Sequential Contrastive and Deep Learning Models to Identify Selective Butyrylcholinesterase Inhibitors.

Butyrylcholinesterase (BChE) is a target of interest in late-stage Alzheimer's Disease (AD) where selective BChE inhibitors (BIs) may offer symptomatic treatment without the harsh side effects of acetylcholinesterase (AChE) inhibitors. In this study, we explore multiple machine learning strategies to identify BIs in silico, optimizing for precision over all other metrics. We compare state-of-the-art supervised contrastive learning (CL) with deep learning (DL) and Random Forest (RF) machine learning, across single and sequential modeling configurations, to identify the best models for BChE selectivity. We used these models to virtually screen a vendor library of 5 million compounds for BIs and tested 20 of these compounds in vitro. Seven of the 20 compounds displayed selectivity for BChE over AChE, reflecting a hit rate of 35% for our model predictions, suggesting a highly efficient strategy for modeling selective inhibition.

Identification of New Modulators and Inhibitors of Palmitoyl-Protein Thioesterase 1 for CLN1 Batten Disease and Cancer.

Palmitoyl-protein thioesterase 1 (PPT1) is an understudied enzyme that is gaining attention due to its role in the depalmitoylation of several proteins involved in neurodegenerative diseases and cancer. PPT1 is overexpressed in several cancers, specifically cholangiocarcinoma and esophageal cancers. Inhibitors of PPT1 lead to cell death and have been shown to enhance the killing of tumor cells alongside known chemotherapeutics. PPT1 is hence a viable target for anticancer drug development. Furthermore, mutations in PPT1 cause a lysosomal storage disorder called infantile neuronal ceroid lipofuscinosis (CLN1 disease). Molecules that can inhibit, stabilize, or modulate the activity of this target are needed to address these diseases. We used PPT1 enzymatic assays to identify molecules that were subsequently tested by using differential scanning fluorimetry and microscale thermophoresis. Selected compounds were also tested in neuroblastoma cell lines. The resulting PPT1 screening data was used for building machine learning models to help select additional compounds for testing. We discovered two of the most potent PPT1 inhibitors reported to date, orlistat (IC50 178.8 nM) and palmostatin B (IC50 11.8 nM). When tested in HepG2 cells, it was found that these molecules had decreased activity, indicating that they were likely not penetrating the cells. The combination of in vitro enzymatic and biophysical assays enabled the identification of several molecules that can bind or inhibit PPT1 and may aid in the discovery of modulators or chaperones. The molecules identified could be used as a starting point for further optimization as treatments for other potential therapeutic applications outside CLN1 disease, such as cancer and neurological diseases.

Computational drug repositioning identifies niclosamide and tribromsalan as inhibitors of Mycobacterium tuberculosis and Mycobacterium abscessus.

Tuberculosis (TB) is still a major global health challenge, killing over 1.5 million people each year, and hence, there is a need to identify and develop novel treatments for Mycobacterium tuberculosis (M. tuberculosis). The prevalence of infections caused by nontuberculous mycobacteria (NTM) is also increasing and has overtaken TB cases in the United States and much of the developed world. Mycobacterium abscessus (M. abscessus) is one of the most frequently encountered NTM and is difficult to treat. We describe the use of drug-disease association using a semantic knowledge graph approach combined with machine learning models that has enabled the identification of several molecules for testing anti-mycobacterial activity. We established that niclosamide (M. tuberculosis IC90 2.95 µM; M. abscessus IC90 59.1 µM) and tribromsalan (M. tuberculosis IC90 76.92 µM; M. abscessus IC90 147.4 µM) inhibit M. tuberculosis and M. abscessus in vitro. To investigate the mode of action, we determined the transcriptional response of M. tuberculosis and M. abscessus to both compounds in axenic log phase, demonstrating a broad effect on gene expression that differed from known M. tuberculosis inhibitors. Both compounds elicited transcriptional responses indicative of respiratory pathway stress and the dysregulation of fatty acid metabolism.

Machine learning-aided search for ligands of P2Y6 and other P2Y receptors.

