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Palmitoylation of the KATP channel Kir6.2 subunit promotes channel opening by regulating PIP2 sensitivity

Yang, Hua-Qian; Martinez-Ortiz, Wilnelly; Hwang, JongIn; Fan, Xuexin; Cardozo, Timothy J; Coetzee, William A
A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K+ (KATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the KATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys166 prevented these effects. Clinical variants in KCNJ11 that affect Cys166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP2 Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys166 enhanced the PIP2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of KATP channels by α1-adrenoreceptor stimulation. The Cys166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.
PMID: 32332165
ISSN: 1091-6490
CID: 4402522

SARS-CoV-2 viral spike G614 mutation exhibits higher case fatality rate

Becerra-Flores, Manuel; Cardozo, Timothy
AIM/OBJECTIVE:The COVID pandemic is caused by infection with the SARS-CoV-2 virus. The major mutation detected to date in the SARS-CoV-2 viral envelope spike protein, which is responsible for virus attachment to the host and is also the main target for host antibodies, is a mutation of an aspartate (D) at position 614 found frequently in Chinese strains to a glycine (G). We sought to infer health impact of this mutation. RESULT/RESULTS:Increased case fatality rate correlated strongly with the proportion of viruses bearing G614 on a country by country basis. The amino acid at position 614 occurs at an internal protein interface of the viral spike, and the presence of G at this position was calculated to destabilize a specific conformation of the viral spike, within which the key host receptor binding site is more accessible. CONCLUSION/CONCLUSIONS:These results imply that G614 is a more pathogenic strain of SARS-CoV-2, which may influence vaccine design. The prevalence of this form of the virus should also be included in epidemiologic models predicting the COVID-19 health burden and fatality over time in specific regions. Physicians should be aware of this characteristic of the virus to anticipate the clinical course of infection.
PMID: 32374903
ISSN: 1742-1241
CID: 4437192

Modulating extracellular TCR-CD3 interaction to identify new immunotherapy targets against cancer [Meeting Abstract]

Krogsgaard, M; Natarajan, A; Velmurugu, Y; Yuan, Z; Ge, C; Nadarajah, V; Cardozo, T; Bracken, W C; Zhu, C
T cell recognition of antigen and resulting proximal signaling are key steps in the initiation of the adaptive immune response. Previous studies targeting antigen binding site for enhancing T-cell responses to tumor antigens often lead to off-target effects and toxicity. Recently, we used nuclear magnetic resonance (NMR) spectroscopy, mutational analysis and computational docking to derive a 3D structure of the extracellular TCRCD3 assembly. Further, biomolecular force probe (BFP) measurements allowed us to determine how 2D affinity and force-modulated TCR-pMHC kinetics depend on TCR-CD3 interaction sites and affect transduction of extracellular pMHC-TCR ligation into T cell function. Based on our TCR-CD3 structural model and binding data, we generated TCR libraries for a melanoma-specific TCR (DMF5) using site-specific mutagenesis in the Cbhelix 3 and helix 4-F strand regions of the TCR to optimize the TCR-CD3 interaction and to select for mutants with enhanced T-cell effector function. One Cb helix 4-F strand mutant, NP202203AA showed increased T cell response to antigen and showed enhanced TCR-pMHC bond lifetime (catch-bonds) in BFP assays leading to prolonged T cell signaling. In the future, DMF5 TCR with reengineered CD3 binding regions will be used in tumor rejection in pre-clinical mouse melanoma models for eficacy and toxicity to develop more effective T cell therapies for human targets
ISSN: 1550-6606
CID: 4638822

Unbiased identification of immunogenic Staphylococcus aureus leukotoxin B-cell epitopes

Hernandez, David N; Tam, Kayan; Shopsin, Bo; Radke, Emily; Kolahi, Pegah; Copin, Richard; Stubbe, François-Xavier; Cardozo, Timothy; Torres, Victor J; Silverman, Gregg J
Unbiased identification of individual, immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as little as a single major pathogen antigen. Using the bi-component pore-forming leukocidin (Luks) exotoxins of the major pathogen Staphylococcus aureus (Sa) as a prototype, we randomly fragmented and separately ligated the Hemolysin gamma A (HlgA) and LukS genes into a custom-built, phage-display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk mAb recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino acid sequence that is highly conserved in four Luk toxin subunits, and is ubiquitous in representation within Sa clinical isolates. The isolated 11-amino acid peptide probe was predicted to retain the native 3D-conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk mAb, and using mutated peptides we showed that a particular amino acid side-chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG-responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment, phage-display based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much needed Sa-protective subunit vaccine.
PMID: 32014894
ISSN: 1098-5522
CID: 4301262

How polypharmacologic is each chemogenomics library?

