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.

Refractory hepatocellular carcinoma (HCC) perpetuates metastasis or recurrence through anti-cancer drug resistance, necessitating more effective and reliable therapeutic strategies. We propose a new therapeutic approach involving the discovery of novel small molecules through target identification and validation in a patient-derived metastatic HCC model. We showed that calcium/calmodulin-dependent protein kinase 2 alpha (CaMK2α)-mediated enhancement of sarco/endoplasmic reticulum (ER) calcium ATPase 1 (SERCA1) expression level was pivotal events under anti-cancer drug treated conditions in patient-derived metastatic HCC cells. Increased SERCA1 was regulates to overloaded free calcium. SERCA is widely recognized as a key regulator of cytosolic free calcium under severe ER stress conditions. Though a cardiac dysfunction was unavoidable in vivo because of non-specific inhibition of SERCA isoforms by standard SERCA inhibitors. Based on the molecular structure of SERCA1, we discovered and synthesized two SERCA1-specific inhibitors, candidate 56 and 62. These compounds significantly reduced tumor size in the metastatic HCC xenograft tumor model without cardiac contractile dysfunction. This study first showed survival mechanism of patient-derived metastatic HCC cell, and propose a new therapeutic approach by the new small molecules, candidate 56 and 62, which are SERCA1 isoform-specific inhibitors without cardiac dysfunction by SERCA1 selectively inhibition.

Discovery of novel small molecules targeting TGF-β signaling for the treatment of non-small cell lung cancer.

Soil microbial communities are pivotal to plant health and nutrient acquisition. It is becoming increasingly clear that many interactions, both among and between microbes and plants, are governed by small bioactive molecules or "secondary metabolites" that can aid in communication, competition, and nutrient uptake. Yet, secondary metabolite biogeography - who makes what, where, and why-is in its infancy. Further, secondary metabolite biosynthesis genes are often silent or weakly expressed under standard laboratory conditions, making it incredibly difficult to study these small molecules. To begin to address these dual challenges, we focused on redox-active metabolites (RAMs), a specific class of small molecules, and took advantage of recent findings that many RAMs aid in acquiring phosphorus and that their production is frequently stimulated by stress for this macronutrient. We developed a screen for RAM-producing bacteria that leverages phosphorus limitation to stimulate metabolite biosynthesis and uses a colorimetric (ferrozine) iron-reduction assay to identify redox activity. We isolated 557 root-associated bacteria from grasses collected at sites across the United States (Santa Rita Experimental Range [AZ], Konza Prairie Biological Station [KS], and Harvard Forest [MA]) and from commercial tomato plants and screened them for RAM production. We identified 128 soil isolates of at least 19 genera across Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes that produced RAMs under phosphorus stress. Our work reveals that the production of RAMs under phosphorus stress is common across diverse soil bacteria and provides an approach to screen for these small molecules rapidly.IMPORTANCEBy secreting secondary metabolites, bacteria at the plant root can defend against diseases and help acquire essential nutrients. However, the genes that synthesize secondary metabolites are typically inactive or are weakly expressed under standard laboratory conditions. This fact makes it difficult to study these small molecules and hinders the discovery of novel small molecules that may play crucial roles in agricultural and biomedical settings. Here, we focus on redox-active metabolites (RAMs), a class of secondary metabolites that can help bacteria solubilize phosphorus and are often produced when phosphorus is limited. We developed a screen that rapidly identifies RAM-producing bacteria by utilizing a colorimetric iron-reduction assay in combination with phosphorus limitation to stimulate biosynthesis. The screen reveals that RAM-producing bacteria are far more prevalent in soil than previously appreciated and that this approach can be used to identify RAM producers.