The P2Y6 receptor, activated by uridine diphosphate (UDP), is a target for antagonists in inflammatory, neurodegenerative, and metabolic disorders, yet few potent and selective antagonists are known to date. This prompted us to use machine learning as a novel approach to aid ligand discovery, with pharmacological evaluation at three P2YR subtypes: initially P2Y6 and subsequently P2Y1 and P2Y14. Relying on extensive published data for P2Y6R agonists, we generated and validated an array of classification machine learning model using the algorithms deep learning (DL), adaboost classifier (ada), Bernoulli NB (bnb), k-nearest neighbors (kNN) classifier, logistic regression (lreg), random forest classifier (rf), support vector classification (SVC), and XGBoost (XGB) classifier models, and the common consensus was applied to molecular selection of 21 diverse structures. Compounds were screened using human P2Y6R-induced functional calcium transients in transfected 1321N1 astrocytoma cells and fluorescent binding inhibition at closely related hP2Y14R expressed in CHO cells. The hit compound ABBV-744, an experimental anticancer drug with a 6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine scaffold, had multifaceted interactions with the P2YR family: hP2Y6R inhibition in a non-surmountable fashion, suggesting that noncompetitive antagonism, and hP2Y1R enhancement, but not hP2Y14R binding inhibition. Other machine learning-selected compounds were either weak (experimental anti-asthmatic drug AZD5423 with a phenyl-1H-indazole scaffold) or inactive in inhibiting the hP2Y6R. Experimental drugs TAK-593 and GSK1070916 (100 µM) inhibited P2Y14R fluorescent binding by 50% and 38%, respectively, and all other compounds by < 20%. Thus, machine learning has led the way toward revealing previously unknown modulators of several P2YR subtypes that have varied effects.

1-Sulfonyl-3-amino-1H-1,2,4-triazoles as Yellow Fever Virus Inhibitors: Synthesis and Structure-Activity Relationship.

Yellow fever virus (YFV) transmitted by infected mosquitoes causes an acute viral disease for which there are no approved small-molecule therapeutics. Our recently developed machine learning models for YFV inhibitors led to the selection of a new pyrazolesulfonamide derivative RCB16003 with acceptable in vitro activity. We report that the N-phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine class, which was recently identified as active non-nucleoside reverse transcriptase inhibitors against HIV-1, can also be repositioned as inhibitors of yellow fever virus replication. As compared to other Flaviviridae or Togaviridae family viruses tested, both compounds RCB16003 and RCB16007 demonstrate selectivity for YFV over related viruses, with only RCB16007 showing some inhibition of the West Nile virus (EC50 7.9 µM, CC50 17 µM, SI 2.2). We also describe the absorption, distribution, metabolism, and excretion (ADME) in vitro and pharmacokinetics (PK) for RCB16007 in mice. This compound had previously been shown to not inhibit hERG, and we now describe that it has good metabolic stability in mouse and human liver microsomes, low levels of CYP inhibition, high protein binding, and no indication of efflux in Caco-2 cells. A single-dose oral PK study in mice has a T1/2 of 3.4 h and Cmax of 1190 ng/mL, suggesting good availability and stability. We now propose that the N-phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine class may be prioritized for in vivo efficacy testing against YFV.

Synthesis and Evaluation of 9-Aminoacridines with SARS-CoV-2 Antiviral Activity.

There have been relatively few small molecules developed with direct activity against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two existing antimalarial drugs, pyronaridine and quinacrine, display whole cell activity against SARS-CoV-2 in A549 + ACE2 cells (pretreatment, IC50 = 0.23 and 0.19 µM, respectively) with moderate cytotoxicity (CC50 = 11.53 and 9.24 µM, respectively). Moreover, pyronaridine displays in vitro activity against SARS-CoV-2 PLpro (IC50 = 1.8 µM). Given their existing antiviral activity, these compounds are strong candidates for repurposing against COVID-19 and prompt us to study the structure-activity relationship of the 9-aminoacridine scaffold against SARS-CoV-2 using traditional medicinal chemistry to identify promising new analogs. Our studies identified several novel analogs possessing potent in vitro activity in U2-OS ACE2 GFP 1-10 and 1-11 (IC50 < 1.0 µM) as well as moderate cytotoxicity (CC50 > 4.0 µM). Compounds such as 7g, 9c, and 7e were more active, demonstrating selectivity indices SI > 10, and 9c displayed the strongest activity (IC50 ≤ 0.42 µM, CC50 ≥ 4.41 µM, SI > 10) among them, indicating that it has potential as a new lead molecule in this series against COVID-19.

In silico ADME/tox comes of age: twenty years later.

