Small Molecule Approaches Research Articles

Mechanisms of podocyte injury in genetic kidney disease.

Glomerular diseases are a leading cause of chronic kidney disease worldwide. Both acquired and hereditary glomerulopathies frequently share a common final disease mechanism: disruption of the glomerular filtration barrier, podocyte injury, and ultimately podocyte death and detachment. Over 70 monogenic causes of proteinuric kidney disease have been identified, and most of these genes are highly expressed in podocytes, regulating key processes such as maintenance of the slit diaphragm, regulation of actin cytoskeleton remodeling, and modulation of downstream transcriptional pathways. Collectively, these are increasingly being referred to as hereditary "podocytopathies," in which podocyte injury is the central feature driving proteinuria and kidney dysfunction. In this review, we provide an overview of the monogenic podocytopathies and discuss the molecular mechanisms by which single-gene defects lead to podocyte injury and ultimately glomerulosclerosis. We review how advances in genomic technology and a better understanding of the cell biological basis of disease have led to the development of more targeted and personalized therapeutic strategies, including an overview of small molecule and gene therapy approaches.

Polymers for Disrupting Protein-Protein Interactions: Where Are We and Where Should We Be?

Protein-protein interactions (PPIs) are central to the cellular signaling and regulatory networks that underlie many physiological and pathophysiological processes. It is challenging to target PPIs using traditional small molecule or peptide-based approaches due to the frequent lack of well-defined binding pockets at the large and flat PPI interfaces. Synthetic polymers offer an opportunity to circumvent these challenges by providing unparalleled flexibility in tuning their physiochemical properties to achieve the desired binding properties. In this review, we summarize the current state of the field pertaining to polymer-protein interactions in solution, highlighting various polyelectrolyte systems, their tunable parameters, and their characterization. We provide an outlook on how these architectures can be improved by incorporating sequence control, foldability, and machine learning to mimic proteins at every structural level. Advances in these directions will enable the design of more specific protein-binding polymers and provide an effective strategy for targeting dynamic proteins, such as intrinsically disordered proteins.

Aspects of complexity in quality and safety assessment of peptide therapeutics and peptide-related impurities. A regulatory perspective

In recent years, a number of therapeutic peptides have been authorized in the EU market, and several others are in the clinical development phase or under assessment for full dossier or generic applications. Quality and safety guidelines specific to peptides are limited, and some aspects have to be considered. In particular, concerns relate to the analytical investigation for impurities and the toxicological assessment of these substances. The guidelines and the compendial pharmacopoeias provide certain references but that may be questionable if interpreted according to whether therapeutic peptides are considered chemical or biological entities, large or small. The characterization of peptide-related impurities cannot follow the small molecule approach but should consider aspects closely linked to the complex mechanisms of action that these large molecules can exert in the human body. Although direct genotoxic mechanisms cannot be excluded, hazardous interactions on biological systems cannot be ruled out, as in the case of natural peptide toxins and their specific interactions with cellular or membrane targets. From a regulatory perspective, only after specific risk identification and characterization should an equally specific safety threshold in relation to potential toxicity be defined.

Dopaminergic progenitors generated by small molecule approach survived, integrated, and promoted functional recovery in (6-OHDA) mouse model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder resulting from the loss of dopamine-producing neurons in the brain, causing motor symptoms like tremors and stiffness. Although current treatments like medication and deep brain stimulation can alleviate symptoms, they don't address the root cause of neuron loss. Therefore, cell replacement therapy emerges as a promising treatment strategy. However, the generation of engraftable dopaminergic (DA) cells in clinically relevant quantities is still a challenge. Recent advances in cell reprogramming technologies open up vast possibilities to produce patient-specific cells of a desired type in therapeutic quantities. The main cell reprogramming strategies involve the enforced expression of individual or sets of genes through viral transduction or transfection, or through small molecules, known as the chemical approach, which is a much easier and safer method. In our previous studies, using a small molecule approach (combinations of epigenetic modifiers and SMAD inhibitors such asDorsomorphin and SB431542), we have been able to generate DA progenitors from human mesenchymal stem cells (hMSCs). The aim of this study was to further improve the method for the generation of DA progenitors and to test their therapeutic effect in an animal model of Parkinson's. The results showed that the addition of an autophagy enhancer (AE) to our DA cell induction protocol further increased the yield of DA progenitor cells. The results also showed that DA progenitors transplanted into the mouse model of PD survived, integrated, and improved PD motor symptoms. These data suggest that chemically-produced DA cells can be very promising and safe cellular therapeutics for PD.

