Search Results (34)

Fragment-based drug discovery (FBDD) has emerged as a primary approach for identifying low molecular weight leads that can be systematically optimized into high-affinity compounds. Because fragments bind inherently weakly, their detection relies on highly sensitive biophysical tools. Nuclear magnetic resonance (NMR) spectroscopy is uniquely qualified for fragment screening due to its capability in detecting weak interactions across a broad affinity range while providing site-specific binding information that supports structure-guided optimization. While FBDD is a mature field for protein targets, structured and disease-relevant RNAs have transitioned from ‘undruggable’ molecules to viable therapeutic targets for small-molecule intervention. Recent studies demonstrate that NMR-based screening can identify authentic RNA binders and guide their evolution into potent, selective ligands. This review summarizes the practical and methodological pipelines for RNA-targeted small molecule NMR screening, covering RNA construct design, sample preparation, and library pooling strategies. We evaluate both ligand- and RNA-observed NMR assays for primary hit screening and validation, integration of NMR restraints with structural modeling, and representative case studies. Finally, we discuss current bottlenecks in the field and highlight emerging strategies to accelerate the discovery of RNA-directed therapeutics. Full article

Bromodomain-containing protein 9 (BRD9) belongs to the non-canonical BAF chromatin remodeling complex and represents a relevant therapeutic target in pathologies featuring dysregulated epigenetic control. The absence of clinically validated inhibitors and the need for diversified chemical entities highlight the interest in identifying new scaffolds targeting this protein. In this study, Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR) was employed to assess its suitability for characterizing BRD9–ligand interactions within a fragment-based discovery framework. STD NMR conditions were first optimized using the known BRD9 ligand 1, verifying the presence of interaction signals. A pharmacophore-based virtual screening campaign was then performed using libraries of commercially available fragments, leading to the selection of a novel isatin derivative, i.e., compound 2, whose binding was demonstrated in AlphaScreen assays. STD NMR experiments provided epitope mapping consistent with the predicted binding mode, thus supporting the stability of the interaction in solution. Moreover, a competitive STD experiment demonstrated displacement of 2 by a reference ligand, confirming the binding within the canonical BRD9 pocket. Overall, this study establishes STD NMR as a reliable approach for probing BRD9–ligand interactions and for the identification and validation of BRD9-targeting scaffolds suitable for future structure-guided optimization. Full article

Background: SY0916 is a novel PAF receptor antagonist used to treat chronic immune-inflammatory diseases and is currently undergoing phase II clinical trials. However, SY0916 is rapidly transformed in vivo, suggesting a demand for metabolite screening. Methods: According to the similar MS fragmentation patterns of SY0916 and its five reported metabolites (M01, M02, M03, M05, and M06), a strategy based on two characteristic ions of m/z 170 and m/z 142 was employed to identify the potential metabolites in precursor screening in vivo, then LC-HRMS and NMR were applied to the metabolites characterization. Results: Two phase I metabolites (M07 and M08) were identified using LC-HRMS and NMR. Eight phase II metabolites, including six glutathione conjugates (M09-M14) and two sulfonate conjugates (M15 and M16), were identified using LC-HRMS. The possible metabolic pathways of SY0916 and fragmentation regularities of mass spectra of its metabolites were summarized. Conclusions: We preliminarily determined the metabolic profile of SY0916 in rats using LC-MS, providing insight into the metabolism of SY0916 in vivo and a basis for clinical studies and future applications. Full article

