Citation: Roth, H.E.; De Lima Leite, A.; Palermo, N.Y.; Powers, R. Leveraging the Structure of DNAJA1 to Discover Novel Potential Pancreatic Cancer Therapies. Biomolecules 2022, 12, 1391. https:// doi.org/10.3390/biom12101391 Academic Editors: Steven R. Van Doren and Martin Lawrence Received: 18 August 2022 Accepted: 26 September 2022 Published: 29 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomolecules Article Leveraging the Structure of DNAJA1 to Discover Novel Potential Pancreatic Cancer Therapies Heidi E. Roth 1 , Aline De Lima Leite 1,2 , Nicolas Y. Palermo 3 and Robert Powers 1,2, * 1 Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA 2 Nebraska Center for Integrated Biomolecular Communication, University of Nebraska-Lincoln, Lincoln, NE 68588, USA 3 Computational Chemistry Core Facility, VCR Cores, University of Nebraska Medical Center, Omaha, NE 68198, USA * Correspondence: rpowers3@unl.edu; Tel.: +1-(402)-472-3039; Fax: +1-(402)-472-9402 Abstract: Pancreatic cancer remains one of the deadliest forms of cancer with a 5-year survival rate of only 11%. Difficult diagnosis and limited treatment options are the major causes of the poor outcome for pancreatic cancer. The human protein DNAJA1 has been proposed as a potential therapeutic target for pancreatic cancer, but its cellular and biological functions remain unclear. Previous studies have suggested that DNAJA1 ′ s cellular activity may be dependent upon its protein binding partners. To further investigate this assertion, the first 107 amino acid structures of DNAJA1 were solved by NMR, which includes the classical J-domain and its associated linker region that is proposed to be vital to DNAJA1 functionality. The DNAJA1 NMR structure was then used to identify both protein and ligand binding sites and potential binding partners that may suggest the intracellular roles of DNAJA1. Virtual drug screenings followed by NMR and isothermal titration calorimetry identified 5 drug-like compounds that bind to two different sites on DNAJA1. A pull-down assay identified 8 potentially novel protein binding partners of DNAJA1. These proteins in conjunction with our previously published metabolomics study support a vital role for DNAJA1 in cellular oncogenesis and pancreatic cancer. Keywords: DNAJA1; pancreatic cancer; NMR structural biology; drug discovery; Hsp40 1. Introduction According to the American Cancer Society, 62,210 people in the USA will be diagnosed with pancreatic cancer in 2022 with 49,830 resulting in fatalities [1]. Pancreatic cancer survival rates have seen minimal improvement in the last thirty years. The stage-one 5-year survival rate is only 11%, with just a few patients being diagnosed at such an early stage of the disease. Early diagnosis of pancreatic cancer is often difficult as the initial stages tend to be asymptomatic and tumor presence is not outwardly apparent [1]. Un- fortunately, the 5-year survival rate decreases with stage progression hitting a low of 3% by stage three [1]. These poor outcomes are due to a delayed diagnosis, rapid acquisi- tion of drug resistance [2,3], and limited options for treatment. Developing treatments for pancreatic cancer is an extremely daunting challenge that has enormously profound implications for human health. Identifying a therapeutic target could lead to improvements in disease treatment and improve pancreatic cancer survival rates, which have otherwise remained stagnant. Towards this end, we describe the discovery of novel, drug-like compounds that bind Human protein DnaJ homolog subfamily A member 1 (DNAJA1) and the identification of potential in vivo protein binding partners of DNAJA1. The solution structure of the first 107 amino acids of DNAJA1 was solved using nuclear magnetic resonance (NMR). The NMR structure of DNAJA1 was then leveraged to aid in identifying chemical leads and the location of their binding sites. A virtual ligand screen employed Molegro Virtual Docker Biomolecules 2022, 12, 1391. https://doi.org/10.3390/biom12101391 https://www.mdpi.com/journal/biomolecules