Leuven innovatie VZW

Aldolase A (ALDOA) is a key enzyme in glycolysis, catalyzing the reversible conversion of fructose-1,6-diphosphate (FBP) to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). The aberrant overexpression of ALDOA is associated with the development of various solid tumors. Here, we developed an in vitro enzymatic coupling reaction assay, which demonstrates high cost-efficiency and is suitable for high-throughput screening (HTS). With this assay we identified two potential ALDOA inhibitors, merbromin and ellagic acid, from our in-house compound library. Merbromin and ellagic acid exhibit significant inhibitory activities with IC₅₀ values of 8.49 ± 0.62 μM and 19.87 ± 2.03 μM, respectively. The nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) experiments further confirmed their high affinities to ALDOA, with the dissociation constants (K_d) of 0.49 ± 0.10 μM and 0.64 ± 0.10 μM, respectively. Enzyme kinetics experiment revealed that both compounds act as noncompetitive inhibitors of ALDOA. Our study showed that the enzymatic coupling reaction-based assay established here is highly effective and offers a promising approach for the development of ALDOA inhibitors.

A series of thioamide-linked spiropyrrolidine derivatives were synthesized and evaluated against SARS-CoV-2 3CL^pro. Among these compounds, 9g demonstrated remarkable inhibition of 3CL^pro (IC₅₀ = 35 nM), surpassing the inhibitory potency of the approved antiviral drug nirmatrelvir (IC₅₀ = 54 nM). In cellular assays against HCoV-OC43 in Huh7 cells, 9g exhibited significant antiviral efficacy (EC₅₀ = 0.52 μM) and low cytotoxicity (CC₅₀ > 10 μM). Furthermore, 9g exhibited pharmacokinetic profiles comparable to that of nirmatrelvir, making it a promising candidate for further development.

The 3-chymotrypsin-like protease (3CL^pro) is a crucial enzyme for the replication of coronaviruses, notable for its high conservation across viral species and the lack of human analogs. These characteristics make it a prime target for the development of broad-spectrum antiviral medications. In this work, we incorporated the zolinium as a hydrophilic group and a ketone as the covalent warhead to develop novel agents targeting SARS-CoV-2 3CL^pro. We designed and synthesized 60 derivatives to systematically study their structure-activity relationships (SAR). Of these, compound 46 demonstrated the most potent inhibition against 3CL^pro (IC₅₀ = 1.75 ± 0.039 μM) and good selectivity against other five enzymes, with reasonable chemical stability and rapid reactivity with cysteine. Mass spectrometry-based peptide mapping revealed that the ketone group of compound 46 covalently modified Cys44 of SARS-CoV-2 3CL^pro. The inactivation kinetics indicated that compound 46 reduced the 3CL^pro activity in a time- and dose-dependent manner, with an inactivation efficiency constant (k_inact/K_i) of 0.011 min^-1 μM^-1. Further covalent docking and molecular dynamics simulations elucidated the binding mechanism involving the disruption of protein's dimer interface and stability, which was partially validated by Native-PAGE analysis. Moreover, compound 46 exhibited negligible cytotoxicity and good metabolic stability in liver microsome assays, positioning it as a promising covalent lead for the advancement of broad-spectrum anti-coronavirus therapies.