Katie J. Simmons

Adhirons are small, antibody-like proteins expressed in bacteria that bind to biological targets with high affinity. Since their discovery, Adhirons have been isolated against over 350 targets, and they can be used for many research applications. However, their therapeutic application is at present in its early stages, hence the interest in developing novel small molecules to mimic their effect. We develop a pipeline for small molecule discovery where Adhirons act as starting templates in a ligand-based virtual screening workflow. For proof of concept, we use Adhirons against two viral systems: Crimean-Congo Haemorrhagic Fever Virus and Influenza A Virus. In both cases, a hit was identified from the mimics using biophysical and cell-based assays, highlighting how Adhirons, commercial libraries and structural approaches can be combined to accelerate small molecule inhibitor discovery of viruses.

Severe drug-resistant childhood epilepsy is caused by KCNT1 gain-of-function genetic variants, resulting in increased K _Na 1.1 channel activity. KCNT1 -associated epilepsy is thought to affect around 1 in 300,000 births worldwide. Current treatment for KCNT1 epilepsy only provides mild symptomatic relief and uses a cocktail of experimental medications which must be personalised for the individual and are often poorly tolerated. Critically, with many patients, no therapeutic benefit is achieved. We sought to address this by using large-scale virtual screening to accelerate the development of a molecule which binds directly to KCNT1 to supress overactivity. We purchased a total of 71 compounds and using a combination of fluorescent thallium flux assays and patch clamp electrophysiology, identified a series of eight structurally diverse, novel inhibitors of the K _Na 1.1 channel with potency in the low micromolar range. These provide potential starting points for further development of drugs to treat KCNT1 -associated epilepsy. Highlights: We have discovered a range of structurally distinct inhibitors of the KCNT1 ion channel using large-scale virtual screening These compound exhibit selectivity for the KCNT1 channel over other related ion channels These compounds could provide starting points for new treatments for KCNT1 related Epilepsy. Graphical

Objective: Hyperactive KCNT1 potassium channels, caused by gain‐of‐function mutations, are associated with a range of epilepsy disorders. Patients typically experience drug‐resistant seizures and, in cases with infantile onset, developmental regression can follow. KCNT1‐related disorders include epilepsy of infancy with migrating focal seizures and sleep‐related hypermotor epilepsy. There are currently no effective treatments for KCNT1 epilepsies, but suppressing overactive channels poses a potential strategy. Methods: Using the KCNT1 channel structure we in silico screened a library of known drugs for those predicted to block the channel pore to inhibit channel activity. Cellular KCNT1 channel inhibition was analyzed using electrophysiology and Drosophila bang‐sensitive assays were used to analyze seizure suppression. Brain penetration of one drug was analyzed using liquid chromatography–mass spectrometry in a mouse. Results: Eight known drugs were investigated in vitro for their effects on patient‐specific mutant KCNT1 channels, with 4 drugs showing significant reduction of K ⁺ current amplitudes. The action of the 4 drugs was then analyzed in vivo and 2 were found to reduce the seizure phenotype in humanized Drosophila KCNT1 epilepsy models. One drug, antrafenine, was shown to cross the blood–brain barrier in mice. Interpretation: This study identified a known drug, antrafenine, that reduces KCNT1 channel activity, reduces seizure activity in Drosophila , and crosses the blood–brain barrier in the mouse, suggesting its potential applicability as a new treatment for KCNT1 epilepsy. The sequential in silico, in vitro, and in vivo mechanism‐based drug selection strategy used here may have broader application for other human disorders where a disease mechanism has been identified. ANN NEUROL 2025;98:1261–1274

Objective: Hyperactive KCNT1 potassium channels, caused by gain-of-function mutations, are associated with a range of epilepsy disorders. Patients typically experience drug-resistant seizures and in cases with infantile onset, developmental regression can follow. KCNT1-related disorders include epilepsy of infancy with migrating focal seizures and sleep related hypermotor epilepsy. There are currently no effective treatments for KCNT1-epilepsies, but suppressing over-active channels poses a potential strategy. Methods: Using KCNT1 channel structural data, we in silico screened a library of known drugs for those predicted to block the channel pore to reduce the current amplitude and inhibit channel activity. Results: Eight known drugs were investigated in vitro for their effects on patient-specific mutant KCNT1 channels, with four drugs showing significant reduction of K ⁺ current amplitudes. The action of the four drugs was then analyzed in vivo and two were found to reduce the seizure phenotype in humanized Drosophila KCNT1-epilepsy models. Interpretation: This study identified two known drugs, antrafenine and nelfinavir mesylate, that reduce KCNT1 channel activity and reduce seizure activity in whole animals, suggesting their potential use as new treatments for KCNT1-epilepsy. The sequential in silico , in vitro and in vivo mechanism-based drug selection strategy used here may have broader application for other human disorders where a disease mechanism has been identified.

