Search Results (168)

Signaling proteins in eukaryotes usually comprise a catalytic domain coupled to one or several interaction domains, such as SH2 and SH3 domains. An additional class of proteins critically involved in cellular communication are adapter or scaffold proteins, which fulfill their purely non-enzymatic functions by organizing protein–protein interactions. Intriguingly, certain signaling enzymes, e.g., kinases and phosphatases, have been demonstrated to promote particular cellular functions by means of their interaction domains only. In this review, we will refer to such a function as "the adapter function of an enzyme". Though many stories can be told, we will concentrate on several proteins executing critical adapter functions in cells of the immune system, such as Bruton´s tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K), and SH2-containing inositol phosphatase 1 (SHIP1), as well as in cancer cells, such as proteins of the rat sarcoma/extracellular signal-regulated kinase (RAS/ERK) mitogen-activated protein kinase (MAPK) pathway. We will also discuss how these adaptor functions of enzymes determine or even undermine the efficacy of targeted therapy compounds, such as ATP-competitive kinase inhibitors. Thereby, we are highlighting the need to develop pharmacological approaches, such as proteolysis-targeting chimeras (PROTACs), that eliminate the entire protein, and thus both enzymatic and adapter functions of the signaling protein. We also review how genetic knock-out and knock-in approaches can be leveraged to identify adaptor functions of signaling proteins. Full article

Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome’s core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome activity. Full article

PROTACs, proteolysis targeting chimeras, are bifunctional molecules inducing protein degradation through a unique proximity-based mode of action. While offering several advantages unachievable by classical drugs, PROTACs have unfavorable physicochemical properties that pose challenges in application and formulation. In this study, we show the solubility enhancement of two PROTACs, ARV-110 and SelDeg51, using Poly(vinyl alcohol). Hereby, we apply a three-fluid nozzle spray drying set-up to generate an amorphous solid dispersion with a 30% w/w drug loading with the respective PROTACs and the hydrophilic polymer. Dissolution enhancement was achieved and demonstrated for t = 0 and t = 4 weeks at 5 °C using a phosphate buffer with a pH of 6.8. A pH shift study on ARV-110-PVA is shown, covering transfer from simulated gastric fluid (SGF) at pH 2.0 to fasted-state simulated intestinal fluid (FaSSIF) at pH 6.5. Additionally, activity studies and binding assays of the pure SelDeg51 versus the spray-dried SelDeg51-PVA indicate no difference between both samples. Our results show how modern enabling formulation technologies can partially alleviate challenging physicochemical properties, such as the poor solubility of increasingly large ‘small’ molecules. Full article

Bruton’s tyrosine kinase (BTK), a non-receptor tyrosine kinase crucial for B cell development and function, acts downstream of the B cell receptor (BCR) in the BCR pathway. Other kinases involved downstream of the BCR besides BTK such as Syk, Lyn, PI3K, and Mitogen-activated protein (MAP) kinases also play roles in relaying signals from the BCR to provide pro-survival, activation, and proliferation cues. BTK signaling is implicated in various B-cell lymphomas such as mantle cell lymphoma, Waldenström Macroglobulinemia, follicular lymphoma, and diffuse large B cell lymphoma, leading to the development of transformative treatments like ibrutinib, the first-in-class covalent BTK inhibitor, and pirtobrutinib, the first-in-class noncovalent BTK inhibitor. However, kinase-deficient mutations C481F, C481Y, C481R, and L528W in the BTK gene confer resistance to both covalent and non-covalent BTK inhibitors, facilitating B cell survival and lymphomagenesis despite kinase inactivation. Further studies have revealed BTK’s non-catalytic scaffolding function, mediating the assembly and activation of proteins including Toll-like receptor 9 (TLR9), vascular cell adhesion protein 1 (VCAM-1), hematopoietic cell kinase (HCK), and integrin-linked kinase (ILK). This non-enzymatic role promotes cell survival and proliferation independently of kinase activity. Understanding BTK’s dual roles unveils opportunities for therapeutics targeting its scaffolding function, promising advancements in disrupting lymphomagenesis and refining B cell lymphoma treatments. Full article

Botulinum neurotoxins are some of the most potent natural toxins known; they cause flaccid paralysis by inhibiting synaptic vesicle release. Some serotypes, notably serotype A and B, can cause persistent paralysis lasting for several months. Because of their potency and persistence, botulinum neurotoxins are now used to manage several clinical conditions, and there is interest in expanding their clinical applications using engineered toxins with novel substrate specificities. It will also be beneficial to engineer toxins with tunable persistence. We have investigated the potential use of small-molecule proteolysis-targeting chimeras (PROTACs) to vary the persistence of modified recombinant botulinum neurotoxins. We also describe a complementary approach that has potential relevance for botulism treatment. This second approach uses a camelid heavy chain antibody directed against botulinum neurotoxin that is modified to bind the PROTAC. These strategies provide proof of principle for the use of two different approaches to fine tune the persistence of botulinum neurotoxins by selectively targeting their catalytic light chains for proteasomal degradation. Full article

