Herein, we show compelling evidence of structural remodeling in mitochondrial membranes of cancerous colon cells when compared to their noncancerous counterparts. The physical characteristics of cancerous and noncancerous mitochondria were systematically investigated using small-angle neutron scattering (SANS), cryogenic electron tomography (cryo-ET), neutron spin-echo (NSE) spectroscopy, and high-throughput lipidomics. Our results exhibit evidence of both mechanical softening of the mitochondrial membrane, as seen by NSE, and ultrastructure rearrangements of the inner mitochondrial membranes, as seen by SANS and cryo-ET, in cancerous cells. These differences in properties seem to be correlated with changes in the lipidome, suggesting the changes are due to the limited number of lipid classes immediately available during the rapid proliferation of cancerous cells. Finally, we highlight a mechanism by which pancratistatin (PST), a natural anticancer therapeutic candidate, may selectively induce apoptosis by altering the mechanical properties and ultrastructure of cancerous mitochondria while sparing noncancerous mitochondria. Discovery of these structural anomalies will lead to improvements in diagnostics, therapeutic development, and the advancement of targeted approaches for traditionally complex cancers which evade our current treatments.
Antibody drug conjugates (ADCs) enable selective delivery of highly potent small molecules, yet most clinically validated payloads target solely cell-division pathways with several drawbacks such as high off-target toxicity and a lack of efficacy on nondividing tumor cells (e.g., tumor stem cells). Metabolic targets such as nicotinamide phosphoribosyltransferase (NAMPT) offer a complementary mode of action and are predestined as ADC payloads since systemic toxicities have prevented clinical use of NAMPT inhibitors (NAMPTi) as free drugs. Here we show that highly hydrophobic NAMPTi, especially cyanoguanidine-containing inhibitors, are chemically and metabolically suboptimal for ADC deployment due to limited efficacy likely due to lysosomal conversion to inactive guanylureas. Guided by structure-based design, and molecular dynamics simulations, we developed two next-generation NAMPT inhibitors featuring (i) a tertiary alcohol to balance hydrophilicity and (ii) an isoindoline-urea group to improve lysosomal stability in comparison to the cyanoguanidine. The optimized inhibitors retained high affinity to the NAMPT enzyme and showed strong cellular activity upon targeted delivery through ADCs. When conjugated to anti-CD30, anti-HER2, or anti-TROP2 antibodies, the resulting ADCs showed durable responses in hematologic and solid tumor models, including complete regressions in the metabolically stringent NCI-N87 gastric carcinoma xenograft after a single 2 mg/kg dose. These findings highlight physicochemical tuning and lysosomal stability as key design principles for NAMPT-based payloads and support NAMPT inhibition as a very promising mode of action (MoA) for next-generation ADC therapeutics.
G-quadruplexes (G4s) are noncanonical, four-stranded nucleic acid structures formed in guanine-rich regions that influence many biological processes, including transcription. We previously developed a yeast-based system to study the transcriptional modulation of p53 family proteins by G4-forming sequences (G4FS) using artificial constructs. Here, motivated by the observation of genome-wide enrichment of G4FS in proximity to p53 and NF-κB response elements (REs), we examined how native combinations of G4FS and REs derived from human promoters influence the activity of p53 or NF-κB family proteins. To this end, five pairs of isogenic reporter strains were developed, and the propensity of G4FS to adopt G4 structures was confirmed by in vitro assays. Results in yeast showed that the presence of G4FS enhanced the transcriptional output of partial-function p53 mutants, p63, and p73 relative to wild-type p53, suggesting a general boost in the activity of weaker transcription factor proteins. Conversely, the effect of G4FS on transactivation by NF-κB proteins was context-dependent and mainly inhibitory. In conclusion, our findings highlight the importance of DNA topology in transcriptional regulation by both p53 and NF-κB proteins and demonstrate that the yeast-based system serves as a valuable tool for isolating the contribution of G4FS within a controlled genomic context.
