Stephanie S. Kim

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

Over the last year, there have been substantial improvements in protein structure prediction, particularly in methods like DeepMind’s AlphaFold2 (AF2) that exploit deep learning strategies. Here we report a new CATH-Assign protocol which is used to analyse the first tranche of AF2 models predicted for 21 model organisms and discuss insights these models bring on the nature of protein structure space. We analyse good quality models and those with no unusual structural characteristics, i.e., features rarely seen in experimental structures. For the ∼370,000 models that meet these criteria, we observe that 92% can be assigned to evolutionary superfamilies in CATH. The remaining domains cluster into 2,367 putative novel superfamilies. Detailed manual analysis on a subset of 618 of those which had at least one human relative revealed some extremely remote homologies and some further unusual features, but 26 could be confirmed as novel superfamilies and one of these has an alpha-beta propeller architectural arrangement never seen before. By clustering both experimental and predicted AF2 domain structures into distinct ‘global fold’ groups, we observe that the new AF2 models in CATH increase information on structural diversity by 36%. This expansion in structural diversity will help to reveal associated functional diversity not previously detected. Our novel CATH-Assign protocol scales well and will be able to harness the huge expansion (at least 100 million models) in structural data promised by DeepMind to provide more comprehensive coverage of even the most diverse superfamilies to help rationalise evolutionary changes in their functions.

Highly accurate structure prediction methods are generating an avalanche of publicly available protein structures. Searching through these structures is becoming the main bottleneck in their analysis. Foldseek enables fast and sensitive comparisons of large structure sets. It reaches sensitivities similar to state-of-the-art structural aligners while being at least 20,000 times faster. Foldseek is free open-source software available at foldseek.com and as a webserver at search.foldseek.com. Contact soeding{at}mpinat.mpg.de , martin.steinegger{at}snu.ac.kr

Background: Given the diverse roles of cyclin A2 both in cell cycle regulation and in DNA damage response, identifying small molecule regulators of cyclin A2 activity carries significant potential to regulate diverse cellular processes in both ageing/neurodegeneration and in cancer. Objective: Based on cyclin A2’s recently discovered role in DNA repair, we hypothesized that small molecule inhibitors that were predicted to bind to both cyclin A2 and CDK2 will be useful as a radiosensitizer of cancer cells. In this study, we used structure-based drug discovery to find inhibitors that target both cyclin A2 and CDK2. Method: Molecular dynamics simulations were used to generate diverse binding pocket conformations for application of the relaxed complex scheme. We then used structure-based virtual screening to find potential dual cyclin A2 and CDK2 inhibitors. Based on a consensus score of docked poses from Glide and AutoDock Vina, we identified about 40 promising hit compounds, where all PAINS scaffolds were removed from consideration. A biochemical luminescence assay of cyclin A2-CDK2 function was used for experimental verification. Result: Four lead inhibitors of cyclin A2-CDK2 complex have been identified using a relaxed complex scheme virtual screen have been verified in a biochemical luminescence assay of cyclin A2- CDK2 function. Two of the four lead inhibitors had inhibitory concentrations in the nanomolar range. Conclusion: The four cyclin A2-CDK2 complex inhibitors are the first reported inhibitors that were specifically designed not to target the cyclin A2-CDK2 protein-protein interface. Overall, our results highlight the potential of combined advanced computational tools and biochemical verification to discover novel binding scaffolds.

Background: Arginine (Arg) deprivation is a promising therapeutic approach for tumors with low argininosuccinate synthetase 1 (ASS1) expression. However, its efficacy as a single agent therapy needs to be improved as resistance is frequently observed. Methods: A tissue microarray was performed to assess ASS1 expression in surgical specimens of pancreatic ductal adenocarcinoma (PDAC) and its correlation with disease prognosis. An RNA-Seq analysis examined the role of ASS1 in regulating global gene transcriptome. A high throughput screen of FDA-approved oncology drugs identified synthetic lethality between histone deacetylase (HDAC) inhibitors and Arg deprivation in PDAC cells with low ASS1 expression. We examined HDAC inhibitor panobinostat (PAN) and Arg deprivation in a panel of human PDAC cell lines, in ASS1-high and -knockdown/knockout isogenic models, in both anchorage-dependent and -independent cultures, and in multicellular complex cultures that model the PDAC tumor microenvironment. We examined the effects of combined Arg deprivation and PAN on DNA damage and the protein levels of key DNA repair enzymes. We also evaluate the efficacy of PAN and ADI-PEG20 (an Arg-degrading agent currently in Phase II clinical trials) in xenograft models with ASS1-low and -high PDAC tumors. Results: Low ASS1 protein level is a negative prognostic indicator in PDAC. Arg deprivation in ASS1-deficient PDAC cells upregulated asparagine synthetase (ASNS) which redirected aspartate (Asp) from being used for de novo nucleotide biosynthesis, thus causing nucleotide insufficiency and impairing cell cycle S-phase progression. Comprehensively validated, HDAC inhibitors and Arg deprivation showed synthetic lethality in ASS1-low PDAC cells. Mechanistically, combined Arg deprivation and HDAC inhibition triggered degradation of a key DNA repair enzyme C-terminal-binding protein interacting protein (CtIP), resulting in DNA damage and apoptosis. In addition, S-phase-retained ASS1-low PDAC cells (due to Arg deprivation) were also sensitized to DNA damage, thus yielding effective cell death. Compared to single agents, the combination of PAN and ADI-PEG20 showed better efficacy in suppressing ASS1-low PDAC tumor growth in mouse xenograft models. Conclusion: The combination of PAN and ADI-PEG20 is a rational translational therapeutic strategy for treating ASS1-low PDAC tumors through synergistic induction of DNA damage. Citation Format: Stephanie S Kim, JING CUI, Shili Xu, Soumya Poddar, Thuc M. Le, Luyi Li, Nanping Wu, Alexandra Moore, Lei Zhou, Alice Yu, Amanda M. Dann, Irmina A. Elliott, Evan R. Abt, Woosuk Kim, David W. Dawson, Caius G. Radu, Timothy R. Donahue. Histone deacetylase inhibition is synthetically lethal with arginine deprivation in pancreatic cancers with low argininosuccinate synthetase 1 expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 554.

A 42-year-old man with Addison disease presented to the emergency department with unsteady gait for 1 year. A half-brother had epilepsy and difficulty walking, and died at 6 years of age. Another half-brother has a longstanding gait disorder. Over 3 to 4 months, his gait has become worse with marked stiffness, ataxia, and dysarthria. Neurologic examination demonstrated saccadic pursuit, ocular flutter, severe spasticity throughout, peripheral neuropathy, hyperreflexia, clonus, with bilateral extensor plantar reflexes, marked dysmetria, and dysdiadochokinesia. He stands with a marked forward stoop and flexed knees. MRI is shown in figures 1 and 2. The plasma concentration of very-long-chain fatty acids, which is a general test for peroxisomal disorders, was elevated, confirming the diagnosis of adrenoleukodystrophy (ALD). Very-long-chain fatty acids were also elevated in the surviving brother's plasma. Adult cerebral ALD is extremely rare. It is an X-linked peroxisomal disorder caused by mutation in the ABCD1 gene located at Xq28 and involves the nervous system, adrenal cortex, and Leydig cells in the testes.1,2