Adenylate cyclase 6 targets in Mucociliary Clearance

Background. Mucociliary clearance (MCC) is a frontline airway defense mechanism that relies on the appropriate interactions between the epithelium, ciliary beat frequency, and the quantity and quality of mucus. MCC is severely impaired in cystic fibrosis (CF) and drives lung function decline and ultimate respiratory failure and death. A great need remains to identify the key molecular players that regulate MCC in CF outside the scope of FDA approved CFTR therapy and to specifically target MCC dysfunction in CF using small molecules. Results. We discover a novel key role of cAMP-synthesizing enzyme Adenylate cyclase 6 (AC6) in MCC via (a) regulation of CFTR chloride channels in the secretory cells that generate airway surface liquid (ASL) component of MCC and (b) AC6 mediated regulation of ciliary beat frequency in the ciliated cells that is required for the mechanical clearance. Specific activators of AC6 (C20: Top candidate) were developed and improved MCC process ex vivo in CF explant tissues and cells. Together these mechanisms and small molecule approach that improve airway fluid balance and cilia function could be beneficial in CF and other respiratory diseases. Experimental Approach. We performed CFTR functional assessment in normal and CF patient derived airway epithelial cells by adapting patch-sequencing approach and using the specific activators of AC6. The effect of AC6 activators on MCC will be evaluated ex vivo in patient derived lung explant tissues , in vivo (micro-CT) in CF rat models and in vitro (ciliary beat frequency and bead flow measurements) in air-liquid interface airway epithelial cultures. Overall, these studies will be mechanistic and translational involving human disease specimens, animal models, drug development and computational analysis. Significance of the results. The proposed research can improve our understanding of epithelial biology and advance activators of AC6 as therapeutic agents to treat mucus clearance defects. These findings can be leveraged to modulate CFTR function and cilia-generate flow, presenting a potential therapeutic avenue for conditions that involve CFTR and cilia dysregulation associated with several respiratory diseases. National Institutes of Health (NIH) DK080834 and P30-DK117467 and HL147351 (to APN). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PSMA-reactive NB7 single domain antibody fragment: A potential scaffold for developing prostate cancer theranostics