Background: Sphingosine kinase (SphK) overexpression is observed in many cancers, including breast, renal and leukaemia, which leads to increased cellular proliferation, survival and growth. SphK inhibition has been an attractive target for anticancer drug development for the past decade, with SphK inhibitors such as PF-543 and opaganib exhibiting clinical antitumour effects. By exploiting both CB5468139 and PF-543 as structural leads, we hereby report on the first quinoline-5,8-dione-based SphK inhibitor using a fragment-based approach. Methods: The quinoline-5,8-dione framework was developed to incorporate two defined regions, namely a polar quinoline core, which links to an aryl lipophilic chain. All synthetic molecules were characterized by NMR and HRMS and assayed against SphK 1 and 2, and molecular docking studies were performed. A subset of compounds was screened for anticancer activity. Results: As the binding site of SphK accommodates the lipophilic tail of sphingosine, we initially set out to explore the substitution of the C(7) aryl moiety to attain eight novel C(7) ether-linked quinoline-5,8-diones, which were screened for SphK1 and SphK2 activity with good potency identified. To improve SphK binding, structural fragments were adapted from PF-543 to participate in hydrogen bonding within the binding site of SphK1. A model study was performed to yield novel compounds through activated C(2) formyl intermediates. Two pyrrolidine-based quinoline-5,8-diones were assayed for SphK activity, with 21 revealing an improvement of SphK1 binding efficacy relative to the parent compound and 20 (and its precursor 4). Molecular modelling on the pyrrolidine quinoline-5,8-dione construct revealed favourable docking, low binding energies and opportunities for further improvement. Conclusions: Although the screening of anticancer activity was inconclusive, low micromolar dual SphK1/2 inhibition with the quinoline-5,8-dione framework has been identified for the first time, and a plausible new binding mode has been identified. Full article

Fragment-based drug discovery is a powerful approach in drug discovery, applicable to a wide range of targets. This method enables the discovery of potent compounds that can modulate target functions, starting from fragment compounds that bind weakly to the targets. While biochemical, biophysical, and cell-based assays are commonly used to identify fragments, ¹⁹F-NMR spectroscopy has emerged as a powerful tool for exploring interactions between biomolecules and ligands. Because fluorine atoms are not naturally present in biological systems, ¹⁹F-NMR serves as a sensitive method for fragment screening against diverse targets. Herein, we reviewed the applications of ¹⁹F-NMR in fragment screening, highlighting its effectiveness in identifying fragments that bind weakly to various targets such as proteins and RNA. The accumulated evidence suggests that ¹⁹F-NMR will continue to be a crucial tool in drug discovery. Full article

Lysergic acid diethylamide (LSD) and its analogs are commonly encountered substances at crime scenes due to their misuse as hallucinogenic compounds. Modern methods have led to synthesizing different LSD analogs with pronounced physiological effects. Theoretical methods can be a valuable tool for predicting the spectra and stability of novel substances, especially when experimental data are partially available. The current work describes the application of theoretical methods in predicting IR, NMR, UV–VIS, and MS spectra of LSD based on the optimized structure at the M05-2X/6-311++G(d,p) level of theory. A suitable functional has been determined by comparison of the theoretically obtained geometrical parameters with the experimental ones based on the crystallographic structure. The MAE values for the structure optimized at M05-2X/6-311++G(d,p) level of theory were 0.0436 Å (bond lengths) and 2.70° (bond angles). The IR spectra of LSD and LSD tartrate have been described in detail, with the prominent bands being well reproduced (the difference between experimental and theoretical C=O stretching vibration wavenumbers was lower than 11 cm⁻¹). Detailed assignment of ¹³C NMR spectra led to a high correlation factor (0.999) and low mean absolute error (2.0 ppm) between experimental and theoretical chemical shifts. Optimizing the ground and excited states allowed for the calculation of the energy difference of 330 nm, which reproduced the observed band position in the UV–VIS spectrum of LSD. The most abundant fragments in the experimental mass spectrum (at 323, 221, 207, 181, and 72 m/z) have been optimized, and their stability has been discussed from the structural point of view. This methodology has been validated by comparison with the experimental GC-MS spectra of sample seized at the crime screen and by structure optimization and computation of NMR spectra of common LSD analogs. The theoretical methods for the structure determination and prediction of spectra show great potential in the fast-developing world of new psychedelics. Full article

Melanoma differentiation-associated gene 9 (MDA-9) is a small adaptor protein with tandem PDZ domains that promotes tumor progression and metastasis in various human cancers. However, it is difficult to develop drug-like small molecules with high affinity due to the narrow groove of the PDZ domains of MDA-9. Herein, we identified four novel hits targeting the PDZ1 and PDZ2 domains of MDA-9, namely PI1A, PI1B, PI2A, and PI2B, using a protein-observed nuclear magnetic resonance (NMR) fragment screening method. We also solved the crystal structure of the MDA-9 PDZ1 domain in complex with PI1B and characterized the binding poses of PDZ1-PI1A and PDZ2-PI2A, guided by transferred paramagnetic relaxation enhancement. The protein–ligand interaction modes were then cross-validated by the mutagenesis of the MDA-9 PDZ domains. Competitive fluorescence polarization experiments demonstrated that PI1A and PI2A blocked the binding of natural substrates to the PDZ1 and PDZ2 domains, respectively. Furthermore, these inhibitors exhibited low cellular toxicity, but suppressed the migration of MDA-MB-231 breast carcinoma cells, which recapitulated the phenotype of MDA-9 knockdown. Our work has paved the way for the development of potent inhibitors using structure-guided fragment ligation in the future. Full article