This study investigated the intraspecific and interspecific variability in the venom effects of Agkistrodon viperid snake species and subspecies (eleven venoms total) on plasma clotting times, fibrinogen levels, and fibrin clot strength. Significant delays in plasma clotting time were observed for A. conanti, A. contortrix mokasen, A. contortrix phaeogaster, A. howardgloydi, A. piscivorus leucostoma, and A. piscivorus piscivorus. Notably, the phylogenetically disjunct lineages A. conanti, A. contortrix mokasen, and A. howardgloydi exhibited the most potent anticoagulant effects, indicating the independent amplification of a basal trait. Inhibition assays with the activated clotting enzymes Factors XIa, IXa, Xa, and IIa (thrombin) revealed that FXa inhibition is another basal trait amplified independently on multiple occasions within the genus, but with A. howardgloydi, notably more potent than all others. Phospholipid degradation and zymogen destruction were identified as mechanisms underlying the variability in venom effects observed experimentally and in previous clinical reports. Thromboelastography demonstrated that the venoms did not clot fibrinogen directly but affected fibrin clot strength by damaging fibrinogen and that thrombin was subsequently only able to cleave into weak, unstable clots. The ability to activate Protein C, an endogenous anticoagulant enzyme, varied across species, with some venoms exceeding that of A. contortrix contortrix, which previously yielded the protein diagnostic agent Protac^®. Phylogenetic analysis suggested that both fibrinogen degradation and Protein C activation were each amplified multiple times within the genus, albeit with negative correlation between these two modes of action. This study highlights the evolutionary, clinical, and biodiscovery implications of venom variability in the Agkistrodon species, underscoring their dynamic evolution, emphasising the need for tailored clinical approaches, and highlighting the potential for novel diagnostic and therapeutic developments inspired by the unique properties of snake venoms. Full article

Current drug development tends towards complex chemical molecules, referred to as “beyond rule of five” (bRo5) compounds, which often exhibit challenging physicochemical properties. Measuring Caco-2 permeability of those compounds is difficult due to technical limitations, including poor recovery and detection sensitivity. We implemented a novel assay, with optimized incubation and analytics, to measure permeability close to equilibrium. In this setup an appropriate characterization of permeability for bRo5 compounds is achievable. This equilibrated Caco-2 assay was verified with respect to data validity, compound recovery, and in vitro to in vivo correlation for human absorption. Compared to a standard assay, it demonstrated comparable performance in predicting the human fraction absorbed (f_a) for reference compounds. The equilibrated assay also successfully characterized the permeability of more than 90% of the compounds analyzed, the majority of which were bRo5 (68%). These compounds could not be measured using the standard assay. Permeability and efflux ratio (ER) were highly predictive for in vivo absorption for a large set of internal bRo5 compounds. Reference cut-offs enabled the correct classification of high, moderate, and low absorption. This optimized equilibrated Caco-2 assay closes the gap for a high-throughput cellular permeability method in the bRo5 chemical space. Full article

Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinomas (PDACs) harbor activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilized proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. The PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, the inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. The concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (Kras^G12D; Trp53^R172H; Pdx1-Cre) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions, and the IGF-1 growth stimulation effect was AKT-dependent. The RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth, and the pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer. Full article

Cyclophilin A (CypA), the cellular receptor of the immunosuppressant cyclosporin A (CsA), is an abundant cytosolic protein and is involved in a variety of diseases. For example, CypA supports cancer proliferation and mediates viral infections, such as the human immunodeficiency virus 1 (HIV-1). Here, we present the design of PROTAC (proteolysis targeting chimera) compounds against CypA to induce its intracellular proteolysis and to investigate their effect on immune cells. Interestingly, upon connecting to E3 ligase ligands, both peptide-based low-affinity binders and CsA-based high-affinity binders can degrade CypA at nM concentration in HeLa cells and fibroblast cells. As the immunosuppressive effect of CsA is not directly associated with the binding of CsA to CypA but the inhibition of phosphatase calcineurin by the CypA:CsA complex, we investigated whether a CsA-based PROTAC compound could induce CypA degradation without affecting the activation of immune cells. P3, the most efficient PROTAC compound discovered from this study, could deplete CypA in lymphocytes without affecting cell proliferation and cytokine production. This work demonstrates the feasibility of the PROTAC approach in depleting the abundant cellular protein CypA at low drug dosage without affecting immune cells, allowing us to investigate the potential therapeutic effects associated with the endogenous protein in the future. Full article

Dysregulation of the insulin-like growth factor (IGF) system determines the onset of various pathological conditions, including cancer. Accordingly, therapeutic strategies have been developed to block this system in tumor cells, but the results of clinical trials have been disappointing. After decades of research in the field, it is safe to say that one of the major reasons underlying the poor efficacy of anti-IGF-targeting agents is derived from an underestimation of the molecular complexity of this axis. Genetic, transcriptional, post-transcriptional and functional interactors interfere with the activity of canonical components of this axis, supporting the need for combinatorial approaches to effectively block this system. In addition, cancer cells interface with a multiplicity of factors from the extracellular compartment, which strongly affect cell destiny. In this review, we will cover novel extracellular mechanisms contributing to IGF system dysregulation and the implications of such dangerous liaisons for cancer hallmarks and responses to known and new anti-IGF drugs. A deeper understanding of both the intracellular and extracellular microenvironments might provide new impetus to better decipher the complexity of the IGF axis in cancer and provide new clues for designing novel therapeutic approaches. Full article

