In silico approach to the discovery of potential non-hydroxamate LpxC inhibitors against pseudomonas aeruginosa and the synthesis and characterization of the precursor compounds of avocadyne analogs

Date of Publication

7-8-2026

Document Type

Bachelor's Thesis

Degree Name

Bachelor of Science in Biochemistry

Subject Categories

Biochemistry

College

College of Science

Department/Unit

Chemistry

Thesis Advisor

Glenn Kelly U. Tan
Faith Marie G. Lagua, co-adviser
Stephani Joy Y. Macalino, co-adviser

Defense Panel Member

Searle Aichelle S. Duay
Virgilio D. Ebajo Jr.

Abstract (English)

Part I.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen and is the second-most critical antibiotic-resistant gram-negative bacteria (GNB). The lipopolysaccharide (LPS) layer on P. aeruginosa’s outer membrane serves as a protective barrier against antimicrobial agents and has an essential lipid A component which has been targeted for drug development against multidrug-resistant GNB. Current research on UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC), an essential enzyme in the biosynthesis of lipid A, focuses on compounds with hydroxamate chelating groups which have been found to have off-target effects and potential toxicity. Thus, this study aimed to discover a potential non-hydroxamate P. aeruginosa LpxC enzyme (PaLpxC) inhibitor by screening metalloenzyme chelators from Life Chemicals Chelator Focused Library via series of virtual screening steps.

The 12,020 non-hydroxamate compounds from the Life Chemicals Chelator Focused Library were subjected to a series of screening steps consisting of ADME screening, pharmacophore-based screening, and molecular docking. The PaLpxC-bound hit compounds were then simulated through molecular dynamics to analyze their physical movements over time. Finally, the essential protein movements of PaLpxC were determined through principal component analysis.

Upon ADME screening, only 259 out of 12,020 non-hydroxamate ligands passed, and only 54 compounds shared similar chemical features with the shared PaLpxC pharmacophore model. The molecular docking binding affinity and 2D PaLpxC-ligand interaction analysis reduced the number of ligands to four hit compounds–L45, L9, L44, and L35. The binding of these ligands was found to preserve the structural integrity of the PaLpxC enzyme throughout the simulation. However, through binding free energy measurements, L35 was found to have the best binding affinity to the PaLpxC enzyme (–40.29 kcal/mol) and was thus considered as the best candidate ligand among the 12,020 screened compounds. The electrostatic interactions of L35 are the primary contributor to ligand binding, and its sulfamoyl and carboxylate groups interact directly with the active site zinc ion. L35 changed the primary mechanism of the PaLpxC enzyme from hinge-bending to collective breathing motion, potentially burying the active site, resulting in longer reaction time and reduced catalytic activity. Considering this, L35 has the potential to inhibit PaLpxC’s biological activity of Lipid A synthesis.

Part II.

Acute myeloid leukemia (AML) is the most prevalent type of leukemia affecting adults. Despite AML only contributing to 1% of all cancer cases, incidences of AML continue to rise worldwide as the global population ages, with 22,720 estimated new cases as of 2026. Leukemic stem cells in acute myeloid leukemia (AML) rely on fatty acid oxidation (FAO) for energy production. Avocatin B from the avocado fruit (Persea americana) causes AML cell death by inhibiting the enzyme very long chain acyl-CoA dehydrogenase (VLCAD), and structural studies have shown that the binding channel can accommodate fatty-acyls with chain lengths as long as 24 carbons, but the effect of chain length on the bioactivity of avocadyne has yet to be explored. The synthetic route entailed by Cunha and colleagues (2019) utilizes a methyl ketone precursor that can be varied in chain length to produce different avocadyne analogs. Thus, this study aimed to contribute to the development of the synthetic route of avocadyne analogs by establishing different routes to synthesize the methyl ketone precursors, specifically by testing various oxidants to determine which will generate the target methyl ketone.

The target methyl ketones were successfully synthesized over a three-step synthesis process. The synthesis began with the oxidation of an alkynol (a) to an alkynal (b), which was followed by a Grignard reaction to form a secondary alcohol (c). Finally, oxidation of the secondary alcohol produced the target methyl ketone precursor (d). The synthesized compounds were characterized via Mass Spectrometry (HRMS-ESI), 1H and 13C NMR Spectroscopy. The yields of the synthesized precursors are as follows: tetradec-13-yn-2-one (1d) (194 mg, 76%), dodec-11-yn-2-one (2d) (283 mg, 65%), dec-9-yn-2-one (3d) (351 mg, 77%), tridec-12-yn-2-one (4d) (78 mg, 42%), undec-10-yn-one (5d) (171 mg, 17% over three steps), and non-8-yn-2-one (6d) (198 mg, 39%).

Abstract Format

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Abstract (Filipino)

Part I. 