IntroductionSingle domain antibody fragments (sdAbs) are an appealing scaffold for radiopharmaceutical development due to their small size (~15 kDa), high solubility, high stability, and excellent tumor penetration. Previously, we developed NB7 sdAb, which has very high affinity for an epitope on PSMA that is different from those targeted by small molecule PSMA inhibitors. Herein, we evaluated NB7 after radioiodination using [*I]SGMIB (1,3,4-isomer) and iso-[*I]SGMIB (1,3,5-isomer), as well as their 211At-labeled analogues. Methods[*I]SGMIB, iso-[*I]SGMIB, [211At]SAGMB, and iso-[211At]SAGMB conjugates of NB7 sdAb were synthesized and their binding affinity, cell uptake and internalization were assessed in PSMA+ PC3 PIP and PSMA− PC3 flu cells. Biodistribution studies were performed in mice bearing PSMA+ PC3 PIP xenografts. First, a single-label experiment evaluated the tissue distribution of a NB7 bearing a His6-tag (NB7H6) and labeled with iso-[125I]SGMIB. Three paired-label experiments then were performed to compare: a) NB7 labeled using [*I]SGMIB and iso-[*I]SGMIB, b) 131I- vs 211At-labeled NB7 conjugates and c) [125I]SGMIB-NB7H6 to the small molecule PSMA inhibitor [131I]YF2. ResultsAll NB7 radioconjugates bound specifically to PSMA with dissociation constants, Kd, in the low nM range (1.4–6.4 nM). An initial biodistribution study demonstrated good tumor uptake for iso-[125I]SGMIB-NB7H6 (7.2 ± 1.5 % ID/g at 1 h) and no deleterious effect of the His6-tag on renal activity levels, which declined to 3.1 ± 1.1 % ID/g by 4 h. Paired-label biodistribution found no distinction between the two SGMIB isomer NB7 conjugates with the [131I]SGMIB-NB7-to-iso-[125I]SGMIB-NB7 tumor uptake ratios not significantly different from unity: 1.06 ± 0.08 at 1 h, 1.04 ± 0.12 at 4 h, and 1.07 ± 0.09 at 24 h. Both isomer conjugates cleared rapidly from normal tissues and exhibited very low uptake in thyroid, lacrimal and salivary glands. Paired-label biodistribution of [131I]SGMIB-NB7H6 and [211At]SAGMB-NB7H6 demonstrated similar tumor uptake and kidney clearance for the two radioconjugates. However, levels of 211At in thyroid, stomach, salivary and lacrimal glands were significantly higher (P < 0.05) that those for 131I suggesting greater dehalogenation for [211At]SAGMB-NB7H6. Finally, co-administration of [125I]SGMIB-NB7H6 and [131I]YF2 demonstrated good tumor uptake for both with considerably more rapid renal clearance for the NB7 radioconjugate. ConclusionNB7 radioconjugates exhibited good accumulation in PSMA-positive xenografts with rapid clearance from kidney and other normal tissues. We conclude that NB7 is a potentially useful scaffold for developing PSMA-targeted theranostics with different characteristics than current small molecule and antibody-based approaches.

How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a functional assessment.

Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic disorders. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs remain key challenges to study human nociception in vitro. Here, we report a detailed functional characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Anatomic's commercially available RealDRG™ were further characterized for both functional and expression phenotyping of key nociceptor markers. Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Manual patch clamp was used to functionally characterize both control and patient-derived neurons. High throughput techniques were further used to demonstrate that RealDRGs™ derived from the Anatomic protocol are amenable to high throughput technologies for disease modelling. The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. Chambers protocol results in predominantly tonic firing when compared to Anatomic protocol. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. RealDRG™ sensory neurons show heterogeneity of nociceptive markers indicating that the cells may be useful as a humanized model system for translational studies. We validated the efficiency of two differentiation protocols and their potential application for functional assessment and thus understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.

A small-molecule approach to restore female sterility phenotype targeted by a homing suppression gene drive in the fruit pest Drosophila suzukii.

CRISPR-based gene drives offer promising prospects for controlling disease-transmitting vectors and agricultural pests. A significant challenge for successful suppression-type drive is the rapid evolution of resistance alleles. One approach to mitigate the development of resistance involves targeting functionally constrained regions using multiple gRNAs. In this study, we constructed a 3-gRNA homing gene drive system targeting the recessive female fertility gene Tyrosine decarboxylase 2 (Tdc2) in Drosophila suzukii, a notorious fruit pest. Our investigation revealed only a low level of homing in the germline, but feeding octopamine restored the egg-laying defects in Tdc2 mutant females, allowing easier line maintenance than for other suppression drive targets. We tested the effectiveness of a similar system in Drosophila melanogaster and constructed additional split drive systems by introducing promoter-Cas9 transgenes to improve homing efficiency. Our findings show that genetic polymorphisms in wild populations may limit the spread of gene drive alleles, and the position effect profoundly influences Cas9 activity. Furthermore, this study highlights the potential of conditionally rescuing the female infertility caused by the gene drive, offering a valuable tool for the industrial-scale production of gene drive transgenic insects.