Discovery of novel small molecules targeting hepatitis B virus core protein from marine natural products with HiBiT-based high-throughput screening

Discovery of novel small molecules targeting the USP21/JAK2/STAT3 axis for the treatment of triple-negative breast cancer

Synthesis and preliminary evaluation of aminophenol derivatives as molecular glues blocking PD-1/PD-L1 interaction

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising therapeutic target for the treatment of hyperlipidemia. In discovery of novel small molecules that interfere PCSK9/LDLR protein-protein interaction (PPI), structural modification was performed based on our previously derived compounds. A series of [5,5'-bibenzo[d][1,3]dioxol]-6-amine analogs were designed and synthesized for the activity evaluation. In the PCSK9/LDLR PPI impairing test, molecules D28 and D29, exhibited remarkable inhibitory potency with IC50 values of 8.30 and 6.70 μM compared with SBC-115337 (17.89 μM), respectively. Molecular docking predicted the binding pattern of compounds D28 and D29 in the LDLR binding site of PCSK9. Hydrophobic interactions play an important role in the binding of aromatic molecular fragments to the pockets in the PCSK9/LDLR binding interface. Further LDLR expression and LDL uptake studies revealed that both D28 and D29 restored LDLR expression on the surface of hepatic HepG2 cells and improved extracellular LDL uptake in the presence of PCSK9. It is significant that molecules D28 and D29 exhibited potential for the treatment of hyperlipidemia in current in vitro investigations. Generally, lead compounds with novel structures were developed in the present study for further design of lipid-lowering molecules by targeting PCSK9/LDLR PPI.

225 (PB105) - Discovery of novel small molecules that recruit DCAF11 for selective degradation of BRD4

Origin of cancer is strongly related to the unusual epigenetic regulation of gene function as indicated by recent reports. The covalent modifications to DNA or histones without affecting genomes that finally lead to phenotypical changes in cells or organisms are referred as "Epigenetics." The possibility to reprogram the epigenetics in the cancer epigenome is the most important target for cancer treatment and drug resistance. The development of epigenetic drugs holds a great potential for the current cancer therapeutic approaches. Nevertheless, targeting cancer epigenetic pathways is still exciting due to the lack of selective and effective small molecule compounds or drug molecules. Therefore, the current book chapter highlights epigenetic pathways for cancer and potential small molecule inhibitors and epidrugs targeting DNA methyltransferase, histone modification, and more new therapies with nanomaterials and imaging to improve the effectiveness of cancer treatment. The structural aspects on discovery of novel small molecules or drugs targeting epigenetic pathways in cancer exploration as promising strategies will be also discussed.

γ-Secretase is an intramembrane aspartyl protease that is important in regulating normal cell physiology via cleavage of over 100 transmembrane proteins, including Amyloid Precursor Protein (APP) and Notch family receptors. However, aberrant proteolysis of substrates has implications in the progression of disease pathologies, including Alzheimer’s disease (AD), cancers, and skin disorders. While several γ-secretase inhibitors have been identified, there has been toxicity observed in clinical trials associated with non-selective enzyme inhibition. To address this, γ-secretase modulators have been identified and pursued as more selective agents. Recent structural evidence has provided an insight into how γ-secretase inhibitors and modulators are recognized by γ-secretase, providing a platform for rational drug design targeting this protease. In this study, docking- and pharmacophore-based screening approaches were evaluated for their ability to identify, from libraries of known inhibitors and modulators with decoys with similar physicochemical properties, γ-secretase inhibitors and modulators. Using these libraries, we defined strategies for identifying both γ-secretase inhibitors and modulators incorporating an initial pharmacophore-based screen followed by a docking-based screen, with each strategy employing distinct γ-secretase structures. Furthermore, known γ-secretase inhibitors and modulators were able to be identified from an external set of bioactive molecules following application of the derived screening strategies. The approaches described herein will inform the discovery of novel small molecules targeting γ-secretase.

The acquired immunodeficiency syndrome (AIDS), caused by the human immunodeficiency virus (HIV), has been a global public health challenge for several decades. The majority of HIV infection is caused by the human immunodeficiency virus type 1 (HIV-1), which enters and infects a host cell via the cell surface proteins of CD4 as the primary receptor, and chemokine receptors CXCR4 or CCR5 as the coreceptor-then undergoing replication using the cell's intracellular machinery. Whereas many drugs targeting CCR5-mediated entry or HIV-1 replication via reverse transcriptase or proteases have long been used clinically, agents targeting CXCR4 are yet to be advanced to clinical application. Here in this review we highlight some of the strategies for and progress made in the discovery of novel small molecules, peptides, and larger molecules that target CXCR4, and their future prospects for translation into the clinic as a new class of anti-HIV therapeutics.

