Bcl-2-associated athanogene 3 (BAG3) is a multifunctional co-chaperone protein that regulates apoptosis, autophagy, and proteostasis through interactions with HSP70 and other partners. Overexpression of BAG3 contributes to tumor cell survival, metastasis, and chemotherapy resistance, making it an appealing but challenging anticancer target due to its intrinsic disorder and lack of structural data. Here, we report a fragment-based drug discovery (FBDD) approach to identify novel small molecules targeting human BAG3. A fragment library of 783 compounds was screened using a thermal shift assay (TSA) against recombinant BAG3 expressed in mammalian cells, followed by hit validation through ligand-observed NMR (WaterLOGSY). Eleven fragments stabilized the protein, and seven were confirmed as binders. Among them, a 6-chloro-2-oxindole fragment ( Fr1 ) exhibited the strongest interaction, with a dissociation constant (K D ) of 97.8 ± 11.1 μM. Structure–activity relationship (SAR) studies focused on maintaining the 6-chloro-2-oxindole core and optimizing substitutions at position 3, identified derivative 7 as a promising lead. Derivative 7 bound BAG3 with improved affinity (K D ≈ 22 μM), as confirmed by grating-coupled interferometry, and displaced Fr1 in competition NMR assays. This work demonstrates the feasibility of applying FBDD to intrinsically disordered and structurally unresolved proteins such as BAG3, providing a validated chemical starting point for the development of selective BAG3 inhibitors. These findings expand the druggability landscape of BAG3 and highlight fragment-based methodologies as powerful tools to explore protein–protein interaction targets previously considered intractable.

Fragment-based discovery of novel STING agonists validated with free energy calculations

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.

Fragment-based discovery and optimization of Zika virus NS3 helicase ligands as antiviral hit candidates.

Despite the improvement of therapeutic options, melanoma patients with advanced metastatic disease are still in high need of durable treatments. Analysis of clinical data from patients receiving targeted and/or immunotherapy, along with genetic and functional studies in preclinical melanoma models, demonstrates the key role of the microphthalmia-associated transcription factor (MITF) throughout disease progression, and provides a solid rationale for its therapeutic inhibition. However, direct targeting of MITF or other basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors is unprecedented. Here, we report on the discovery of ligands for the DNA binding domain of MITF, using fragment-based screening (FBS) by nuclear magnetic resonance (NMR). Initial fragments, binding the kink pocket of MITF very weakly, are optimized to sub-micromolar affinities by structure-based design enabled by X-ray crystallography and biophysics. Furthermore, NMR experiments and molecular dynamics simulations reveal a dynamic conformational exchange between helices in the asymmetric homodimer, a phenomenon that is perturbed by ligand binding. This work advances our knowledge on direct targeting of bHLH-LZ DNA binding domains and sets the basis to further explore pharmacological inhibition of MITF.

Fragment-based discovery, dynamics simulation and pharmacological study of 2-amino-pyrimidine derivative as HIPK2 inhibitor.

Flap endonuclease 1 (FEN1) is a long-standing target of interest in the DNA damage response (DDR) field due to its therapeutic potential in BRCA mutant cancers. To-date there have only been a handful of FEN1 inhibitors reported in the literature, most of which display modest selectivity and/or weak cellular activity. As such, there is a need for more advanced pharmacological tools to probe the biology of FEN1. Here, we report the discovery of MSC778, the first potent, selective, and orally bioavailable FEN1 inhibitor. We describe our metal-chelating fragment screening approach and structure-based optimization to identify MSC778, using structural insights to drive design. Consistent with FEN1 inhibition, MSC778 selectively kills BRCA2-deficient cells and potentiates the activity of PARPi niraparib in vivo to induce tumor stasis in a BRCA2 KO DLD-1 mouse xenograft. Furthermore, we illustrate how development of this approach has the potential for addressing nucleases as a target class.

Fragment-based discovery of TopBP1 inhibitors integrated with AI-driven molecular docking

Fragment-based drug discovery typically relies on specialized spectrometric methods to identify low-affinity compounds that bind to biomolecules. Here, we report a proof-of-concept study on the development of a streamlined fragment-based screening platform for small molecules targeting RNA. This method employs low molecular weight fragments appended with a diazirine reactive moiety and an alkyne tag. Upon photolysis and click chemistry with an azide-containing fluorophore, these compounds can be visualized for binding to the r(CUG) repeat expansion [r(CUG)exp] implicated in myotonic dystrophy type 1 (DM1). Fragments were found to bind the 1 × 1 nucleotide U/U internal loops formed when r(CUG)exp folds, guiding the design of homodimeric compounds capable of interacting with adjacent internal loops in a single molecule. One dimeric compound exhibited enhanced affinity and was converted into a proximity-induced covalent binder for prolonged target occupancy. This work establishes a versatile platform for targeting structured RNAs with potential applications across a variety of disease-relevant RNA targets.

Calcitonin gene-related peptide (CGRP) receptor antagonists have demonstrated clinical efficacy in the treatment of migraine. In this study, we performed surface plasmon resonance-based fragment screening to identify various site binders and estimated their binding modes based on surface plasmon resonance and nuclear magnetic resonance studies using mutated CGRP receptors. Two fragment hits were merged and optimized to create compound 15, which showed good oral bioavailability in rats, attractive preclinical properties overall, and robust activity in a primate model of CGRP-induced facial blood flow.

