{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,28]],"date-time":"2026-02-28T04:24:12Z","timestamp":1772252652459,"version":"3.50.1"},"reference-count":27,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2023,1,4]],"date-time":"2023-01-04T00:00:00Z","timestamp":1672790400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R21AI57392"],"award-info":[{"award-number":["R21AI57392"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>The \u201cblock and lock\u201d strategy is one approach that might elicit a sterilizing cure for HIV-1 infection. The \u201cblock\u201d refers to a compound\u2019s ability to inhibit latent HIV-1 proviral transcription, while the \u201clock\u201d refers to its capacity to induce permanent proviral silencing. We previously identified PF-3758309, a pan-isoform inhibitor of p21-activated kinases (PAKs), as a potent inhibitor of HIV-1 latency reversal. The goal of this study was to define the mechanism(s) involved. We found that both 24ST1NLESG cells (a cell line model of HIV-1 latency) and purified CD4+ na\u00efve and central memory T cells express high levels of PAK2 and lower levels of PAK1 and PAK4. Knockdown of PAK1 or PAK2, but not PAK4, in 24ST1NLESG cells resulted in a modest, but statistically significant, decrease in the magnitude of HIV-1 latency reversal. Overexpression of PAK1 significantly increased the magnitude of latency reversal. A phospho-protein array analysis revealed that PF-3758309 down-regulates the NF-\u03baB signaling pathway, which provides the most likely mechanism by which PF-3758309 inhibits latency reversal. Finally, we used cellular thermal shift assays combined with liquid chromatography and mass spectrometry to ascertain whether PF-3758309 off-target binding contributed to its activity. In 24ST1NLESG cells and in peripheral blood mononuclear cells, PF-3758309 bound to mitogen-activated protein kinase 1 and protein kinase A; however, knockdown of either of these kinases did not impact HIV-1 latency reversal. Collectively, our study suggests that PAK1 and PAK2 play a key role in the maintenance of HIV-1 latency.<\/jats:p>","DOI":"10.3390\/biom13010100","type":"journal-article","created":{"date-parts":[[2023,1,4]],"date-time":"2023-01-04T04:08:07Z","timestamp":1672805287000},"page":"100","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Mechanism by Which PF-3758309, a Pan Isoform Inhibitor of p21-Activated Kinases, Blocks Reactivation of HIV-1 Latency"],"prefix":"10.3390","volume":"13","author":[{"given":"Benni","family":"Vargas","sequence":"first","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James","family":"Boslett","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nathan","family":"Yates","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA"},{"name":"Department of Chemistry, University of Pittsburgh School of Medicine; Pittsburgh, PA 15260, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicolas","family":"Sluis-Cremer","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1146\/annurev-pathol-050520-112001","article-title":"In Vivo Dynamics of the Latent Reservoir for HIV-1: New Insights and Implications for Cure","volume":"17","author":"Siliciano","year":"2022","journal-title":"Annu. Rev. Pathol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1093\/infdis\/jiaa649","article-title":"Low Inducibility of Latent Human Immunodeficiency Virus Type 1 Proviruses as a Major Barrier to Cure","volume":"223","author":"Siliciano","year":"2021","journal-title":"J. Infect. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Chitrakar, A., Sanz, M., Maggirwar, S.B., and Soriano-Sarabia, N. (2022). HIV Latency in Myeloid Cells: Challenges for a Cure. Pathogens, 11.","DOI":"10.3390\/pathogens11060611"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Moranguinho, I., and Valente, S.T. (2020). Block-And-Lock: New Horizons for a Cure for HIV-1. Viruses, 12.","DOI":"10.3390\/v12121443"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Vansant, G., Bruggemans, A., Janssens, J., and Debyser, Z. (2020). Block-And-Lock Strategies to Cure HIV Infection. Viruses, 12.","DOI":"10.3390\/v12010084"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"328","DOI":"10.1016\/j.virol.2014.02.008","article-title":"Transcriptional control of HIV latency: Cellular signaling pathways, epigenetics, happenstance and the hope for a cure","volume":"454\u2013455","author":"Mbonye","year":"2014","journal-title":"Virology"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e01744-18","DOI":"10.1128\/AAC.01744-18","article-title":"Inhibitors of Signaling Pathways That Block Reversal of HIV-1 Latency","volume":"63","author":"Vargas","year":"2019","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"9446","DOI":"10.1073\/pnas.0911863107","article-title":"Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth","volume":"107","author":"Murray","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.gene.2016.12.014","article-title":"Structure, biochemistry, and biology of PAK kinases","volume":"605","author":"Kumar","year":"2017","journal-title":"Gene"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"126","DOI":"10.4161\/cl.20222","article-title":"PAK in pathogen-host interactions","volume":"2","author":"Semblat","year":"2012","journal-title":"Cell. Logist."