Research Article Targeting Essential Hypothetical Proteins of Pseudomonas aeruginosa PAO1 for Mining of Novel Therapeutics: An In Silico Approach Atikur Rahman , 1 Md. Takim Sarker , 1 Md Ashiqul Islam, 2 Mohammad Uzzal Hossain, 3 Mahmudul Hasan, 4 and Tasmina Ferdous Susmi 1 1 Department of Genetic Engineering and Biotechnology, Faculty of Biological Science and Technology, Jashore University of Science and Technology, Jashore 7408, Bangladesh 2 Department of Chemistry and Biochemistry, University of Windsor, Canada 3 Bioinformatics Division, National Institute of Biotechnology, Ganakbari, Ashulia, Savar, Dhaka 1349, Bangladesh 4 Department of Pharmaceuticals and Industrial Biotechnology, Sylhet Agricultural University, Sylhet 3100, Bangladesh Correspondence should be addressed to Tasmina Ferdous Susmi; tsusmi7@gmail.com Received 26 June 2022; Revised 24 January 2023; Accepted 6 February 2023; Published 11 April 2023 Academic Editor: Luis Morales-Quintana Copyright © 2023 Atikur Rahman et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. As an omnipresent opportunistic bacterium, Pseudomonas aeruginosa PAO1 is responsible for acute and chronic infection in immunocompromised individuals. Currently, this bacterium is on WHO’s red list where new antibiotics are urgently required for the treatment. Finding essential genes and essential hypothetical proteins (EHP) can be crucial in identifying novel druggable targets and therapeutics. This study is aimed at characterizing these EHPs and analyzing subcellular and physiochemical properties, PPI network, nonhomologous analysis against humans, virulence factor and novel drug target prediction, and finally structural analysis of the identified target employing around 42 robust bioinformatics tools/databases, the output of which was evaluated using the ROC analysis. The study discovered 18 EHPs from 336 essential genes, with domain and functional annotation revealing that 50% of these proteins belong to the enzyme category. The majority are cytoplasmic and cytoplasmic membrane proteins, with half being stable proteins subjected to PPIs network analysis. The network contains 261 nodes and 269 edges for 9 proteins of interest, with 11 hubs containing at least three nodes each. Finally, a pipeline builder predicts 7 proteins with novel drug targets, 5 nonhomologous proteins against human proteome, human antitargets, and human gut flora, and 3 virulent proteins. Among these, homology modeling of NP_249450 and NP_251676 was done, and the Ramachandran plot analysis revealed that more than 94% of the residues were in the preferred region. By analyzing functional attributes and virulence characteristics, the findings of this study may facilitate the development of innovative antibacterial drug targets and drugs of Pseudomonas aeruginosa PAO1. 1. Introduction Pseudomonas aeruginosa, often termed as an opportunistic pathogen, is a rod-shaped, motile, Gram-negative, and non- fermenting bacteria found ubiquitously in soil and water as well as found in colonies on the animate part of plants and animals including humans [1, 2]. Isolates collected from diverse environments reported 272 species of the Pseudomo- nas genus in which Pseudomonas aeruginosa PA01 is one of the most commonly used laboratory strains as well as employed to generate publicly accessible genomic resources [2, 3]. Pseudomonas aeruginosa PA01 is the first-ever strain of its species having a completely sequenced genome from a chronic lesion isolate dated from the 1950s. The genome is 6.3 Mbp long that includes 5570 ORFs, roughly 89.4% cod- ing regions, and 0.4% stable RNAs. This was the largest bac- terial genome available during the year 2000 when sequenced. However, despite the same species, different Hindawi BioMed Research International Volume 2023, Article ID 1787485, 28 pages https://doi.org/10.1155/2023/1787485