In the early 2000s pharmaceutical drug discovery was beginning to use computational approaches for absorption, distribution, metabolism, excretion and toxicity (ADME/Tox, also known as ADMET) prediction. This emphasis on prediction was an effort to reduce the risk of later stage failures from ADME/Tox.Much has been written in the intervening twenty plus years and significant expenditure has occurred in companies developing these in silico capabilities which can be gleaned from publications. It is therefore an appropriate time to briefly reflect on what was proposed then and what the reality is today.20 years ago, we tended to optimise bioactivity and perhaps one ADME/Tox property at a time. Previously pharmaceutical companies needed a whole infrastructure for models - in silico and in vitro experts, IT, champions on a project team, educators and management support. Now we are in the age of generative de novo design where bioactivity and many ADME/Tox properties can be optimised and large language model technologies are available.There are also some challenges such as the focus on very large molecules which may be outside of current ADME/Tox models.We provide an opportunity to look forward with the increasing public data for ADME/Tox as well as expanded types of algorithms available.

Learning from COVID-19: How drug hunters can prepare for the next pandemic.

Over 3 years, the SARS-CoV-2 pandemic killed nearly 7 million people and infected more than 767 million globally. During this time, our very small company was able to contribute to antiviral drug discovery efforts through global collaborations with other researchers, which enabled the identification and repurposing of multiple molecules with activity against SARS-CoV-2 including pyronaridine tetraphosphate, tilorone, quinacrine, vandetanib, lumefantrine, cetylpyridinium chloride, raloxifene, carvedilol, olmutinib, dacomitinib, crizotinib, and bosutinib. We highlight some of the key findings from this experience of using different computational and experimental strategies, and detail some of the challenges and strategies for how we might better prepare for the next pandemic so that potential antiviral treatments are available for future outbreaks.

Discovery of PLpro and Mpro Inhibitors for SARS-CoV-2.

There are very few small-molecule antivirals for SARS-CoV-2 that are either currently approved (or emergency authorized) in the US or globally, including remdesivir, molnupiravir, and paxlovid. The increasing number of SARS-CoV-2 variants that have appeared since the outbreak began over three years ago raises the need for continual development of updated vaccines and orally available antivirals in order to fully protect or treat the population. The viral main protease (Mpro) and the papain-like protease (PLpro) are key for viral replication; therefore, they represent valuable targets for antiviral therapy. We herein describe an in vitro screen performed using the 2560 compounds from the Microsource Spectrum library against Mpro and PLpro in an attempt to identify additional small-molecule hits that could be repurposed for SARS-CoV-2. We subsequently identified 2 hits for Mpro and 8 hits for PLpro. One of these hits was the quaternary ammonium compound cetylpyridinium chloride with dual activity (IC50 = 2.72 ± 0.09 µM for PLpro and IC50 = 7.25 ± 0.15 µM for Mpro). A second inhibitor of PLpro was the selective estrogen receptor modulator raloxifene (IC50 = 3.28 ± 0.29 µM for PLpro and IC50 = 42.8 ± 6.7 µM for Mpro). We additionally tested several kinase inhibitors and identified olmutinib (IC50 = 0.54 ± 0.04 µM), bosutinib (IC50 = 4.23 ± 0.28 µM), crizotinib (IC50 = 3.81 ± 0.04 µM), and dacominitinib (IC50 = IC50 3.33 ± 0.06 µM) as PLpro inhibitors for the first time. In some cases, these molecules have also been tested by others for antiviral activity for this virus, or we have used Calu-3 cells infected with SARS-CoV-2. The results suggest that approved drugs can be identified with promising activity against these proteases, and in several cases we or others have validated their antiviral activity. The additional identification of known kinase inhibitors as molecules targeting PLpro may provide new repurposing opportunities or starting points for chemical optimization.

Transporter Inhibition Profile for the Antivirals Tilorone, Quinacrine and Pyronaridine.