The pain target NaV1.7 is expressed late during human iPS cell differentiation into sensory neurons as determined in high-resolution imaging

Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel NaV1.7, which is a validated pain target. NaV1.7 deficiency leads to pain insensitivity, whereas NaV1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag NaV1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface NaV1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust NaV1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes NaV1.7 surface expression. A clinically effective NaV1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.

Locked Nucleic Acid Oligonucleotides Facilitate RNA•LNA-RNA Triple-Helix Formation and Reduce MALAT1 Levels.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-β (MENβ) are two long noncoding RNAs upregulated in multiple cancers, marking these RNAs as therapeutic targets. While traditional small-molecule and antisense-based approaches are effective, we report a locked nucleic acid (LNA)-based approach that targets the MALAT1 and MENβ triple helices, structures comprised of a U-rich internal stem-loop and an A-rich tract. Two LNA oligonucleotides resembling the A-rich tract (i.e., A9GCA4) were examined: an LNA (L15) and a phosphorothioate LNA (PS-L15). L15 binds tighter than PS-L15 to the MALAT1 and MENβ stem loops, although both L15 and PS-L15 enable RNA•LNA-RNA triple-helix formation. Based on UV thermal denaturation assays, both LNAs selectively stabilize the Hoogsteen interface by 5-13 °C more than the Watson-Crick interface. Furthermore, we show that L15 and PS-L15 displace the A-rich tract from the MALAT1 and MENβ stem loop and methyltransferase-like protein 16 (METTL16) from the METTL16-MALAT1 triple-helix complex. Human colorectal carcinoma (HCT116) cells transfected with LNAs have 2-fold less MALAT1 and MENβ. This LNA-based approach represents a potential therapeutic strategy for the dual targeting of MALAT1 and MENβ.

Small molecule approaches to targeting RNA.

The development of innovative methodologies to identify RNA binders has attracted enormous attention in chemical biology and drug discovery. Although antibiotics targeting bacterial ribosomal RNA have been on the market for decades, the renewed interest in RNA targeting reflects the need to better understand complex intracellular processes involving RNA. In this context, small molecules are privileged tools used to explore the biological functions of RNA and to validate RNAs as therapeutic targets, and they eventually are to become new drugs. Despite recent progress, the rational design of specific RNA binders requires a better understanding of the interactions which occur with the RNA target to reach the desired biological response. In this Review, we discuss the challenges to approaching this underexplored chemical space, together with recent strategies to bind, interact and affect biologically relevant RNAs.

Cellular Visualization of G-Quadruplex RNA via Fluorescence- Lifetime Imaging Microscopy.

Over the past decade, appreciation of the roles of G-quadruplex (G4) structures in cellular regulation and maintenance has rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro; however, achieving sufficient selectivity to detect G4s in a dense and structurally diverse cellular environment is challenging. The use of fluorescent probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G4s that does not rely on fluorescence intensity switch-on and, thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independent of the local probe concentration. Furthermore, TOR-G4 primarily colocalizes with RNA in the cytoplasm and nucleoli of cells, making it the first lifetime-based probe validated for exploring the emerging roles of RNA G4s in cellulo.

Functional and pharmacological analyses of visual habituation learning in larval zebrafish

Habituation allows animals to learn to ignore persistent but inconsequential stimuli. Despite being the most basic form of learning, a consensus model on the underlying mechanisms has yet to emerge. To probe relevant mechanisms, we took advantage of a visual habituation paradigm in larval zebrafish, where larvae reduce their reactions to abrupt global dimming (a dark flash). We used Ca2+ imaging during repeated dark flashes and identified 12 functional classes of neurons that differ based on their rate of adaptation, stimulus response shape, and anatomical location. While most classes of neurons depressed their responses to repeated stimuli, we identified populations that did not adapt or that potentiated their response. These neurons were distributed across brain areas, consistent with a distributed learning process. Using a small-molecule screening approach, we confirmed that habituation manifests from multiple distinct molecular mechanisms, and we have implicated molecular pathways in habituation, including melatonin, oestrogen, and GABA signalling. However, by combining anatomical analyses and pharmacological manipulations with Ca2+ imaging, we failed to identify a simple relationship between pharmacology, altered activity patterns, and habituation behaviour. Collectively, our work indicates that habituation occurs via a complex and distributed plasticity processes that cannot be captured by a simple model. Therefore, untangling the mechanisms of habituation will likely require dedicated approaches aimed at sub-component mechanisms underlying this multidimensional learning process.