Ang Pseudomonas aeruginosa (P. aeruginosa) ay isang uri ng bacteria na nagdudulot ng sakit sa mga taong mayroong mahinang resistensya. Tinagurian ito bilang pangalawang pinaka-kritikal na gram negative bacteria (GNB). Mayroon itong lipopolysaccharide (LPS) na pumoprotekta laban sa mga antibiotic, at isa sa mga bumubuo sa LPS ay ang Lipid A na madalas maging target ng drug development labas sa multidrug-resistance GNB. Ang mga kasalukuyang pananaliksik sa UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC), isang importanteng bahagi ng LPS, ay mas pinagtutuunan ng pansin ang pag-aaral sa compounds na mayroong hydroxamate chelating groups na napatunayang mayroong hindi kagustuhang target at posibleng may kaakibat na toksisidad. Sa gayon, layunin ng pag-aaral na itong makadiskubre ng potensyal na inhibitor ng non-hydroxamate P. aeruginosa LpxC enzyme (PaLpxC). Susuriin ang mga metalloenzyme chelators galing sa Life Chemicals Chelator Focused Library gamit ang sunod-sunod na virtual screening steps.

Matapos ang ADME screening, 259 compounds na lamang mula sa 12,020 non-hydroxamate ligands ang pumasa, at 54 compounds ang mayroong kahawig na chemical features sa PaLpxC na shared pharmacophore model. Pagkatapos kalkulahin ang molecular docking binding affinity at analisahin and 2D interkasyon ng compounds sa PaLpxC-ligand, bumaba sa apat ang bilang ng potensyal na inhibitors (L45, L9, L44, at L35). Napag-alamang napreserba ang istruktura ng PaLpxC enzyme matapos ang pagbigkis ng apat na ligand sa buong simulasyon. Gayunpaman, ang L35 compound ang mayroong pinakamataas na binidng affinity sa PaLpxC enzyme (–40.29 kcal/mol), kung kaya masasabing ito ang pinaka-epektibo sa lahat ng 12,020 na sinuring compound. Ang mga electrostatic na interaksyon ang pangunahing kontributor sa pagbikis ng L35 compound sa PaLpxC enzyme. Nadiskubreng mayroong direktang interaksyon ang sulfamoyl at carboxylate groups ng L35 compound sa zinc ion ng PaLpxC enzyme. Mula sa hinge-bending na paggalaw, espisipiko sa pagbukas at pagsara ng active site, ito ay naging collective breathing na paggalaw. Ang pagbabagong ito ay maaaring magdulot ng pagbaon ng active site ng enzyme, magresulta sa mahabang reaction time, at mabagal na catalytic activity. Alinsunod dito, napagtanto na ang pagbigkis ng L35 compound ay posibleng mag-inhibit sa biolohikal na aktibidad ng PaLpxC enzyme sa paggawa ng Lipid A.

Part II. 

Ang acute myeloid leukemia (AML) ang pinakakaraniwang uri ng leukemia na nakaaapekto sa mga nasa hustong gulang. Bagaman isang porsyento (1%) lamang sa kabuuang kaso ng kanser ang naiuugnay sa AML, patuloy na tumataas ang bilang ng mga kaso sa buong mundo kasabay ang pagtanda ng pandaigdigang populasyon, na may tinatayang 22,720 bagong kaso noong 2026. Fatty acid oxidation (FAO) ang pinagkukunan ng enerhiya ng leukemic stem cells ng AML. Ang avocatin B mula sa prutas na avocado (Persea americana) ay nagdudulot ng pagkamatay ng AML cell sa pamamagitan ng inhibition sa enzyme na very long chain acyl-CoA dehydrogenase (VLCAD). Nagpag-aralang posible ang pagbigkis ng fatty-acyls na mayroong haba hanggang 24 carbons sa binding channel ng VLCAD, ngunit hindi pa kompirmado ang epekto ng haba ng fatty-acyl sa bioactivity ng avocadyne. Ang synthetic route na ginawa ni Cunha et al. (2019) ay nangangailangan ng methyl ketone bilang pangunang compound na maaaring baguhin ang haba upang makabuo ng iba’t ibang analog ng avocadyne. Sa gayon, layunin ng pag-aaral na itong makatulong sa paggawa ng synthetic route ng avocadyne analogs sa pamamagitan ng pagsubok ng iba’t ibang oxidant upang madiskubre ang iba’t ibang paraan upang makabuo ng methyl ketone precursors.

Naging matagumpay ang pagbuo ng methyl ketones matapos magsagawa ng tatlong hakbang sa pagbuo nito– ang unang hakbang ay oxidation ng alkynol (a) upang mabuo ang alkynal (b), na sinundan ng Grignard reaction upang mabuo ang secondary alcohol (c), at ang huling hakbang ay oxidation ng secondary alcohol upang mabuo ang methyl ketone precursor (d). Nakumpirma ang mga nabuong compound gamit ang Mass Spectrometry (HRMS-ESI), 1H and 13C NMR Spectroscopy. Ang yield ng bawat precursor ay ang sumusunod: tetradec-13-yn-2-one (1d) (194 mg, 76%), dodec-11-yn-2-one (2d) (283 mg, 65%), dec-9-yn-2-one (3d) (351 mg, 77%), tridec-12-yn-2-one (4d) (78 mg, 42%), undec-10-yn-one (5d) (171 mg, 17% over three steps), and non-8-yn-2-one (6d) (198 mg, 39%).

Abstract Format

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Language

English

Format

Electronic

Keywords

Pseudomonas aeruginosa

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8-28-2026

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