Structure-Activity Relationship and Target Investigation of Thiophen-2-yl-Pyrimidines against <i>Schistosoma</i> species

bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-08

ABSTRACT Chemotherapeutic options for schistosomiasis, a prevalent infectious disease of poverty, are limited to just one drug, praziquantel (PZQ), and alternatives are needed. Our previous studies identified thiophen-2-yl pyrimidines (TPPs), which are structurally derived from microtubule (MT)-active phenylpyrimidines, as potent paralytics of Schistosoma mansoni . Although relatively non-toxic to mammalian cells, the progenitor compound, 3 , had poor aqueous solubility and was lipophilic potentially hindering preclinical advancement. To address these issues and expand on the structure-activity and structure-property relationships, 43 new TPP analogs were designed and synthesized, their lipophilicity calculated (cLogP), and their anti-schistosomal activity evaluated in culture. This effort yielded compound 38 , which possessed an oxetane-containing amine moiety at C5, and an ortho, ortho- difluoroaniline at C6 of the TPP scaffold. Compared to 3 , compound 38 had better aqueous solubility (46 vs. &lt; 0.5 µM) and decreased lipophilicity (logP calc. 4.48 vs. 6.81), with toxicity CC 50 values &gt; 20 µM against three mammalian cell lines. Further, paralytic potency, as measured by the EC 50 value for adult S. mansoni motility, was increased 14.5-fold (538 vs. 37 nM), and plasma half-life (t 1/2 ) was improved 3-fold, from 0.48 to 1.51 h for a 40% loss in maximum plasma concentration (C max ). In washout experiments, 38 produced a sustained paralysis of both juvenile and adult S. mansoni, possibly suggesting a broader in vitro efficacy spectrum compared to PZQ, which is inactive against the juvenile parasite. Also, the two other medically important species, Schistosoma haematobium and Schistosoma japonicum, were susceptible to 38 . Finally, to identify potential protein targets, we synthesized a TPP photoaffinity labeling (PAL) probe that labeled several S. mansoni proteins by SDS-PAGE fluorescence analysis, although, notably, not tubulin, suggesting that the antischistosomal activity of 38 is a function of engaging other targets. Future work with the TPP series will aim to decrease toxicity further while improving PK properties to better support in vivo efficacy testing.

<i>d</i>₄-Cystamine: A Deuterated Cystamine Derivative with Improved Anti-Inflammatory and Anti-Fibrotic Activities in a Murine Model of Fibrosing Steatohepatitis

ACS Pharmacology & Translational Science · 2025-02-24 · 2 citations

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a multifactorial chronic disease that can progress to metabolic dysfunction-associated steatohepatitis (MASH) and liver fibrosis, ultimately leading to liver cirrhosis and hepatocellular carcinoma. Oxidative stress is believed to play an important role in the development of MASH. Small aminothiol compounds such as cysteamine and its oxidized precursor, cystamine, are known pleiotropic compounds that exhibit relatively potent antioxidant and other effects. Herein, we evaluate the efficacy of cystamine, as well as two deuterated derivatives, in a choline-deficient, L-amino acid-defined, high-fat-diet (CDAA-HFD) mouse model of rapidly progressing liver fibrosis. Compared to control mice, daily oral administration of isotopically reinforced cystamine derivatives (200 mg/kg) led to a significant reduction of liver fibrosis and inflammation as well as oxidative stress. Moreover, the efficacy of treatment appeared to increase with the deuteration state of cystamine, with the tetradeuterated derivative, d4-cystamine, being the most effective. These results indicate that deuterated cystamine derivatives hold promise as potential candidates for the treatment of MASH.

Photoaffinity labeling of protein targets in a complex metazoan: proof-of-concept using a probe for <i>Schistosoma mansoni</i> tubulin

bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-06 · 1 citations

ABSTRACT Treatment of schistosomiasis, a prevalent neglected tropical disease, relies precariously on a single drug. The discovery and development of alternative anti-schistosomal small molecules most often relies on phenotypic (whole-organism) screening, whereas target- or “protein-first”-based discovery options are hampered by a paucity of genetic interrogation strategies and a complex biology. Here, we demonstrate the application of photoaffinity labeling (PAL) as a chemical biology strategy to probe protein target-ligand interactions in the schistosome. Using a triazolopyrimidine (TPD) probe that binds tubulin, we established a PAL workflow with living Schistosoma mansoni worms. The probe elicited deleterious phenotypic responses consistent with the TPD series and, upon UV light activation, covalently labeled tubulin as identified by proteomics. Specific concentration-dependent engagement of tubulin was confirmed using a photostable competitor TPD. When applied directly to worm lysates, the PAL workflow produced non-specific labeling, suggesting that the conformation of the protein target is important for ligand binding. The successful application of PAL for a metazoan is, to our knowledge, novel, and the platform should prove generally applicable to identifying potential drug targets, and exploring protein-ligand interactions, in schistosomes and other organisms.