Functional and pharmacological analyses of visual habituation learning in larval zebrafish.

Habituation allows animals to learn to ignore persistent but inconsequential stimuli. Despite being the most basic form of learning, a consensus model on the underlying mechanisms has yet to emerge. To probe relevant mechanisms, we took advantage of a visual habituation paradigm in larval zebrafish, where larvae reduce their reactions to abrupt global dimming (a dark flash). We used Ca2+ imaging during repeated dark flashes and identified 12 functional classes of neurons that differ based on their rate of adaptation, stimulus response shape, and anatomical location. While most classes of neurons depressed their responses to repeated stimuli, we identified populations that did not adapt or that potentiated their response. These neurons were distributed across brain areas, consistent with a distributed learning process. Using a small-molecule screening approach, we confirmed that habituation manifests from multiple distinct molecular mechanisms, and we have implicated molecular pathways in habituation, including melatonin, oestrogen, and GABA signalling. However, by combining anatomical analyses and pharmacological manipulations with Ca2+ imaging, we failed to identify a simple relationship between pharmacology, altered activity patterns, and habituation behaviour. Collectively, our work indicates that habituation occurs via a complex and distributed plasticity processes that cannot be captured by a simple model. Therefore, untangling the mechanisms of habituation will likely require dedicated approaches aimed at sub-component mechanisms underlying this multidimensional learning process.

A first-in-class pan-lysyl oxidase inhibitor impairs stromal remodeling and enhances gemcitabine response and survival in pancreatic cancer.

The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.

Small-Molecule Drugs in Immunotherapy.

Immunotherapy has been increasingly used in the treatment of cancer. Compared with chemotherapy, immunotherapy relies on the autoimmune system with fewer side effects. Small molecule immune-oncological medicines usually have good bioavailability, higher tissue and tumor permeability, and a reasonable half-life. In this work, we summarize the current advances in the field of small molecule approaches in tumor immunology, including small molecules in clinical trials and preclinical studies, containing PD1/PD-L1 small molecule inhibitors, IDO inhibitor, STING activators, RORγt agonists, TGF-β inhibitors, etc. PD-1/DP-L1 is the most attractive target at present. Some small molecule drugs are being in clinical trial studies. Among them, CA-170 has attracted much attention as an oral small molecule drug. IDO is another popular target after PD-1/PDL1. The dual IDO and PD-1 inhibitor can improve the low response of PD-1 and has a good synergistic effect. STING is a protein that occurs naturally in the human body and can enhance the body's immunity. RORγt is mainly expressed in cells of the immune system. It promotes the differentiation of Th17 cells and produces the key factor IL-17, which plays a key role in the development of autoimmune diseases. TGFβ signaling exhibits potent immunosuppressive activity on the coordinate innate and adaptive immunity, impairing the antitumor potential of innate immune cells in the tumor microenvironment. It is worth mentioning that immunotherapy drugs can often achieve better effects when used in combination, which will help defeat cancer.

Oscillations in PP1 activity are essential for accurate progression through mammalian oocyte meiosis

ABSTRACT Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in PP1 activity driving mitotic M-phase. Evidence from a variety of experimental systems also points to roles in meiosis. Here, we report that PP1 is important for M-phase transitions through mouse oocyte meiosis. We employed a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis. These studies show that temporal control of PP1 activity is essential for the G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at the G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in female fertility, and more broadly, M-phase regulation.