Tissue fibrosis is a hallmark of systemic sclerosis (SSc) and results from the excessive production and deposition of collagen and other extracellular matrix proteins by resident fibroblasts. This excessive connective tissue accumulation leads to tissue disruption and subsequent dysfunction in the skin, lungs and other internal organs. Recent studies highlight a role for the matricellular protein Tenascin C in SSc, whereby its stimulation of Toll-like receptor 4 triggers self-sustaining fibroblast activation and ensuing fibrosis. We have utilised Adhiron guided ligand discovery to generate small molecules that target the fibrinogen-like globe domain of Tenascin C, a region involved in Toll-like receptor 4 activation and have demonstrated a reduction in the profibrotic phenotype of human dermal fibroblasts. These studies may facilitate the development of effective targeted therapy for fibrosis in SSc and other fibrotic diseases. Moreover, it highlights the utility of Adhiron guided ligand discovery to generate small molecule inhibitors to selectively modulate proteins. Highlights Adhiron screening was undertaken to identify binders to the FBG domain of Tenascin C and the crystal structure of the FBG-Adhiron binding was resolved. Adhiron guided ligand discovery was used to generate small molecule inhibitors that selectively target the FBG domain of Tenascin C. SPR was used to show binding of these molecules to the FBG domain of Tenascin C The small molecule inhibitors generated attenuated TGFβ-mediated fibrosis in dermal fibroblasts

Adhirons are small, antibody-like proteins expressed in bacteria that bind to biological targets with high affinity. Since their discovery, Adhirons have been isolated against over 350 targets, and they can be used for many research applications. However, their therapeutic application is at present in its early stages, hence the interest in developing novel small molecules to mimic their effect. We developed a pipeline for small molecule discovery where Adhirons act as starting templates in a ligand-based virtual screening workflow. For proof of concept, we used Adhirons against two viral systems: Crimean-Congo Haemorrhagic Fever Virus and Influenza A Virus. In both cases, a hit was identified from the mimics using biophysical and cell-based assays, highlighting how Adhirons, commercial libraries and structural approaches can be combined to accelerate small molecule inhibitor discovery of viruses.

During recent decades, changes in lifestyle have led to widespread nutritional obesity and its related complications. Remodelling adipose tissue as a therapeutic goal for obesity and its complications has attracted much attention and continues to be actively explored. The endothelium lines all blood vessels and is close to all cells, including adipocytes. The endothelium has been suggested to act as a paracrine organ. We explore the role of endothelial insulin-like growth factor-1 receptor (IGF-1R), as a paracrine modulator of white adipose phenotype. We show that a reduction in endothelial IGF-1R expression in the presence of high-fat feeding in male mice leads to depot-specific beneficial white adipose tissue remodelling, increases whole-body energy expenditure and enhances insulin sensitivity via a non-cell-autonomous paracrine mechanism. We demonstrate that increased endothelial malonate may be contributory and that malonate prodrugs have potentially therapeutically relevant properties in the treatment of obesity-related metabolic disease.

Hypofibrinolysis is a documented abnormality in conditions with high risk of vascular occlusion. A key inhibitor of fibrinolysis is α2-antiplasmin (α2AP), and we hypothesize that the Affimer technology, comprising small conformational proteins with 2 9-amino-acid variable regions, can be used to modulate α2AP activity and facilitate fibrinolysis. Using a phage display system, a library of Affimers was screened against α2AP. A total of 28 α2AP-specific Affimers were isolated, of which 1, termed Affimer A11, inhibited protein function and enhanced fibrinolysis. Affimer A11 displayed a monomeric form and consistently reduced the lysis time of clots made from plasma samples of individuals with type 2 diabetes mellitus (n = 15; from 150.8 ± 100.9 to 109.8 ± 104.8 minutes) and those with cardiovascular disease (n = 15; 117.6 ± 40.6 to 79.7 ± 33.3 minutes; P < .01 for both groups). The effects of A11 on fibrinolysis were maintained when clots were made from whole blood samples. Mechanistic studies demonstrated that A11 did not affect clot structure or interfere with the incorporation of α2AP into fibrin networks but significantly enhanced plasmin activity and accelerated plasmin generation. Affimer A11 reduced α2AP binding to plasmin(ogen), whereas molecular modeling demonstrated interactions with α2AP in an area responsible for binding to plasminogen, explaining the effects on both plasmin activity and generation. Affimer A11, at 0.15 to 0.60 mg/mL, had the ability to bind 70% to 90% of plasma α2AP. In conclusion, we demonstrate that Affimers are viable tools for inhibiting α2AP function and facilitating fibrinolysis, making them potential future therapeutic agents to reduce thrombosis risk.

Bacteria are known to be constantly adapting to become resistant to antibiotics. Currently, efficient antibacterial compounds are still available; however, it is only a matter of time until these compounds also become inefficient. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, and many of them are essential for microbial survival. Members of the PNPase and RNase II families of exoribonucleases have been implicated in virulence in many pathogens and, as such, are valid targets for the development of new antibacterials. In this paper, we describe the use of virtual high-throughput screening (vHTS) to identify chemical compounds predicted to bind to the active sites within the known structures of RNase II and PNPase from Escherichia coli. The subsequent in vitro screening identified compounds that inhibited the activity of these exoribonucleases, with some also affecting cell viability, thereby providing proof of principle for utilizing the known structures of these enzymes in the pursuit of new antibacterials.