Kaempferia parviflora extract and its methoxyflavones as potential anti-Alzheimer assessing in vitro, integrated computational approach, and in vivo impact on behaviour in scopolamine-induced amnesic mice

PLoS ONE · 2025-03-10 · 6 citations

Alzheimer's disease (AD), a growing global challenge, lacks effective preventive and therapeutic strategies. This study explored the promising potential of the Kaempferia parviflora (KP) and its methoxyflavones (MFs) against the disease. We evaluated KP extract and its five MFs for antioxidant capacity, cholinesterase inhibition (AChE, and BChE), amyloid plaque (Aβ) reduction, neuroprotection, and memory improvement in a mouse model. HPLC quantified the five MFs in KP extract, with 5,7-dimethoxyflavone (F1) being the most abundant. 5,7,4'-Trimethoxyflavone (F3) and 5-hydroxy-3,7-dimethoxyflavone (F4) exhibited the strongest AChE and BChE inhibitory activities, respectively. MFs hindered Aβ1-42 aggregation and destabilized fibrils, with F3 showing the potent anti-aggregation and the strongest fibril destabilization. They also protected SH-SY5Y cells from Aβ1-42-induced damage. Notably, F3 combined anti-cholinesterase and anti-Aβ activities, suggesting its potential as a multi-target agent. KP extract ameliorated scopolamine-induced memory deficits in mice, suggesting its potential for cognitive improvement. These findings revealed that KP can be a promising candidate for herbal medicine development against AD. Its multi-target MFs offered a unique advantage by targeting multiple AD pathways. KP may have a great potential to modify the disease and overcome the challenge of drug development as cognitive enhancing herbal medicine.

Design, Synthesis, and Evaluation of An Anti‐trypanosomal [1,2,4]Triazolo[1,5‐<i>a</i>]pyrimidine Probe for Photoaffinity Labeling Studies

ChemMedChem · 2024-01-26 · 6 citations

Studies have shown that depending on the substitution pattern, microtubule (MT)-targeting 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) can produce different cellular responses in mammalian cells that may be due to these compounds interacting with distinct binding sites within the MT structure. Selected TPDs are also potently bioactive against the causative agent of human African trypanosomiasis, Trypanosoma brucei, both in vitro and in vivo. So far, however, there has been no direct evidence of tubulin engagement by these TPDs in T. brucei. Therefore, to enable further investigation of anti-trypanosomal TPDs, a TPD derivative amenable to photoaffinity labeling (PAL) was designed, synthesized, and evaluated in PAL experiments using HEK293 cells and T. brucei. The data arising confirmed specific labeling of T. brucei tubulin. In addition, proteomic data revealed differences in the labeling profiles of tubulin between HEK293 and T. brucei, suggesting structural differences between the TPD binding site(s) in mammalian and trypanosomal tubulin.

Clickable Probes for Pathogen Proteasomes: Synthesis and Applications

ACS Omega · 2024-08-02 · 1 citations

The 20S proteasome is a multimeric protease complex that is essential for proteostasis in the cell. Small molecule proteasome inhibitors are approved drugs for various cancers and are advancing clinically as antiparasitics. Although tools and technologies to study the 20S proteasome have advanced, only one probe is commercially available to image proteasome activity. This probe consists of a fluorescently labeled, peptidyl vinyl sulfone that binds to one or more of the catalytic proteasome subunits. Here, we synthesized two, active site-directed epoxyketone probes, LJL-1 and LJL-2, that were based on the peptidyl backbones of the anticancer drugs, carfilzomib and bortezomib, respectively. Each probe was conjugated, via click chemistry, to a bifunctional group comprising 5-carboxytetramethylrhodamine (TAMRA) and biotin to, respectively, visualize and enrich the 20S proteasome from protein extracts of two eukaryotic pathogens, Leishmania donovani and Trichomonas vaginalis. Depending on species, each probe generated a different subunit-binding profile by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the biotin tag enabled the enrichment of the bound subunits which were then formally identified by proteomics. Species differences in the order of electrophoretic migration by the β subunits were also noted. Finally, both probes reacted specifically with the 20S subunits in contrast to the commercial vinyl sulfone probe that cross reacted with cysteine proteases. LJL-1 and LJL-2 should find general utility in the identification and characterization of pathogen proteasomes, and serve as reagents to evaluate the specificity and mechanism of binding of new antiparasitic proteasome inhibitors.