The variable domains of heavy chain only antibodies, also called nanobodies, have provided powerful new tools for biomedical research. Their rapid development across multiple fields has helped address emerging scientific and clinical challenges, establishing single-domain antibodies (sdAbs) as highly adaptable platforms for a wide range of biotechnological and therapeutic applications. In this study, we identified a nanobody from an alpaca immune library, sdAbCM1, that binds to the maltose-maltodextrin binding protein, MBP, with picomolar affinity. Biophysical investigations showed that sdAbCM1 recognizes a yet unreported epitope and interacts with MBP in its closed conformation when maltose is present. The high affinity, epitope specificity, and ability to tolerate the ligand-bound state render sdAbCM1 a valuable and versatile biotechnological tool with potential applications such as detection, localization, and purification of MBP fusion proteins and protein complex engineering for single-particle microscopy.
Abstract Xenobiotic detoxification is the biotransformation of exogenous compounds entering the human body through enzymatic catalysis in order to assist their eventual elimination. In the past, static active-site models that mainly focus on substrate binding and oxidation chemistry have been employed to understand the xenobiotic functionality of CYP450s. However, modern computational approaches in the field of enzymology have changed this perspective, which show that the P450 machinery utilizes significant structural flexibility and electrostatic control to achieve its function. This review aims to unify these developments into an integrated mechanistic framework that encompasses substrate orientation, conformational gating, catalytic-site organization, transient water-channel assembly, and modulation of the electronic-structure landscape via classical electrostatic and steric confinement. Special focus has been given on how flexible loops/helices, gating residues, transient access channels, along with second-shell residues, dictate xenobiotic accommodation, regio-, stereo-, and chemoselectivity across various members of the CYP450 superfamily.
Understanding how glycoside hydrolases structurally heterogeneous polysaccharides remains a central challenge due to the limited availability of well-defined substrates. Here, we establish a chemically defined, probe-based platform to systematically dissect the hydrolytic specificity of an endo-β-1,3-glucanase using a systematically designed library of branched β-1,3-glucan oligosaccharides with precisely controlled branch position and length. Synthetic access to these glycans enabled their conversion into fluorescent probes for sensitive, site-specific analysis of enzymatic cleavage. Our results revealed that β-1,6-linked branching acts as a key structural determinant governing both cleavage efficiency and positional selectivity in a branched length-dependent manner. While monosaccharide branches are broadly accommodated, disaccharide branches introduce steric constraints that significantly suppress hydrolysis. Notably, analysis using an extended octasaccharide substrate uncovered a dominant cleavage at the central β-1,3-glycosidic bond, allowing refinement and expansion of the enzyme subsite model beyond that accessible with shorter substrates. Collectively, these findings define branch architecture as a critical regulator of substrate orientation and cleavage-site selection, and establish chemically defined glycan libraries as a generalizable platform for mechanistic interrogation of glycoside hydrolases.
Moraxella catarrhalis is an emerging human respiratory pathogen responsible for various infectious diseases. Recent transposon sequencing analysis identified yggW (renamed hemW) as essential for M. catarrhalis growth under iron-limiting conditions, mimicking host-imposed nutritional immunity. HemW is annotated as a putative radical S-adenosylmethionine (SAM) enzyme and belongs to the HemN-like subfamily, but its biochemical properties remain unclear. Here, we report on the first experimental characterization of M. catarrhalis HemW (McHemW). Our bioinformatic analysis confirmed its evolutionary relationship with the putative heme-binding radical SAM enzyme HemW in Escherichia coli. We experimentally demonstrated that McHemW contains a catalytically active [4Fe-4S] cluster and binds heme through biochemical assays and spectroscopy. Using hydrogen-deuterium exchange mass spectrometry, we demonstrated, for the first time, that the heme-binding site resides within the C-terminal auxiliary domain unique to HemW. These findings provide mechanistic insight into the molecular basis of heme recognition in McHemW and establish a foundation for understanding its role in the pathogenicity of M. catarrhalis under iron-limited conditions.