Sirtuins are class III histone deacetylases, whose enzymatic activity is dependent on NAD+ as a cofactor. Sirtuins are reported to modulate numerous activities by controlling gene expression, DNA repair, metabolism, oxidative stress response, mitochondrial function, and biogenesis. Deregulation of their expression and/or action may lead to tissue-specific degenerative events involved in the development of several human pathologies, including cancer, neurodegeneration, and cardiovascular disease. The most studied member of this class of enzymes is sirtuin 1 (SIRT1), whose expression is associated with increasing insulin sensitivity. SIRT1 has been implicated in both tumorigenic and anticancer processes, and is reported to regulate essential metabolic pathways, suggesting that its activation might be beneficial against disorders of the metabolism. Via regulation of p53 deacetylation and modulation of autophagy, SIRT1 is implicated in cellular response to caloric restriction and lifespan extension. In recent years, scientific interest focusing on the identification of SIRT1 modulators has led to the discovery of novel small molecules targeting SIRT1 activity. This review will examine compounds of natural origin recently found to upregulate SIRT1 activity, such as polyphenolic products in fruits, vegetables, and plants including resveratrol, fisetin, quercetin, and curcumin. We will also discuss the potential therapeutic effects of these natural compounds in the prevention and treatment of human disorders, with particular emphasis on their metabolic impact.

Bladder cancer is the 10th most commonly diagnosed cancer worldwide. Although the incidence in men is 4 times higher than that in women, the diagnoses are worse for women. Over the past 30 years, the treatment for bladder cancer has not achieved a significant positive effect, and the outlook for mortality rates due to muscle-invasive bladder cancer and metastatic disease is not optimistic. Phytochemicals found in plants and their derivatives present promising possibilities for cancer therapy with improved treatment effects and reduced toxicity. In this study, we summarize the promising natural products of plant origin with anti-bladder cancer potential, and their anticancer mechanisms—especially apoptotic induction—are discussed. With the developments in immunotherapy, small-molecule targeted immunotherapy has been promoted as a satisfactory approach, and the discovery of novel small molecules against immune targets for bladder cancer treatment from products of plant origin represents a promising avenue of research. It is our hope that this could pave the way for new ideas in the fields of oncology, immunology, phytochemistry, and cell biology, utilizing natural products of plant origin as promising drugs for bladder cancer treatment.

BackgroundThe skin micro-environment varies across the body, but all sites are host to microorganisms that can impact skin health. Some of these organisms are true commensals which colonize a unique niche on the skin, while open exposure of the skin to the environment also results in the transient presence of diverse microbes with unknown influences on skin health. Culture-based studies of skin microbiota suggest that skin microbes can affect skin properties, immune responses, pathogen growth, and wound healing.ResultsIn this work, we greatly expanded the diversity of available commensal organisms by collecting > 800 organisms from 3 body sites of 17 individuals. Our collection includes > 30 bacterial genera and 14 fungal genera, with Staphylococcus and Micrococcus as the most prevalent isolates. We characterized a subset of skin isolates for the utilization of carbon compounds found on the skin surface. We observed that members of the skin microbiota have the capacity to metabolize amino acids, steroids, lipids, and sugars, as well as compounds originating from personal care products.ConclusionsThis collection is a resource that will support skin microbiome research with the potential for discovery of novel small molecules, development of novel therapeutics, and insight into the metabolic activities of the skin microbiota. We believe this unique resource will inform skin microbiome management to benefit skin health.63JN7tcpwHsG48Gd-WoakTVideo abstract.

