describe the experimentally determined structure of the isolated CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex at 3.4 Å global resolution using cryogenic electron microscopy. This structure supports previous analysis in which p27 was found to be structurally dynamic, transitioning from disordered to nascent secondary structure on target binding. We employed 3D variability analysis to further explore the conformational space of the hexameric complex and uncovered a previously unidentified hinge motion centred on CKS1. This flexibility gives rise to open and closed conformations of the hexameric complex that we propose may contribute to p27 regulation by facilitating recognition with SCFSKP2. This 3D variability analysis further informed particle subtraction and local refinement approaches to enhance the local resolution of the complex.

Variations of pain medication use for patients with acute extremity pain in an emergency department: A quality improvement project.

OBJECTIVE: The opioid epidemic continues to take over 50,000 lives annually. At least 75 percent of patients present to an emergency department (ED) for pain. The objective of this study is to describe the characteristic(s) for receiving opioid, non-opioid, and combination analgesics in an ED for acute extremity pain. METHODS: A single-site, retrospective chart audit was conducted at a community-based teaching hospital. Patients ≥ 18 years old who were discharged from the ED with acute extremity pain and received at least one analgesic were included. Primary goal included determining characteristics associated with the prescribing of analgesics. Secondary goals included amount of pain score reduction, frequency of prescribing, and discharge prescription patterns among each group. Analyses consisted of univariate and multivariate general linear models analyses. RESULTS: There were 878 patients identified as having acute extremity pain between February and April 2019. A total of 335 patients met inclusion criteria and were separated into three groups: nonopioids (n = 200), opioids (n = 97), and combination analgesics (n = 38). The individual characteristics showing statistical differences (p < 0.05) between the groups were (1) an allergy to specific analgesics, (2) diastolic blood pressure > 90 mmHg, (3) heart rate > 100 bpm, (4) opioid use prior to ED admission, (5) prescriber level, and (6) discharge diagnosis. Multivariate analyses showed combination therapy (regardless of which two analgesics were administered) had a significant difference in mean pain score reduction compared to nonopioids (p < 0.05). CONCLUSION: There are patient, prescriber, and environment-specific characteristics that are associated with analgesic selection in an ED. Combination therapy had the greatest reduction in pain regardless of the two medications received.

Phenyl Dihydrouracil: An Alternative Cereblon Binder for PROTAC Design.

Thalidomide and its analogues are frequently used in PROTAC design. However, they are known to be inherently unstable, undergoing hydrolysis even in commonly utilized cell culture media. We recently reported that phenyl glutarimide (PG)-based PROTACs displayed improved chemical stability and, consequently, improved protein degradation efficacy and cellular potency. Our optimization efforts, aiming to further improve the chemical stability and eliminate the racemization-prone chiral center in PG, led us to the development of phenyl dihydrouracil (PD)-based PROTACs. Here we describe the design and synthesis of LCK-directing PD-PROTACs and compare their physicochemical and pharmacological properties to those of the corresponding IMiD and PG analogues.

Mapping Ligand Interactions of Bromodomains BRD4 and ATAD2 with FragLites and PepLites─Halogenated Probes of Druglike and Peptide-like Molecular Interactions.

The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions. We extend the FragLite set with analogous compounds derived from amino acids (termed PepLites) that mimic the interactions of peptides. The output of the FragLite maps is shown to enable the development of ligands with leadlike potency. This work establishes the use of FragLite and PepLite screening at an early stage in ligand discovery allowing the rapid assessment of tractability of protein targets and informing downstream hit-finding.

Structural and Biochemical Features of Human Serum Albumin Essential for Eukaryotic Cell Culture.

Serum albumin physically interacts with fatty acids, small molecules, metal ions, and several other proteins. Binding with a plethora of bioactive substances makes it a critical transport molecule. Albumin also scavenges the reactive oxygen species that are harmful to cell survival. These properties make albumin an excellent choice to promote cell growth and maintain a variety of eukaryotic cells under in vitro culture environment. Furthermore, purified recombinant human serum albumin is mostly free from impurities and modifications, providing a perfect choice as an additive in cell and tissue culture media while avoiding any regulatory constraints. This review discusses key features of human serum albumin implicated in cell growth and survival under in vitro conditions.

The Common Germline TP53-R337H Mutation Is Hypomorphic and Confers Incomplete Penetrance and Late Tumor Onset in a Mouse Model.