ParB, which is a condensate-forming DNA clamp and a CTP switch, is a key member of the bacterial chromosome segregation apparatus. Molecular features that govern the assembly of ParB for partition assembly condensate formation that leads to DNA segregation are sparsely understood. We report that ParB1 from the multipartite bacteria Vibrio cholerae is a CTPase. The AI-predicted structural model of the ParB1-parS1 nucleoprotein complex consistent with small-angle X-ray scattering revealed how parS1 loading primes ParB1 for sliding, with the formation of a lumen for housing the sliding DNA. The CTPase domains in this dimeric ParB1-parS1 structure are accessible for the formation of higher-order assemblies. Furthermore, we showed that a truncated N-terminal segment of ParB1 containing the CTPase domain undergoes concentration-dependent oligomerization. A flexible linker joining this oligomerization-prone N-terminal segment and the C-terminal dimerization domain appears to intrinsically restrain the self-association of full-length ParB1. Our results suggest that the oligomerization propensity of the N-terminal segment of ParB1 is a key factor for higher-order partition assembly formation.
Drug discovery scientists and chemical biologists continually seek to improve strategies for compound development. Here, we present insights derived from multiple studies of the epidermal growth factor receptor (EGFR), illustrating how this well-established target can inform practical aspects of drug discovery. Case studies spanning fragment-based drug design, bivalent inhibitor discovery, and the optimization of irreversible covalent inhibitors demonstrate how focused investigation of a single system can serve as a framework for methodological innovation and generation of broadly applicable knowledge. We propose that these collective efforts exemplify an emerging paradigm in medicinal chemistry, wherein "model drug targets" are deliberately leveraged to yield generalizable design principles and uncover new strategies for ligand discovery and chemical probe development.
Under iron limiting conditions, bacteria biosynthesize and secrete small molecule iron chelators, siderophores, to scavenge this essential metal. Siderophores are biosynthesized by non-ribosomal peptide synthetases (NRPS) or NRPS independent siderophore (NIS) synthetases, the latter of which are significantly less studied. Streptomyces spp. utilize an iterative NIS synthetase, DesD, to produce desferrioxamine type siderophores through dimerization, trimerization, and (in some cases) macrocyclization of monomers such as N 1 -hydroxy- N 1 -succinyl cadaverine (HSC) and N 1 -hydroxy- N 1 -acetyl cadaverine (HAC). Prior work has utilized an acyl-sulfamoyl adenosine (AMS) inhibitor of monomeric HSC (HSC-AMS) to interrogate the initial adenylation reaction of HSC. However, much is still unknown about how the enzyme active site accommodates substrates of varying sizes during further oligomerization reactions. To answer this question, AMS analogs of the monomer (HSC-AMS), dimer (HSC-HSC-AMS), and trimer (HSC-HSC-HSC-AMS) were chemically synthesized. Biochemical results from in vitro DesD reactions, IC 50 assays, and isothermal titration calorimetry along with structural studies conducted via co-crystallization inform an updated mechanistic model for the iterative DesD catalytic cycle. The acyl adenylate motif in the growing substrate chain drives tight binding in the enzyme active site while the N-terminal HSC units dynamically sample conformations en route to terminating macrocyclization of the HSC-HSC-HSC trimer.
Mutations in FLT3 (FMS-like tyrosine kinase 3) are directly related to the development of acute myeloid leukemia (AML), contributing to the dysregulated proliferation and survival of malignant myeloid cells. FLT3 inhibitors (FLT3i), such as gilteritinib and quizartinib, have demonstrated relevant clinical benefits, including symptom relief and increased overall survival in patients with AML. However, the duration of response to FLT3i remains limited due to the emergence of resistance. In this scenario, the development of targeted therapies has emerged as a promising strategy to overcome these limitations, aiming to address the restrictions observed in the previous generation. These approaches range from the combination of FLT3i with other antileukemic agents to the use of multitarget inhibitors capable of simultaneously modulating FLT3 and other relevant molecular targets, as well as the development of next-generation inhibitors that are more selective and capable of maintaining activity against secondary mutations, such as the gatekeeper mutation (F691L), which confers resistance to clinically available drugs. This review article addresses the therapeutic relevance of FLT3i in AML and provides an overview of the binding modes of representative FLT3 inhibitors. Structural and conformational aspects involved in the interaction with this target, with emphasis on the ATP-binding site, as well as the impact of resistance-associated mutations, are discussed. In addition, this work seeks to correlate the binding modes described in the literature with experimental activity data.