Pyronaridine, tilorone and quinacrine are cationic molecules that have in vitro activity against Ebola, SARS-CoV-2 and other viruses. All three molecules have also demonstrated in vivo activity against Ebola in mice, while pyronaridine showed in vivo efficacy against SARS-CoV-2 in mice. We have recently tested these molecules and other antivirals against human organic cation transporters (OCTs) and apical multidrug and toxin extruders (MATEs). Quinacrine was found to be an inhibitor of OCT2, while tilorone and pyronaridine were less potent, and these displayed variability depending on the substrate used. To assess whether any of these three molecules have other potential interactions with additional transporters, we have now screened them at 10 µM against various human efflux and uptake transporters including P-gp, OATP1B3, OAT1, OAT3, MRP1, MRP2, MRP3, BCRP, as well as confirmational testing against OCT1, OCT2, MATE1 and MATE2K. Interestingly, in this study tilorone appears to be a more potent inhibitor of OCT1 and OCT2 than pyronaridine or quinacrine. However, both pyronaridine and quinacrine appear to be more potent inhibitors of MATE1 and MATE2K. None of the three compounds inhibited MRP1, MRP2, MRP3, OAT1, OAT3, P-gp or OATP1B3. Similarly, we previously showed that tilorone and pyronaridine do not inhibit OATP1B1 and have confirmed that quinacrine behaves similarly. In total, these observations suggest that the three compounds only appear to interact with OCTs and MATEs to differing extents, suggesting they may be involved in fewer clinically relevant drug-transporter interactions involving pharmaceutical substrates of the other major transporters tested.

The protein disulfide isomerase inhibitor 3-methyltoxoflavin inhibits Chikungunya virus.

Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever, a (re)emerging arbovirus infection, that causes severe and often persistent arthritis, as well as representing a serious health concern worldwide for which no antivirals are currently available. Despite efforts over the last decade to identify and optimize new inhibitors or to reposition existing drugs, no compound has progressed to clinical trials for CHIKV and current prophylaxis is based on vector control, which has shown limited success in containing the virus. Our efforts to rectify this situation were initiated by screening 36 compounds using a replicon system and ultimately identified the natural product derivative 3-methyltoxoflavin with activity against CHIKV using a cell-based assay (EC50 200 nM, SI = 17 in Huh-7 cells). We have additionally screened 3-methyltoxoflavin against a panel of 17 viruses and showed that it only additionally demonstrated inhibition of the yellow fever virus (EC50 370 nM, SI = 3.2 in Huh-7 cells). We have also showed that 3-methyltoxoflavin has excellent in vitro human and mouse microsomal metabolic stability, good solubility and high Caco-2 permeability and it is not likely to be a P-glycoprotein substrate. In summary, we demonstrate that 3-methyltoxoflavin has activity against CHIKV, good in vitro absorption, distribution, metabolism and excretion (ADME) properties as well as good calculated physicochemical properties and may represent a valuable starting point for future optimization to develop inhibitors for this and other related viruses.

Validation of Acetylcholinesterase Inhibition Machine Learning Models for Multiple Species.

Acetylcholinesterase (AChE) is an important enzyme and target for human therapeutics, environmental safety, and global food supply. Inhibitors of this enzyme are also used for pest elimination and can be misused for suicide or chemical warfare. Adverse effects of AChE pesticides on nontarget organisms, such as fish, amphibians, and humans, have also occurred as a result of biomagnifications of these toxic compounds. We have exhaustively curated the public data for AChE inhibition data and developed machine learning classification models for seven different species. Each set of models were built using up to nine different algorithms for each species and Morgan fingerprints (ECFP6) with an activity cutoff of 1 µM. The human (4075 compounds) and eel (5459 compounds) consensus models predicted AChE inhibition activity using external test sets from literature data with 81% and 82% accuracy, respectively, while the reciprocal cross (76% and 82% percent accuracy) was not species-specific. In addition, we also created machine learning regression models for human and eel AChE inhibition to return a predicted IC50 value for a queried molecule. We did observe an improved species specificity in the regression models, where a human support vector regression model of human AChE inhibition (3652 compounds) predicted the IC50s of the human test set to a better extent than the eel regression model (4930 compounds) on the same test set, based on mean absolute percentage error (MAPE = 9.73% vs 13.4%). The predictive power of these models certainly benefits from increasing the chemical diversity of the training set, as evidenced by expanding our human classification model by incorporating data from the Tox21 library of compounds. Of the 10 compounds we tested that were predicted active by this expanded model, two showed >80% inhibition at 100 µM. This machine learning approach therefore offers the ability to rapidly score massive libraries of molecules against the models for AChE inhibition that can then be selected for future in vitro testing to identify potential toxins. It also enabled us to create a public website, MegaAChE, for single-molecule predictions of AChE inhibition using these models at megaache.collaborationspharma.com.

Structure and neutralization mechanism of a human antibody targeting a complex Epitope on Zika virus.

We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.

Multiple approaches to repurposing drugs for neuroblastoma.