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li-Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up. SIGNIFICANCE: A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation.

The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.

Mechanisms of NT5C2-Mediated Thiopurine Resistance in Acute Lymphoblastic Leukemia.

Relapse remains a formidable challenge for acute lymphoblastic leukemia (ALL). Recently, recurrent mutations in NT5C2 were identified as a common genomic lesion unique in relapsed ALL and were linked to acquired thiopurine resistance. However, molecular mechanisms by which NT5C2 regulates thiopurine cytotoxicity were incompletely understood. To this end, we sought to comprehensively characterize the biochemical and cellular effects of NT5C2 mutations. Compared with wild-type NT5C2, mutant proteins showed elevated 5'-nucleotidase activity with a stark preference of thiopurine metabolites over endogenous purine nucleotides, suggesting neomorphic effects specific to thiopurine metabolism. Expression of mutant NT5C2 mutations also significantly reduced thiopurine uptake in vitro with concomitant increase in efflux of 6-mercaptopurine (MP) metabolites, plausibly via indirect effects on drug transporter pathways. Finally, intracellular metabolomic profiling revealed significant shifts in nucleotide homeostasis induced by mutant NT5C2 at baseline; MP treatment also resulted in global changes in metabolomic profiles with completely divergent effects in cells with mutant versus wild-type NT5C2. Collectively, our data indicated that NT5C2 mutations alter thiopurine metabolism and cellular disposition, but also influence endogenous nucleotide homeostasis and thiopurine-induced metabolomic response. These complex mechanisms contributed to NT5C2-mediated drug resistance in ALL and pointed to potential opportunities for therapeutic targeting in relapsed ALL.

The Pioneering Role of GATA2 in Androgen Receptor Variant Regulation Is Controlled by Bromodomain and Extraterminal Proteins in Castrate-Resistant Prostate Cancer.

The androgen receptor (AR) is a key driver of prostate cancer development. Antiandrogens effectively inactivate the AR, but subsequent AR reactivation progresses the disease to castrate-resistant prostate cancer (CRPC). Constitutively active AR splice variants (AR-V) that function unchallenged by current AR-targeted therapies are key drivers of CRPC. Currently, very little is known about the regulation of AR-Vs at the chromatin level. Here, we show that the pioneer factor GATA2 is a critical regulator of AR-Vs. Furthermore, we demonstrate that the GATA2 cistrome in CRPC shares considerable overlap with bromodomain and extraterminal (BET) proteins and is codependent for DNA binding. GATA2 activity is compromised by BET inhibitors, which attenuates the pioneering role of GATA2 in CRPC. In all, this study indicates that GATA2 is a critical regulator of AR-V-mediated transactivation and is sensitive to BET inhibitors, signifying these agents may be efficacious in patients with CRPC which overexpress GATA2. IMPLICATIONS: We have defined novel mechanisms of AR-V and GATA2 regulation in advanced prostate cancer that could be therapeutically exploited.

Small-molecules that bind to the ubiquitin-binding motif of REV1 inhibit REV1 interaction with K164-monoubiquitinated PCNA and suppress DNA damage tolerance.

REV1 protein is a mutagenic DNA damage tolerance (DDT) mediator and encodes two ubiquitin-binding motifs (i.e., UBM1 and UBM2) that are essential for the DDT function. REV1 interacts with K164-monoubiquitinated PCNA (UbPCNA) in cells upon DNA-damaging stress. By using AlphaScreen assays to detect inhibition of REV1 and UbPCNA protein interactions along with an NMR-based strategy, we identified small-molecule compounds that inhibit the REV1/UbPCNA interaction and that directly bind to REV1 UBM2. In cells, one of the compound prevented recruitment of REV1 to PCNA foci on chromatin upon cisplatin treatment, delayed removal of UV-induced cyclopyrimidine dimers from nuclei, prevented UV-induced mutation of HPRT gene, and diminished clonogenic survival of cells that were challenged by cyclophosphamide or cisplatin. This study demonstrates the potential utility of a small-molecule REV1 UBM2 inhibitor for preventing DDT.

Alteration of RNA Splicing by Small-Molecule Inhibitors of the Interaction between NHP2L1 and U4.