Discovery of small molecules targeting GRP78 for antiangiogenic and anticancer therapy

Discovery of novel small molecule induced selective degradation of the bromodomain and extra-terminal (BET) bromodomain protein BRD4 and BRD2 with cellular potencies

Background:Capsular contracture is a devastating complication that occurs in patients undergoing implant-based breast reconstruction. Ionizing radiation drives and exacerbates capsular contracture in part by activating cytokines, including transforming growth factor-beta (TGF-β). TGF-β promotes myofibroblast differentiation and proliferation, leading to excessive contractile scar formation. Therefore, targeting the TGF-β pathway may attenuate capsular contracture.Methods:A 20,000 small molecule library was screened for anti-TGF-β activity. Structurally diverse anti-TGF-β agents were identified and then tested on primary human capsular fibroblasts. Fibroblasts were irradiated or not, and then treated with both TGF-β and candidate molecules. Resulting cells were then analyzed for myofibroblast activity using myofibroblast markers including alpha-smooth muscle actin, collagen I, Thy1, and periostin, using Western Blot, quantitative real-time polymerase chain reaction, and immunofluorescence.Results:Human capsular fibroblasts treated with TGF-β showed a significant increase in alpha-smooth muscle actin, collagen I, and periostin levels (protein and/or mRNA). Interestingly, fibroblasts treated with latent TGF-β and 10 Gy radiation also showed significantly increased levels of myofibroblast markers. Cells that were treated with the novel small molecules showed a significant reduction in myofibroblast activation, even in the presence of radiation.Conclusions:Several novel small molecules with anti-TGF-β activity can effectively prevent human capsular fibroblast to myofibroblast differentiation in vitro, even in the presence of radiation. These results highlight novel therapeutic options that may be utilized in the future to prevent radiation-induced capsular contracture.

Oncology immunotherapy has gained significant advances in recent years and benefits cancer patients with superior efficacy and superior clinical responses. Currently over ten immune checkpoint antibodies targeting CTLA-4 and PD-1/PD-L1 have received regulatory approval worldwide and over thousands are under active clinical trials. However, compared to the rapid advance of Monoclonal Antibody (mAb), studies on immunotherapeutic small molecules have far lagged behind. Small molecule immunotherapy not only can target immunosuppressive mechanisms similar to mAbs, but also can stimulate intracellular pathways downstream of checkpoint proteins in innate or adaptive immune cells that mAbs are unable to access. Therefore, small molecule immunotherapy can provide an alternative treatment modality either alone or complementary to or synergistic with extracellular checkpoint mAbs to address low clinical response and drug resistance. Fortunately, remarkable progress has achieved recently in the pursuit of small molecule immunotherapy. This review intends to provide a timely highlight on those clinically investigated small molecules targeting PD-1/PD-L1, IDO1, and STING. The most advanced IDO1 inhibitor epacadostat have been aggressively progressed into multiple clinical testings. Small molecule PD-1/PD-L1 inhibitors and STING activators are still in a premature state and their decisive application needs to wait for the ongoing clinical outcomes. Since no small molecule immunotherapy has been approved yet, the future research should continue to focus on discovery of novel small molecules with distinct chemo-types and higher potency, identification of biomarkers to precisely stratify patients, as well as validation of many other immune-therapeutic targets, such as LAG3, KIRs, TIM-3, VISTA, B7-H3, and TIGIT.

The tumor suppressor protein p53 is inactivated in all types of human cancers, either by negative regulation, by mutation or deletion of its gene. Specifically, in tumors that retain wild-type (wt) p53 status, p53 is inactivated by interaction with negative regulators, such as MDM2 and MDMX. These two proteins are found to be overexpressed in several different types of cancers, and the restoration of p53 activity by inhibition of these proteins is now considered an important approach for cancer treatment. The first studies using this strategy to reactivate wt p53 were focused on the development of small molecules that could inhibit MDM2. In this way, p53 could be liberated and act again as a tumor suppressor. From these studies, nine small molecules have reached clinical trials. More recently, MDMX was also identified as an important therapeutic target to efficiently reactivate wt p53, and it is now considered that, for full p53 reactivation, dual inhibition of MDM2 and MDMX is required. In this review we will focus on the most recent advances in the discovery of novel small molecules and stapled peptides that act as selective MDMX inhibitors or as dual MDM2/X inhibitors.