Native American ataxia medicines rescue ataxia-linked mutant potassium channel activity via binding to the voltage sensing domain

There are currently no drugs known to rescue the function of Kv1.1 voltage-gated potassium channels carrying loss-of-function sequence variants underlying the inherited movement disorder, Episodic Ataxia 1 (EA1). The Kwakwaka’wakw First Nations of the Pacific Northwest Coast used Fucus gardneri (bladderwrack kelp), Physocarpus capitatus (Pacific ninebark) and Urtica dioica (common nettle) to treat locomotor ataxia. Here, we show that extracts of these plants enhance wild-type Kv1.1 current, especially at subthreshold potentials. Screening of their constituents revealed that gallic acid and tannic acid similarly augment wild-type Kv1.1 current, with submicromolar potency. Crucially, the extracts and their constituents also enhance activity of Kv1.1 channels containing EA1-linked sequence variants. Molecular dynamics simulations reveal that gallic acid augments Kv1.1 activity via a small-molecule binding site in the extracellular S1-S2 linker. Thus, traditional Native American ataxia treatments utilize a molecular mechanistic foundation that can inform small-molecule approaches to therapeutically correcting EA1 and potentially other Kv1.1-linked channelopathies.

Regenerative engineering of long bones using the small molecule forskolin

Bone grafting procedures have become increasingly common in the United States, with approximately 500,000 cases occurring each year at a societal cost exceeding $2.4 billion. Recombinant human bone morphogenetic proteins (rhBMPs) are therapeutic agents that have been widely used by orthopedic surgeons to stimulate bone tissue formation alone and when paired with biomaterials. However, significant limitations such as immunogenicity, high production cost, and ectopic bone growth from these therapies remain. Therefore, efforts have been made to discover and repurpose osteoinductive small-molecule therapeutics to promote bone regeneration. Previously, we have demonstrated that a single-dose treatment with the small-molecule forskolin for just 24 h induces osteogenic differentiation of rabbit bone marrow-derived stem cells invitro, while mitigating adverse side effects attributed with prolonged small-molecule treatment schemes. In this study, we engineered a composite fibrin-PLGA [poly(lactide-co-glycolide)]-sintered microsphere scaffold for the localized, short-term delivery of the osteoinductive small molecule, forskolin. Invitro characterization studies showed that forskolin released out of the fibrin gel within the first 24 h and retained its bioactivity toward osteogenic differentiation of bone marrow-derived stem cells. The forskolin-loaded fibrin-PLGA scaffold was also able to guide bone formation in a 3-mo rabbit radial critical-sized defect model comparable to recombinant human bone morphogenetic protein-2 (rhBMP-2) treatment, as demonstrated through histological and mechanical evaluation, with minimal systemic off-target side effects. Together, these results demonstrate the successful application of an innovative small-molecule treatment approach within long bone critical-sized defects.

Visualizing the disordered nuclear transport machinery in situ

The approximately 120 MDa mammalian nuclear pore complex (NPC) acts as a gatekeeper for the transport between the nucleus and cytosol1. The central channel of the NPC is filled with hundreds of intrinsically disordered proteins (IDPs) called FG-nucleoporins (FG-NUPs)2,3. Although the structure of the NPC scaffold has been resolved in remarkable detail, the actual transport machinery built up by FG-NUPs—about 50 MDa—is depicted as an approximately 60-nm hole in even highly resolved tomograms and/or structures computed with artificial intelligence4–11. Here we directly probed conformations of the vital FG-NUP98 inside NPCs in live cells and in permeabilized cells with an intact transport machinery by using a synthetic biology-enabled site-specific small-molecule labelling approach paired with highly time-resolved fluorescence microscopy. Single permeabilized cell measurements of the distance distribution of FG-NUP98 segments combined with coarse-grained molecular simulations of the NPC allowed us to map the uncharted molecular environment inside the nanosized transport channel. We determined that the channel provides—in the terminology of the Flory polymer theory12—a ‘good solvent’ environment. This enables the FG domain to adopt expanded conformations and thus control transport between the nucleus and cytoplasm. With more than 30% of the proteome being formed from IDPs, our study opens a window into resolving disorder–function relationships of IDPs in situ, which are important in various processes, such as cellular signalling, phase separation, ageing and viral entry.