Trypanosoma brucei is a species of kinetoplastid causing sleeping sickness in humans and nagana in cows and horses. One of the peculiarities of this species of parasites is represented by their redox metabolism. One of the proteins involved in this redox machinery is the monothiol glutaredoxin 1 (1CGrx1) which is characterized by a unique disordered N-terminal extension exclusively conserved in trypanosomatids and other organisms. This region modulates the binding profile of the glutathione/trypanothione binding site, one of the functional regions of 1CGrx1. No endogenous ligands are known to bind this protein which does not present well-shaped binding sites, making it target particularly challenging to target. With the aim of targeting this peculiar system, we carried out two different screenings: (i) a fragment-based lead discovery campaign directed to the N-terminal as well as to the canonical binding site of 1CGrx1; (ii) a structure-based virtual screening directed to the 1CGrx1 canonical binding site. Here we report a small molecule that binds at the glutathione binding site in which the binding mode of the molecule was deeply investigated by Nuclear Magnetic Resonance (NMR). This compound represents an important step in the attempt to develop a novel strategy to interfere with the peculiar Trypanosoma Brucei redox system, making it possible to shed light on the perturbation of this biochemical machinery and eventually to novel therapeutic possibilities. Full article

Calculations with high accuracy for atomic and inter-atomic properties, such as nuclear magnetic resonance (NMR) spectroscopy and bond dissociation energies (BDEs) are valuable for pharmaceutical molecule structural analysis, drug exploration, and screening. It is important that these calculations should include relativistic effects, which are computationally expensive to treat. Non-relativistic calculations are less expensive but their results are less accurate. In this study, we present a computational framework for predicting atomic and inter-atomic properties by using machine-learning in a non-relativistic but accurate and computationally inexpensive framework. The accurate atomic and inter-atomic properties are obtained with a low dimensional deep neural network (DNN) embedded in a fragment-based graph convolutional neural network (F-GCN). The F-GCN acts as an atomic fingerprint generator that converts the atomistic local environments into data for the DNN, which improves the learning ability, resulting in accurate results as compared to experiments. Using this framework, the ${}^{13}$C/${}^1$H NMR chemical shifts of Nevirapine and phenol O–H BDEs are predicted to be in good agreement with experimental measurement. Full article

By binding to the spliceosomal protein Snu66, the human ubiquitin-like protein Hub1 is a modulator of the spliceosome performance and facilitates alternative splicing. Small molecules that bind to Hub1 would be of interest to study the protein-protein interaction of Hub1/Snu66, which is linked to several human pathologies, such as hypercholesterolemia, premature aging, neurodegenerative diseases, and cancer. To identify small molecule ligands for Hub1, we used the interface analysis, peptide modeling of the Hub1/Snu66 interaction and the fragment-based NMR screening. Fragment-based NMR screening has not proven sufficient to unambiguously search for fragments that bind to the Hub1 protein. This was because the Snu66 binding pocket of Hub1 is occupied by pH-sensitive residues, making it difficult to distinguish between pH-induced NMR shifts and actual binding events. The NMR analyses were therefore verified experimentally by microscale thermophoresis and by NMR pH titration experiments. Our study found two small peptides that showed binding to Hub1. These peptides are the first small-molecule ligands reported to interact with the Hub1 protein. Full article