Web spiders assemble soluble silk proteins, so-called spidroins, into fibers with extraordinary toughness. The spidroin N-terminal domains (NTDs) are highly conserved self-assembly modules that dimerize induced by chemical stimuli within the spinning duct. Spidroins from the flagelliform spinning gland form tough and elastic capture spiral silk. Their NTDs exhibit an unusual content of charged amino acid side chains with an unknown function. Here, we engineered a molecular exciton probe into a flagelliform spidroin NTD to detect the mechanism and strength of binding through kinetic fluorescence in combination with chemical denaturation experiments. Site-directed mutagenesis experiments revealed that salt bridges formed by few additional side chain charges tighten the dimer dramatically, reducing the equilibrium dissociation constant by five to six orders of magnitude. The central helix of the dimerization interface resembles a dipolar electrostatic stalk that, through cooperative effects within an antiparallel dimer, generates an equilibrium dissociation constant in the femtomolar range. The additional electrostatic forces acting between flagelliform spidroin NTDs are likely to contribute to the remarkable mechanical properties of capture spiral silk.
Immunomodulators that simultaneously engage multiple pattern recognition receptors (PRRs) represent a promising strategy for shaping immune responses. Here we report the design and synthesis of covalently linked dual PRR agonists composed of a NOD1-selective ligand conjugated to agonists of TLR4, TLR7, or RIG-I. The resulting chimeric molecules were evaluated for receptor-specific agonist activation, in vitro immunomodulatory activity in human peripheral blood mononuclear cells, and in vivo adjuvant properties. In most cases, conjugation attenuated receptor agonist activity and reduced cytokine responses in vitro compared with mixtures of the corresponding unconjugated agonists. Despite modest in vitro activity, the conjugates, particularly the dual NOD1/TLR7 agonist, displayed robust adjuvant effects in a murine vaccination model. These results highlight the distinct immune signatures of conjugated NOD1-based dual agonists and support their development as next-generation vaccine adjuvants.
The global rise of multidrug-resistant (MDR) bacteria poses a major public health crisis, threatening the effectiveness of modern medicine. Traditional antibiotic development struggles to keep pace with bacterial evolution, largely due to the rapid dissemination of antibiotic resistance genes via horizontal gene transfer (HGT). HGT mechanisms both canonical and noncanonical enable bacteria to acquire resistance traits defining species and even special challenges. In this review, we cover the current understanding of HGT in spreading antibiotic resistance and explore possible strategies to control HGT and slow the spread of antimicrobial resistance. Recent advances highlight the potential of synthetic competence inhibitors, advanced oxidation processes (AOPs), CRISPR-Cas technologies, gene drives, and antiplasmids to disrupt horizontal gene flow and mitigate resistance evolution. Despite promising laboratory results, challenges remain in translating these approaches into clinical and environmental applications. Blocking HGT could complement antimicrobial stewardship programs and traditional antibiotic therapies by curbing the emergence of new resistant strains at their genetic roots. By targeting the foundational mechanisms of resistance acquisition, these strategies offer a proactive pathway to extend the efficacy of existing antibiotics and prevent a "postantibiotic" era. Ongoing research into bacterial pathogenesis, genome defense systems, and innovative gene-editing technologies will be critical to developing effective, scalable solutions for managing MDR infections worldwide.