A new, eco-friendly process utilising the green solvent propylene carbonate (PC) has been developed to perform N-alkylation of N-, O- and/or S-containing heterocyclic compounds. PC in these reactions served as both the reagent and solvent. Importantly, no genotoxic alkyl halides were required. No auxiliary was necessary when using anhydrous PC. Product formation includes nucleophilic substitution with the concomitant loss of water and carbon dioxide. Substrates prepared, including the newly invented PROTAC drugs, are widely used. Full article

Recent advances in the treatment of gastric cancer (GC) with chemotherapy, immunotherapy, anti-angiogenic therapy and targeted therapies have yielded some improvement in survival outcomes; however, metastatic GC remains a lethal malignancy and amongst the leading causes of cancer-related mortality worldwide. Importantly, the ongoing molecular characterisation of GCs continues to uncover potentially actionable molecular targets. Among these, aberrant FGFR2-driven signalling, predominantly arising from FGFR2 amplification, occurs in approximately 3–11% of GCs. However, whilst several inhibitors of FGFR have been clinically tested to-date, there are currently no approved FGFR-directed therapies for GC. In this review, we summarise the significance of FGFR2 as an actionable therapeutic target in GC, examine the recent pre-clinical and clinical data supporting the use of small-molecule inhibitors, antibody-based therapies, as well as novel approaches such as proteolysis-targeting chimeras (PROTACs) for targeting FGFR2 in these tumours, and discuss the ongoing challenges and opportunities associated with their clinical development. Full article

Ovarian cancer is the deadliest gynecologic malignancy. The majority of patients diagnosed with advanced ovarian cancer will relapse, at which point additional therapies can be administered but, for the most part, these are not curative. As such, a need exists for the development of novel therapeutic options for ovarian cancer patients. Research in the field of targeted protein degradation (TPD) through the use of proteolysis-targeting chimeras (PROTACs) has significantly increased in recent years. The ability of PROTACs to target proteins of interest (POI) for degradation, overcoming limitations such as the incomplete inhibition of POI function and the development of resistance seen with other inhibitors, is of particular interest in cancer research, including ovarian cancer research. This review provides a synopsis of PROTACs tested in ovarian cancer models and highlights PROTACs characterized in other types of cancers with potential high utility in ovarian cancer. Finally, we discuss methods that will help to enable the selective delivery of PROTACs to ovarian cancer and improve the pharmacodynamic properties of these agents. Full article

The integration of a multidimensional treatment dominated by active ingredients of traditional Chinese medicine (TCM), including enhanced chemotherapy and synergistically amplification of oxidative damage, into a nanoplatform would be of great significance for furthering accurate and effective cancer treatment with the active ingredients of TCM. Herein, in this study, we designed and synthesized four matrine-proteolysis-targeting chimeras (PROTACs) (depending on different lengths of the chains named LST-1, LST-2, LST-3, and LST-4) based on PROTAC technology to overcome the limitations of matrine. LST-4, with better anti-tumor activity than matrine, still degrades p-Erk and p-Akt proteins. Moreover, LST-4 NPs formed via LST-4 self-assembly with stronger anti-tumor activity and glutathione (GSH) depletion ability could be enriched in lysosomes through their outstanding enhanced permeability and retention (EPR) effect. Then, we synthesized LST-4@ZnPc NPs with a low-pH-triggered drug release property that could release zinc(II) phthalocyanine (ZnPc) in tumor sites. LST-4@ZnPc NPs combine the application of chemotherapy and phototherapy, including both enhanced chemotherapy from LST-4 NPs and the synergistic amplification of oxidative damage, through increasing the reactive oxygen species (ROS) by photodynamic therapy (PDT), causing an GSH decrease via LST-4 mediation to effectively kill tumor cells. Therefore, multifunctional LST-4@ZnPc NPs are a promising method for killing cancer cells, which also provides a new paradigm for using natural products to kill tumors. Full article

Proteolysis-targeting chimeras (PROTACs) are an emerging therapeutic modality that show promise to open a target space not accessible to conventional small molecules via a degradation-based mechanism. PROTAC degraders, due to their bifunctional nature, which is categorized as ‘beyond the Rule of Five’, have gained attention as a distinctive therapeutic approach for oral administration in clinical settings. However, the development of PROTACs with adequate oral bioavailability remains a significant hurdle, largely due to their large size and less than ideal physical and chemical properties. This review encapsulates the latest advancements in orally delivered PROTACs that have entered clinical evaluation as well as developments highlighted in recent scholarly articles. The insights and methodologies elaborated upon in this review could be instrumental in supporting the discovery and refinement of novel PROTAC degraders aimed at the treatment of various human cancers. Full article