A small‐molecule microtubule‐stabilizing agent safely reduces Aβ plaque and tau pathology in transgenic mouse models of Alzheimer's disease

Alzheimer s & Dementia · 2024-06-17 · 11 citations

INTRODUCTION: Intraneuronal inclusions composed of tau protein are found in Alzheimer's disease (AD) and other tauopathies. Tau normally binds microtubules (MTs), and its disengagement from MTs and misfolding in AD is thought to result in MT abnormalities. We previously identified triazolopyrimidine-containing MT-stabilizing compounds that provided benefit in AD mouse models and herein describe the characterization and efficacy testing of an optimized candidate, CNDR-51997. METHODS: CNDR-51997 underwent pharmacokinetic, pharmacodynamic, safety pharmacology, and mouse tolerability testing. In addition, the compound was examined for efficacy in 5XFAD amyloid beta (Aβ) plaque mice and PS19 tauopathy mice. RESULTS: CNDR-51997 significantly reduced Aβ plaques in 5XFAD mice and tau pathology in PS19 mice, with the latter also showing attenuated axonal dystrophy and gliosis. CNDR-51997 was well tolerated at doses that exceeded efficacy doses, with a good safety pharmacology profile. DISCUSSION: CNDR-51997 may be a candidate for advancement as a potential therapeutic agent for AD and/or other tauopathies. Highlights There is evidence of microtubule alterations (MT) in Alzheimer's disease (AD) brain and in mouse models of AD pathology. Intermittent dosing with an optimized, brain-penetrant MT-stabilizing small-molecule, CNDR-51997, reduced both Aβ plaque and tau inclusion pathology in established mouse models of AD. CNDR-51997 attenuated axonal dystrophy and gliosis in a tauopathy mouse model, with a strong trend toward reduced hippocampal neuron loss. CNDR-51997 is well tolerated in mice at doses that are meaningfully greater than required for efficacy in AD mouse models, and the compound has a good safety pharmacology profile.

High-throughput Virtual Screening- and Molecular Docking-based Prediction for Acetylcholinesterase Inhibitors and Exploring its Mechanisms against Alzheimer’s Disease based on Network Pharmacology

Biomedical Sciences and Clinical Medicine · 2024-07-01 · 1 citations

OBJECTIVE This study aimed to identify promising ligands for inhibiting acetylcholinesterase (AChE) activity using virtual screening (VS). METHODS VS was used to identify potential AChE inhibitors from the PubChem database. Ligands with favorable binding pocket interactions were selected. SwissADME and pkCSM tools were used to assess drug-likeness and pharmacokinetic properties. Molecular dynamic (MD) simulations provided insights into binding interactions. Network pharmacology was used to explore interactions between the target molecule and AD-related genes to determine its mechanism of action. RESULTS VS identified promising AChE inhibitor candidates with acridone, carbazole, and xanthone scaffolds. Docking simulations showed strong binding with AChE. These ligands displayed favorable drug-likeness and ADMET properties, with one (M5) lacking predicted hepatotoxicity. MD simulations suggested stable binding of M5 to AChE, potentially affecting both catalytic and peripheral sites, hinting at dual inhibition. M5’s interactions, especially near His440, appeared more favorable than donepezil. Network analysis implicated M5 in targeting multiple pathways in AD, with potential focus on neuroinflammation. CONCLUSIONS This study identified promising AChE inhibitor candidates through virtual screening. Ligand M5 emerged as particularly promising due to its favorable binding characteristics, lack of predicted hepatotoxicity, and potential for targeting multiple pathways in AD. However, further in vitro and in vivo validation is essential for clinical development. KEYWORDS virtual screening, acetylcholinesterase inhibitors, PubChem database, molecular dynamics simulation, gene ontology, KEGG pathways

AI is a viable alternative to high throughput screening: a 318-target study

Scientific Reports · 2024 · 119 citations

• Computer Science
• Computer Science
• Machine Learning

High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery.

Targeted Reversible Covalent Modification of a Noncatalytic Lysine of the Krev Interaction Trapped 1 Protein Enables Site-Directed Screening for Protein–Protein Interaction Inhibitors

ACS Pharmacology & Translational Science · 2023-10-09 · 9 citations

The covalent reversible modification of proteins is a validated strategy for the development of probes and candidate therapeutics. However, the covalent reversible targeting of noncatalytic lysines is particularly challenging. Herein, we characterize the 2-hydroxy-1-naphthaldehyde (HNA) fragment as a targeted covalent reversible ligand of a noncatalytic lysine (Lys720) of the Krev interaction trapped 1 (KRIT1) protein. We show that the interaction of HNA with KRIT1 is highly specific, results in prolonged residence time of >8 h, and inhibits the Heart of glass 1 (HEG1)–KRIT1 protein–protein interaction (PPI). Screening of HNA derivatives identified analogs exhibiting similar binding modes as the parent fragment but faster target engagement and stronger inhibition activity. These results demonstrate that HNA is an efficient site-directing fragment with promise in developing HEG1-KRIT1 PPI inhibitors. Further, the aldimine chemistry, when coupled with templating effects that promote proximity, can produce a long-lasting reversible covalent modification of noncatalytic lysines.