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1128\/JVI.80.1.130-137.2006","article-title":"\u201cUnPAKing\u201d human immunodeficiency virus (HIV) replication: Using small interfering RNA screening to identify novel cofactors and elucidate the role of group I PAKs in HIV infection","volume":"80","author":"Nguyen","year":"2006","journal-title":"J. Virol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1186\/1742-4690-10-47","article-title":"Group I p21-activated kinases facilitate Tax-mediated transcriptional activation of the human T-cell leukemia virus type 1 long terminal repeats","volume":"10","author":"Chan","year":"2013","journal-title":"Retrovirology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"198","DOI":"10.2174\/1570162X113119990039","article-title":"HIV-1 Nef mediates Pak phosphorylation of Mek1 serine298 and elicits an active phospho-state of Pak2","volume":"11","author":"Kouwenhoven","year":"2013","journal-title":"Curr. HIV Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5185","DOI":"10.1128\/AAC.49.12.5185-5188.2005","article-title":"Human immunodeficiency virus type 1 latency model for high-throughput screening","volume":"49","author":"Pacchia","year":"2005","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1420","DOI":"10.1038\/s42003-021-02949-7","article-title":"A specific inhibitor of ALDH1A3 regulates retinoic acid biosynthesis in glioma stem cells","volume":"4","author":"Li","year":"2021","journal-title":"Commun. Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1038\/s41592-020-0801-4","article-title":"Meltome atlas-thermal proteome stability across the tree of life","volume":"17","author":"Jarzab","year":"2020","journal-title":"Nat. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1038\/nmeth.1322","article-title":"Universal sample preparation method for proteome analysis","volume":"6","author":"Zougman","year":"2009","journal-title":"Nat. Methods"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"7324","DOI":"10.4049\/jimmunol.172.12.7324","article-title":"A novel role for p21-activated protein kinase 2 in T cell activation","volume":"172","author":"Chu","year":"2004","journal-title":"J. Immunol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"879","DOI":"10.1016\/j.str.2010.04.011","article-title":"Why an A-loop phospho-mimetic fails to activate PAK1: Understanding an inaccessible kinase state by molecular dynamics simulations","volume":"18","author":"Ng","year":"2010","journal-title":"Structure"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1007\/s11010-013-1928-8","article-title":"PF-3758309, p21-activated kinase 4 inhibitor, suppresses migration and invasion of A549 human lung cancer cells via regulation of CREB, NF-\u03baB, and \u03b2-catenin signalings","volume":"389","author":"Ryu","year":"2014","journal-title":"Mol. Cell. Biochem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"103159","DOI":"10.1016\/j.ebiom.2020.103159","article-title":"NF-\u03baB sub-pathways and HIV cure: A revisit","volume":"63","author":"Wong","year":"2021","journal-title":"EBioMedicine"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1146\/annurev-pharmtox-010715-103715","article-title":"The Cellular Thermal Shift Assay: A Novel Biophysical Assay for In Situ Drug Target Engagement and Mechanistic Biomarker Studies","volume":"56","author":"Nordlund","year":"2016","journal-title":"Annu. Rev. Pharmacol. Toxicol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"917941","DOI":"10.3389\/fviro.2022.917941","article-title":"Toward a Functional Cure for HIV-1 Infection: The Block and Lock therapeutic Approach","volume":"2","author":"Vargas","year":"2022","journal-title":"Front. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1097\/COH.0b013e32834124fd","article-title":"NF-\u03baB\/Rel: Agonist and antagonist roles in HIV-1 latency","volume":"6","author":"Chan","year":"2011","journal-title":"Curr. Opin. HIV AIDS"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3020675","DOI":"10.1155\/2018\/3020675","article-title":"Characterization of Site-Specific Phosphorylation of NF-\u03baB p65 in Retinal Cells in Response to High Glucose and Cytokine Polarization","volume":"2018","author":"Shi","year":"2018","journal-title":"Mediat. Inflamm."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"34036","DOI":"10.1074\/jbc.M111.284216","article-title":"Disruption of IkappaB kinase (IKK)-mediated RelA serine 536 phosphorylation sensitizes human multiple myeloma cells to histone deacetylase (HDAC) inhibitors","volume":"286","author":"Dai","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1021\/acs.jcim.5b00409","article-title":"Model of the Interaction between the NF-\u03baB Inhibitory Protein p100 and the E3 Ubiquitin Ligase \u03b2-TrCP based on NMR and Docking Experiments","volume":"57","author":"Melikian","year":"2017","journal-title":"J. Chem. Inf. Model."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/13\/1\/100\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T17:58:27Z","timestamp":1760119107000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/13\/1\/100"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,1,4]]},"references-count":27,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2023,1]]}},"alternative-id":["biom13010100"],"URL":"https:\/\/doi.org\/10.3390\/biom13010100","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2022.08.02.502581","asserted-by":"object"}]},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,1,4]]}}}