Ni, Eric; Kwon, Eehjoe; Young, Lauren M; Felsovalyi, Klara; Fuller, Jennifer; Cardozo, Timothy
Aim/UNASSIGNED:High-throughput phenotypic screens have emerged as a promising avenue for small-molecule drug discovery. The challenge faced in high-throughput phenotypic screens is target deconvolution once a small molecule hit is identified. Chemogenomics libraries have emerged as an important tool for meeting this challenge. Here, we investigate their target-specificity by deriving a 'polypharmacology index' for broad chemogenomics screening libraries. Methods/UNASSIGNED:All known targets of all the compounds in each library were plotted as a histogram and fitted to a Boltzmann distribution, whose linearized slope is indicative of the overall polypharmacology of the library. Results & conclusion/UNASSIGNED:Comparison of libraries clearly distinguished the most target-specific library, which might be assumed to be more useful for target deconvolution in a phenotypic screen.
PMID: 32149277
ISSN: 2631-3316
CID: 4349602

Modulating the extracellular TCR-CD3 interaction to identify novel immunotherapy targets against melanoma [Meeting Abstract]

Natarajan, Aswin; Velmurugu, Yogambigai; Zhou, Yuan; Ge, Chenghao; Nadarajah, Vidushan; Felsovalyi, Klara; Cardozo, Timothy J.; Bracken, Clay; Zhu, Cheng; Krogsgaard, Michelle
ISSN: 1479-5876
CID: 4345052

Corrigendum to "Triazolo[4,5-d]pyrimidines as validated general control nonderepressible 2 (GCN2) protein kinase inhibitors reduce growth of leukemia cells" [Comput. Struct. Biotechnol. J. 16 (2018) 350-360]

Lough, Lea; Sherman, Dan; Becerra-Flores, Manuel; Vasudevan, Deepika; Lavinda, Olga; Ni, Eric; Wang, Hong; Ryoo, Hyung Don; Tibes, Raoul; Cardozo, Timothy
[This corrects the article DOI: 10.1016/j.csbj.2018.09.003.].
PMID: 32435428
ISSN: 2001-0370
CID: 4444472

Historeceptomics: Integrating a drug's multiple targets (polypharmacology) with their expression pattern in human tissues

Chapter by: Cardozo, Timothy
in: Multi-Target Drug Design Using Chem-Bioinformatic Approaches by
New York : Humana Press, 2018
pp. 517-528
ISBN: 978-1-4939-8732-0
CID: 3566512

Molecular basis of atypicality of bupropion inferred from its receptor engagement in nervous system tissues

Kim, Eric J; Felsovalyi, Klara; Young, Lauren M; Shmelkov, Sergey V; Grunebaum, Michael F; Cardozo, Timothy
Despite decades of clinical use and research, the mechanism of action (MOA) of antidepressant medications remains poorly understood. Selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) are the most commonly prescribed antidepressants-atypical antidepressants such as bupropion have also proven effective, while exhibiting a divergent clinical phenotype. The difference in phenotypic profiles presumably lies in the differences among the MOAs of SSRIs/SNRIs and bupropion. We integrated the ensemble of bupropion's affinities for all its receptors with the expression levels of those targets in nervous system tissues. This "combined target tissue" profile of bupropion was compared to those of duloxetine, fluoxetine, and venlafaxine to isolate the unique target tissue effects of bupropion. Our results suggest that the three monoamines-serotonin, norepinephrine, and dopamine-all contribute to the common antidepressant effects of SSRIs, SNRIs, and bupropion. At the same time, bupropion is unique in its action on 5-HT3AR in the dorsal root ganglion and nicotinic acetylcholine receptors in the pineal gland. These unique tissue-specific activities may explain unique therapeutic effects of bupropion, such as pain management and smoking cessation, and, given melatonin's association with nicotinic acetylcholine receptors and depression, highlight the underappreciated role of the melatonergic system in bupropion's MOA.
PMID: 29961917
ISSN: 1432-2072
CID: 3185572

Chemical probes of Skp2-mediated p27 ubiquitylation and degradation

Lough, Lea; Sherman, Dan; Ni, Eric; Young, Lauren M; Hao, Bing; Cardozo, Timothy
Skp2 is a member of the F-box family of proteins that serve as substrate-specific adaptors in Skp1-CUL1-ROC1-F-box (SCF) E3 ubiquitin ligases. Skp2 (Fbxl1) directly binds to the tumor suppressor p27 in the context of the SCFSkp2 E3 ubiquitin ligase to ubiquitylate and target-phosphorylated p27 for proteasomal degradation. As p27 is a powerful suppressor of growth in a variety of cells, and as Skp2 is also overexpressed in many human cancers, Skp2 is considered an oncogene and an intriguing drug target. However, despite 20 years of investigation, a valid chemical inhibitor of Skp2-mediated degradation of p27 has not been identified. Recently, an increasing number of compounds designed to have this bioactivity have been reported. Here, we conduct a meta-analysis of the evidence regarding bioactivity, structure, and medicinal chemistry in order to evaluate and compare these Skp2 inhibitor compounds. Despite chemically diverse compounds with a wide array of Skp2-mediated p27 ubiquitylation inhibition properties reported by several independent groups, no current chemical probe formally qualifies as a validated pharmaceutical hit compound. This finding suggests that our knowledge of the structural biochemistry of the Skp2-p27 complex remains incomplete and highlights the need for novel modes of inquiry.
PMID: 30108998
ISSN: 2040-2503
CID: 3239202