Antimicrobial resistance (AMR) poses a serious threat to our society from both the medical and economic point of view, while the antibiotic discovery pipeline has been dwindling over the last decades. Targeting non-essential bacterial pathways, such as those leading to antibiotic persistence, a bacterial bet-hedging strategy, will lead to new molecular entities displaying low selective pressure, thereby reducing the insurgence of AMR. Here, we describe a way to target (p)ppGpp (guanosine tetra- or penta-phosphate) signaling, a non-essential pathway involved in the formation of persisters, with a structure-based approach. A superfamily of enzymes called RSH (RelA/SpoT Homolog) regulates the intracellular levels of this alarmone. We virtually screened several fragment libraries against the (p)ppGpp synthetase domain of our RSH chosen model Rel_Seq, selected three main chemotypes, and measured their interaction with Rel_Seq by thermal shift assay and STD-NMR. Most of the tested fragments are selective for the synthetase domain, allowing us to select the aminobenzoic acid scaffold as a hit for lead development. Full article

YEATS (YAF9, ENL, AF9, TAF14, SAS5) family proteins recognize acylated histones and in turn regulate chromatin structure, gene transcription, and stress signaling. The chromosomal translocations of ENL and mixed lineage leukemia are considered oncogenic drivers in acute myeloid leukemia and acute lymphoid leukemia. However, known ENL YEATS domain inhibitors have failed to suppress the proliferation of 60 tested cancer cell lines. Herein, we identified four hits from the NMR fragment-based screening against the AF9 YEATS domain. Ten inhibitors of new chemotypes were then designed and synthesized guided by two complex structures and affinity assays. The complex structures revealed that these inhibitors formed an extra hydrogen bond to AF9, with respect to known ENL inhibitors. Furthermore, these inhibitors demonstrated antiproliferation activities in AF9-sensitive HGC-27 cells, which recapitulated the phenotype of the CRISPR studies against AF9. Our work will provide the basis for further structured-based optimization and reignite the campaign for potent AF9 YEATS inhibitors as a precise treatment for AF9-sensitive cancers. Full article

Tuberculosis (TB) remains a public health crisis, requiring the urgent identification of new anti-mycobacterial drugs. We screened several organic and aqueous marine invertebrate extracts for their in vitro inhibitory activity against the causative organism, Mycobacterium tuberculosis. Here, we report the results obtained for 54 marine invertebrate extracts. The chemical components of two of the extracts were dereplicated, using ¹H NMR and HR-LCMS with GNPS molecular networking, and these extracts were further subjected to an activity-guided isolation process to purify the bioactive components. Hyrtios reticulatus yielded heteronemin 1 and Jaspis splendens was found to produce the bengamide class of compounds, of which bengamides P 2 and Q 3 were isolated, while a new derivative, bengamide S 5, was putatively identified and its structure predicted, based on the similarity of its MS/MS fragmentation pattern to those of other bengamides. The isolated bioactive metabolites and semi-pure fractions exhibited M. tuberculosis growth inhibitory activity, in the range <0.24 to 62.50 µg/mL. This study establishes the bengamides as potent antitubercular compounds, with the first report of whole-cell antitubercular activity of bengamides P 2 and Q 3. Full article

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series. Full article

A series of new tertiary phenothiazine derivatives containing a quinoline and a pyridine fragment was synthesized by the reaction of 1-methyl-3-benzoylthio-4-butylthioquinolinium chloride with 3-aminopyridine derivatives bearing various substituents on the pyridine ring. The direction and mechanism of the cyclization reaction of intermediates with the structure of 1-methyl-4-(3-pyridyl)aminoquinolinium-3-thiolate was related to the substituents in the 2- and 4-pyridine position. The structures of the compounds were analyzed using ¹H, ¹³C NMR (COSY, HSQC, HMBC) and X-ray analysis, respectively. Moreover, the antiproliferative activity against tumor cells (A549, T47D, SNB-19) and a normal cell line (NHDF) was tested. The antibacterial screening of all the compounds was conducted against the reference and quality control strain Staphylococcus aureus ATCC 29213, three clinical isolates of methicillin-resistant S. aureus (MRSA). In silico computation of the intermolecular similarity was performed using principal component analysis (PCA) and hierarchical clustering analysis (HCA) on the pool of structure/property-related descriptors calculated for the novel tetracyclic diazaphenothiazine derivatives. The distance-oriented property evaluation was correlated with the experimental anticancer activities and empirical lipophilicity as well. The quantitative shape-based comparison was conducted using the CoMSA method in order to indicate the potentially valid steric, electronic and lipophilic properties. Finally, the numerical sampling of similarity-related activity landscape (SALI) provided a subtle picture of the SAR trends. Full article