Aggregation of human islet amyloid polypeptide (hIAPP, amylin) into amyloid fibrils is a hallmark of β-cell dysfunction in type 2 diabetes, yet the molecular determinants governing its aggregation pathways remain incompletely understood. Here, we investigate how systematic fluorination of Phe23a key aromatic residue within the amyloidogenic coremodulates intra- and intermolecular interactions and thereby probes hIAPP self-assembly under physiologically relevant acidic and neutral pH conditions. Using a combination of Thioflavin T kinetics, scaling-exponent analysis, pyrene fluorescence, ion mobility-mass spectrometry, circular dichroism, 19F NMR spectroscopy, and molecular dynamics simulations, we show that fluorination of Phe23 reshapes aggregation behavior in a highly nonadditive manner. While wild-type and minimally fluorinated variants, at neutral pH, follow the surface-catalyzed secondary nucleation mechanism discussed well in the literature, higher degrees of fluorination progressively reduce monomer dependence and give rise to pronounced concentration-dependent, self-inhibiting aggregation behavior. Under acidic conditions, protonation of His18 leads to a divergent concentration dependence, with reduced monomer dependence for wild-type and minimally fluorinated peptides and enhanced concentration sensitivity for tetra- and penta-fluorinated variants. These concentration dependencies reflect differences in the conformational accessibility, flexibility, and oligomerization efficiency required for productive fibril formation under each pH condition. Together, these results identify Phe23 as a molecular switch that couples local interactions to global aggregation pathways and demonstrate how subtle chemical and environmental perturbations can modulate the productivity of hIAPP fibril formation.
Alzheimer's disease (AD) involves astrocytic dysfunction characterized by impaired lysosomal activity, defective amyloid clearance, and neuroinflammation, processes strongly regulated by the inflammatory effector CHI3L1. G721-0282, a reported CHI3L1-binding small molecule with demonstrated modulation of downstream signaling pathways including MAPK and STAT3, provides a validated chemical starting point for targeting CHI3L1-driven astrocytic pathology in AD, but it exhibits suboptimal potency and drug-like properties that limit its translational potential. We therefore performed a virtual screening of commercially available analogues of G721-0282 to enable structure-guided optimization, generating a detailed structure-activity map, and prioritizing 24 derivatives. Biophysical analyses identified compound G721-0377 as the most promising candidate, with optimized substitutions resulting in enhanced CHI3L1-binding affinity (K d = 45 μM), representing a ∼3-5-fold improvement over related analogs (K d = 65-236 μM). Compound G721-0377 also exhibited favorable physicochemical and pharmacokinetic properties, including improved solubility, balanced permeability, reduced microsomal clearance, and an enhanced cardiac safety margin. Given their micromolar potency, all functional studies were conducted in vitro. Functionally, G721-0377 uniquely reversed CHI3L1-induced astrocytic dysfunction, restoring amyloid uptake, lysosomal proteolysis and acidification, suppressing CHI3L1 and IL-6 secretion, and inhibiting NF-κB activation to levels comparable to a neutralizing anti-CHI3L1 antibody. Collectively, these findings establish G721-0377 as a promising early stage lead compound with improved affinity, safety, and robust functional efficacy, supporting its further development as a disease-modifying therapeutic for AD.
Microporous antibacterial surfaces offer a promising route for preventing bacterial adhesion and biofilm formation, yet their fabrication often involves complex and energy consuming processes. In this work, we present a sustainable strategy for producing multifunctional antibacterial surfaces by combining a microporous cellulose acetate butyrate (CAB) substrate, a bioinspired polydopamine (PDA) coating, and in situ synthesized silver nanoparticles (AgNPs). CAB, selected for its film-forming capability and ease of microstructuring via spin-coating, provides a tunable platform for topographical control. PDA, formed through the oxidative self-polymerization of dopamine, serves as a conformal adhesive layer and simultaneously acts as a reductive matrix for AgNP formation. Immersion in silver nitrate solution enables the direct deposition of AgNPs without external reducing agents, facilitated by the catechol functionalities of PDA. The resulting CAB-PDA-AgNP surfaces exhibit enhanced antibacterial activity through the combined effects of patterning, surface chemistry, and silver-mediated bactericidal action. The fabrication process is low-energy and solvent-minimized, aligning with principles of sustainable material development and offering a versatile platform for antibacterial coatings.