The phospholipid sensing transcription factor liver receptor homologue 1 (LRH-1) participates in the transcriptional regulation of metabolic balance and inflammation in liver, pancreas, and other tissues. It is an emerging target for metabolic dysfunction, fatty liver disease, and cancer, but LRH-1 modulators are rare and lack drug-like properties. We discovered new LRH-1 ligands with improved physicochemical features in a fragment-based approach and optimized a venlafaxine-related lead for LRH-1 activation. Despite a strict structure-activity relationship, systematic structural variation resulted in a new LRH-1 agonist scaffold with strong activation efficacy, validated direct and cellular target engagement, and anti-inflammatory and ER-stress-resolving properties in functional cellular settings.

Designed polypharmacology aims to exploit additive or synergistic effects of simultaneous multi-target modulation. Multifactorial diseases like metabolic dysfunction requiring multi-drug treatment may significantly benefit from this concept. To identify multi-target lead pharmacophores for the development of designed dual ligands, we performed a focused two-stage screening of fatty acid mimetic fragments for modulation of the nuclear receptors THR, PPAR, FXR and RXR which are involved in transcriptional regulation of metabolic balance. Dual, multiple and pan-agonist hits were retrieved for various combinations of these targets of interest and preliminary SAR evaluation yielded dual agonist and pan-agonist fragments with attractive potency and efficacy as valuable leads for polypharmacology.

Identification of triazolyl KAT6 inhibitors via a templated fragment approach

The PWWP domain of hepatoma-derived growth factor-related protein 2 (HDGFRP2) recognizes methylated histones to initiate the recruitment of homologous recombination repair proteins to damaged silent genes. The combined depletion of HDGFRP2 and its paralog PSIP1 effectively impedes the onset and progression of diffuse intrinsic pontine glioma (DIPG). Here, we discovered varenicline and 4-(4-bromo-1H-pyrazol-3-yl) pyridine (BPP) as inhibitors of the HDGFRP2 PWWP domain through a fragment-based screening method. The complex crystal structures reveal that both Varenicline and BPP engage with the aromatic cage of the HDGFRP2 PWWP domain, albeit via unique binding mechanisms. Notably, BPP represents the first single-digit micromolar inhibitor of the HDGFRP2 PWWP domain with a high ligand efficiency. As a dual inhibitor targeting both HDGFRP2 and PSIP1 PWWP domains, BPP offers an exceptional foundation for further optimization into a chemical tool to dissect the synergetic function of HDGFRP2 and PSIP1 in DIPG pathogenesis.

Chemical probes are an indispensable tool for translating biological discoveries into new therapies, though are increasingly difficult to identify since novel therapeutic targets are often hard-to-drug proteins. We introduce FRASE-based hit-finding robot (FRASE-bot), to expedite drug discovery for unconventional therapeutic targets. FRASE-bot mines available 3D structures of ligand-protein complexes to create a database of FRAgments in Structural Environments (FRASE). The FRASE database can be screened to identify structural environments similar to those in the target protein and seed the target structure with relevant ligand fragments. A neural network model is used to retain fragments with the highest likelihood of being native binders. The seeded fragments then inform ultra-large-scale virtual screening of commercially available compounds. We apply FRASE-bot to identify ligands for Calcium and Integrin Binding protein 1 (CIB1), a promising drug target implicated in triple negative breast cancer. FRASE-based virtual screening identifies a small-molecule CIB1 ligand (with binding confirmed in a TR-FRET assay) showing specific cell-killing activity in CIB1-dependent cancer cells, but not in CIB1-depletion-insensitive cells.

β-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher's disease and are a major genetic risk factor for Parkinson's disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.

MUS81 is a structure-selective endonuclease that cleaves various branched DNA structures arising from natural physiological processes such as homologous recombination and mitosis. Due to this, MUS81 is able to relieve replication stress, and its function has been reported to be critical to the survival of many cancers, particularly those with dysfunctional DNA-repair machinery. There is therefore interest in MUS81 as a cancer drug target, yet there are currently few small molecule inhibitors of this enzyme reported, and no liganded crystal structures are available to guide hit optimization. Here we report the fragment-based discovery of novel small molecule MUS81 inhibitors with sub-μM biochemical activity. These inhibitors were used to develop a novel crystal system, providing the first structural insight into the inhibition of MUS81 with small molecules.

DNA methyl transferases (DNMTs) are one of the crucial epigenetic modulators associated with a wide variety of cancer conditions. Among the DNMT isoforms, DNMT1 is correlated with bladder, pancreatic, and breast cancer, as well as acute myeloid leukemia and esophagus squamous cell carcinoma. Therefore, the inhibition of DNMT1 could be an attractive target for combating cancers and other metabolic disorders. The disadvantages of the existing nucleoside and non-nucleoside DNMT1 inhibitors are the main motive for the discovery of novel promising inhibitors. Here, pharmacophore modeling, 3D-QSAR, and e-pharmacophore modeling of DNMT1 inhibitors were performed for the large fragment database screening. The resulting fragments with high dock scores were combined into molecules. The current study revealed several constitutional pharmacophoric features that can be essential for selective DNMT1 inhibition. The fragment docking and virtual screening identified 10 final hit molecules that exhibited good binding affinities in terms of docking score, binding free energies, and acceptable ADME properties. Also, the modified lead molecules (GL1b and GL2b) designed in this study showed effective binding with DNMT1 confirmed by their docking scores, binding free energies, 3D-QSAR predicted activities and acceptable drug-like properties. The MD simulation studies also suggested that leads (GL1b and GL2b) formed stable complexes with DNMT1. Therefore, the findings of this study can provide effective information for the development/identification of novel DNMT1 inhibitors as effective anticancer agents.

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Advancing structural insights and inhibitor development for RQT complex via a fragment-based discovery platform