Splicing is an important eukaryotic mechanism for expanding the transcriptome and proteome, influencing a number of biological processes. Understanding its regulation and identifying small molecules that modulate this process remain a challenge. We developed an assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to detect the interaction between the protein NHP2L1 and U4 RNA, which are two key components of the spliceosome. We used this assay to identify small molecules that interfere with this interaction in a high-throughput screening (HTS) campaign. Topotecan and other camptothecin derivatives were among the top hits. We confirmed that topotecan disrupts the interaction between NHP2L1 and U4 by binding to U4 and inhibits RNA splicing. Our data reveal new functions of known drugs that could facilitate the development of therapeutic strategies to modify splicing and alter gene function.

Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo.

The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1) capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2) capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction. IMPORTANCE: Acute gastroenteritis, with its sequela diarrhea, is one of the most important causes of childhood morbidity and mortality worldwide. A variety of infectious agents cause gastroenteritis, and in many cases, an enterotoxin produced by the agent is involved in disease manifestations. Although we commonly think of bacteria as a source of toxins, at least one enteric virus, rotavirus, produces a protein with enterotoxigenic activity during viral replication. In these studies, we demonstrate that oral administration of the turkey astrovirus 2 (TAstV-2) structural (capsid) protein induces acute diarrhea, increases barrier permeability, and causes relocalization of NHE3 in the small intestine, suggesting that rotavirus may not be alone in possessing enterotoxigenic activity.

Identification of the first small-molecule inhibitor of the REV7 DNA repair protein interaction.

DNA interstrand crosslink (ICL) repair (ICLR) has been implicated in the resistance of cancer cells to ICL-inducing chemotherapeutic agents. Despite the clinical significance of ICL-inducing chemotherapy, few studies have focused on developing small-molecule inhibitors for ICLR. The mammalian DNA polymerase ζ, which comprises the catalytic subunit REV3L and the non-catalytic subunit REV7, is essential for ICLR. To identify small-molecule compounds that are mechanistically capable of inhibiting ICLR by targeting REV7, high-throughput screening and structure-activity relationship (SAR) analysis were performed. Compound 1 was identified as an inhibitor of the interaction of REV7 with the REV7-binding sequence of REV3L. Compound 7 (an optimized analog of compound 1) bound directly to REV7 in nuclear magnetic resonance analyses, and inhibited the reactivation of a reporter plasmid containing an ICL in between the promoter and reporter regions. The normalized clonogenic survival of HeLa cells treated with cisplatin and compound 7 was lower than that for cells treated with cisplatin only. These findings indicate that a small-molecule inhibitor of the REV7/REV3L interaction can chemosensitize cells by inhibiting ICLR.

Short article: Successful faecal coliform sensitivity-based oral ertapenem therapy for chronic antibiotic-refractory pouchitis: a case series.

Pouchitis is a common complication of restorative proctocolectomy for ulcerative colitis, and a proportion of patients develop a refractory course. The treatment of chronic antibiotic-refractory pouchitis (CARP) is challenging, and treatment failure is often a cause of pouch excision. We report on a series of three patients with CARP who were treated with oral ertapenem following faecal coliform sensitivity testing. There was an improvement in the pouchitis disease activity index in all three patients [pretreatment pouch disease activity index, median 13 (range: 10-14); post-treatment pouch disease activity index, median 1 (range: 1-3)]. Identification of faecal coliform sensitivity and treatment with oral ertapenem might be helpful in patients with CARP.

Multivalent Pneumococcal Protein Vaccines Comprising Pneumolysoid with Epitopes/Fragments of CbpA and/or PspA Elicit Strong and Broad Protection.

Immunization with the pneumococcal proteins pneumolysin (Ply), choline binding protein A (CbpA), or pneumococcal surface protein A (PspA) elicits protective responses against invasive pneumococcal disease in animal models. In this study, we used different mouse models to test the efficacy of a variety of multivalent protein-based vaccines that comprised various combinations of full-length or peptide regions of the immunogens Ply, CbpA, or PspA: Ply toxoid with the L460D substitution (referred to herein as L460D); L460D fused with protective peptide epitopes from CbpA (YPT-L460D-NEEK [YLN]); L460D fused with the CD2 peptide containing the proline-rich region (PRR) of PspA (CD2-L460D); a combination of L460D and H70 (L460D+H70), a slightly larger PspA-derived peptide containing the PRR and the SM1 region; H70+YLN; and other combinations. Each mouse was immunized either intraperitoneally (i.p.) or subcutaneously (s.c.) with three doses (at 2-week intervals) of the various antigen combinations in alum adjuvant and then challenged in mouse models featuring different infection routes with multiple Streptococcus pneumoniae strains. In the i.p. infection sepsis model, H70+YLN consistently provided significant protection against three different challenge strains (serotypes 1, 2, and 6A); the CD2+YLN and H70+L460D combinations also elicited significant protection. Protection against intravenous (i.v.) sepsis (type 3 and 6A challenge strains) was largely dependent on PspA-derived antigen components, and the most protection was elicited by H70 with or without L460D or YLN. In a type 4 intratracheal (i.t.) challenge model that results in progression to meningitis, antigen combinations that contained YLN elicited the strongest protection. Thus, the trivalent antigen combination of H70+YLN elicited the strongest and broadest protection in diverse pneumococcal challenge models.