Different bioconjugation strategies are available for the cysteine (Cys)-specific functionalization of proteins with different payloads, including imaging probes and (pro-)-drugs. Most commonly applied linkers include maleimides (mal); however, because of the sometimes-observed instability of the formed thiosuccinimidyl linkage, its suitability for in vivo applications has been challenged. Consequently, several alternatives have been developed and compared to mal as a benchmark, yet examples of a direct comparison among new methodologies are scarce. We herein report a comparison of the use of mal, phenyloxadiazole methyl sulfone (PODS), and vinylketone (VK) as functional groups for the thiol-specific functionalization of human serum albumin (HSA) via its available free Cys34. Bifunctional chelating agents (BFCA) based on DFO*, identical in all regards but the functional group for bioconjugation, were prepared and conjugated to HSA and the obtained DFO*-HSA conjugates were radiolabeled with Zirconium-89 (89Zr) for positron emission tomography (PET). The efficiency of the conjugation of DFO*-X (X = mal, PODS, VK) to HSA differed significantly, with mal > PODS > VK. Stability studies of the 89Zr-labeled HSA-conjugates indicated good stability for [89Zr]-Zr-DFO*malHSA 11 and [89Zr]-Zr-DFO*-POD-HSA 12 in blood serum but only the latter was found stable in cell culture medium. [89Zr]-Zr-DFO*VK-HSA 13 was excluded from biological experiments due to its surprisingly low stability in all media tested. [89Zr]-Zr-DFO*-POD-HSA 12 was further investigated in CT26-tumor-bearing mice by PET/CT imaging and biodistribution studies. Specific uptake of radioactivity in tumors was high (up to 17% ID/g) and the tumors could be clearly visualized by PET at all time points with excellent tumor-to-background signal (tumor-to-blood ratio 3.2 ± 1.0 after 48 h p.i.). Unexpectedly, the uptake of radioactivity in bones was observed for [89Zr]-Zr-DFO*-POD-HSA 12. Overall, the in vivo performance of [89Zr]-Zr-DFO*-labeled HSA obtained by mal chemistry is the most promising candidate as a companion diagnostic PET imaging probe for the stratification of patients for therapies based on HSA-binding (pro-)-drugs.
The mycobacterial membrane exporter MmpL3 transports trehalose monomycolates (TMM) from the cytosol to the outer membrane of Mycobacterium tuberculosis, making it a potential drug target. Proton influx is believed to drive TMM efflux, suggesting that disrupting proton transfer (PT) could be therapeutic. However, the PT mechanism and its relation to function remain unclear. Recent MmpL3 structures reveal a potential proton channel in its hydrophobic core, which also binds potential antituberculosis compounds. We investigated the PT process using hybrid quantum-mechanical/molecular-mechanical and classical molecular dynamics simulations. We show that transient water chains form in two connected transmembrane cavities that act as proton conduits. Four consecutive PT events are necessary to alter the protonation states of acidic residues in the protein core, triggering conformational changes that affect the TMM binding site. The process begins with the tandem movement of two protons through an upper cavity, protonating two aspartate residues via a classical hydronium migration. After conformational shifts, PT proceeds through a lower cavity, protonating two glutamate residues near the cytosolic opening and inducing further conformational shifts; here, PT occurs sequentially via hydronium and proton-hole migration. The cycle ends with the release of protons into the cytosol. Based on the observed conformational changes, we propose a mechanism for TMM efflux.
Insufficient drug encapsulation, typically at about 10 wt %, remains a major challenge in the design of nanotechnology-based drug delivery systems. In this study, we created cyclodextrin-based nanoparticles (CDNP_PEGs), synthesized by the hyperbranched polymerization of cyclodextrins using a diepoxy poly-(ethylene glycol) linker, which exhibited a high drug encapsulation capacity of up to 54 wt % using the xanthone derivative α-mangostin (MGS) as a model compound. Evaluation in a tumor-bearing mouse model indicated that a single intravenous administration via the tail vein induced a significantly greater antitumor effect compared with free MGS. These results demonstrate the potential of CDNP_PEGs as promising high-capacity carriers for the delivery of anticancer drugs.