Generation of a lentiviral vector producer cell clone for human Wiskott-Aldrich syndrome gene therapy.

We have developed a producer cell line that generates lentiviral vector particles of high titer. The vector encodes the Wiskott-Aldrich syndrome (WAS) protein. An insulator element has been added to the long terminal repeats of the integrated vector to limit proto-oncogene activation. The vector provides high-level, stable expression of WAS protein in transduced murine and human hematopoietic cells. We have also developed a monoclonal antibody specific for intracellular WAS protein. This antibody has been used to monitor expression in blood and bone marrow cells after transfer into lineage negative bone marrow cells from WAS mice and in a WAS negative human B-cell line. Persistent expression of the transgene has been observed in transduced murine cells 12-20 weeks following transplantation. The producer cell line and the specific monoclonal antibody will facilitate the development of a clinical protocol for gene transfer into WAS protein deficient stem cells.

The use of a biological graft for the closure of large abdominal wall defects following excision of soft tissue tumours.

Primary soft tissue tumours arising from the abdominal wall are uncommon and surgical excision of such tumours can result in large abdominal wall defects. There are many techniques available for abdominal wall repair following tumour excision, each having its own advantages and disadvantages. The options range from direct closure to the use of tissue flap reconstructions and/or prosthetic meshes. Currently, synthetic material such as polypropylene mesh is a common choice for closure of abdominal wall defects after tumour excision. Biological meshes are an alternative option for repair, and this report outlines two cases of abdominal wall repair using the porcine intestinal submucosa biological graft following excision of abdominal wall tumours. There was no evidence of infection, recurrence, seroma or hernias at 2-year follow-up. Following excision of soft tissue tumours of the abdominal wall, biological reconstructions can be successfully used to bridge the defect with minimal morbidity.

A site-selective, irreversible inhibitor of the DNA replication auxiliary factor proliferating cell nuclear antigen (PCNA).

Proliferating cell nuclear antigen (PCNA) assumes an indispensable role in supporting cellular DNA replication and repair by organizing numerous protein components of these pathways via a common PCNA-interacting sequence motif called a PIP-box. Given the multifunctional nature of PCNA, the selective inhibition of PIP-box-mediated interactions may represent a new strategy for the chemosensitization of cancer cells to existing DNA-directed therapies; however, promiscuous blockage of these interactions may also be universally deleterious. To address these possibilities, we utilized a chemical strategy to irreversibly block PIP-box-mediated interactions. Initially, we identified and validated PCNA methionine 40 (M40) and histidine 44 (H44) as essential residues for PCNA/PIP-box interactions in general and, more specifically, for efficient PCNA loading onto chromatin within cells. Next, we created a novel small molecule incorporating an electrophilic di-chloro platinum moiety that preferentially alkylated M40 and H44 residues. The compound, designated T2Pt, covalently cross-linked wild-type but not M40A/H44A PCNA, irreversibly inhibited PCNA/PIP-box interactions, and mildly alkylated plasmid DNA in vitro. In cells, T2Pt persistently induced cell cycle arrest, activated ATR-Chk1 signaling and modestly induced DNA strand breaks, features typical of cellular replication stress. Despite sustained activation of the replication stress response by the compound and its modestly genotoxic nature, T2Pt demonstrated little activity in clonogenic survival assays as a single agent, yet sensitized cells to cisplatin. The discovery of T2Pt represents an original effort directed at the development of irreversible PCNA inhibitors and sets the stage for the discovery of analogues more selective for PCNA over other cellular nucleophiles.