Neuroblastoma (NB) is the second leading extracranial solid tumor of early childhood with about two-thirds of cases presenting before the age of 5, and accounts for roughly 15 percent of all pediatric cancer fatalities in the United States. Treatments against NB are lacking, resulting in a low survival rate in high-risk patients. A repurposing approach using already approved or clinical stage compounds can be used for diseases for which the patient population is small, and the commercial market limited. We have used Bayesian machine learning, in vitro cell assays, and combination analysis to identify molecules with potential use for NB. We demonstrated that pyronaridine (SH-SY5Y IC50 1.70 µM, SK-N-AS IC50 3.45 µM), BAY 11-7082 (SH-SY5Y IC50 0.85 µM, SK-N-AS IC50 1.23 µM), niclosamide (SH-SY5Y IC50 0.87 µM, SK-N-AS IC50 2.33 µM) and fingolimod (SH-SY5Y IC50 4.71 µM, SK-N-AS IC50 6.11 µM) showed cytotoxicity against NB. As several of the molecules are approved drugs in the US or elsewhere, they may be repurposed more readily for NB treatment. Pyronaridine was also tested in combinations in SH-SY5Y cells and demonstrated an antagonistic effect with either etoposide or crizotinib. Whereas when crizotinib and etoposide were combined with each other they had a synergistic effect in these cells. We have also described several analogs of pyronaridine to explore the structure-activity relationship against cell lines. We describe multiple molecules demonstrating cytotoxicity against NB and the further evaluation of these molecules and combinations using other NB cells lines and in vivo models will be important in the future to assess translational potential.

Discovery of New Zika Protease and Polymerase Inhibitors through the Open Science Collaboration Project OpenZika.

The Zika virus (ZIKV) is a neurotropic arbovirus considered a global threat to public health. Although there have been several efforts in drug discovery projects for ZIKV in recent years, there are still no antiviral drugs approved to date. Here, we describe the results of a global collaborative crowdsourced open science project, the OpenZika project, from IBM's World Community Grid (WCG), which integrates different computational and experimental strategies for advancing a drug candidate for ZIKV. Initially, molecular docking protocols were developed to identify potential inhibitors of ZIKV NS5 RNA-dependent RNA polymerase (NS5 RdRp), NS3 protease (NS2B-NS3pro), and NS3 helicase (NS3hel). Then, a machine learning (ML) model was built to distinguish active vs inactive compounds for the cytoprotective effect against ZIKV infection. We performed three independent target-based virtual screening campaigns (NS5 RdRp, NS2B-NS3pro, and NS3hel), followed by predictions by the ML model and other filters, and prioritized a total of 61 compounds for further testing in enzymatic and phenotypic assays. This yielded five non-nucleoside compounds which showed inhibitory activity against ZIKV NS5 RdRp in enzymatic assays (IC50 range from 0.61 to 17 µM). Two compounds thermally destabilized NS3hel and showed binding affinity in the micromolar range (Kd range from 9 to 35 µM). Moreover, the compounds LabMol-301 inhibited both NS5 RdRp and NS2B-NS3pro (IC50 of 0.8 and 7.4 µM, respectively) and LabMol-212 thermally destabilized the ZIKV NS3hel (Kd of 35 µM). Both also protected cells from death induced by ZIKV infection in in vitro cell-based assays. However, while eight compounds (including LabMol-301 and LabMol-212) showed a cytoprotective effect and prevented ZIKV-induced cell death, agreeing with our ML model for prediction of this cytoprotective effect, no compound showed a direct antiviral effect against ZIKV. Thus, the new scaffolds discovered here are promising hits for future structural optimization and for advancing the discovery of further drug candidates for ZIKV. Furthermore, this work has demonstrated the importance of the integration of computational and experimental approaches, as well as the potential of large-scale collaborative networks to advance drug discovery projects for neglected diseases and emerging viruses, despite the lack of available direct antiviral activity and cytoprotective effect data, that reflects on the assertiveness of the computational predictions. The importance of these efforts rests with the need to be prepared for future viral epidemic and pandemic outbreaks.

Vandetanib Blocks the Cytokine Storm in SARS-CoV-2-Infected Mice.

The portfolio of SARS-CoV-2 small molecule drugs is currently limited to a handful that are either approved (remdesivir), emergency approved (dexamethasone, baricitinib, paxlovid, and molnupiravir), or in advanced clinical trials. Vandetanib is a kinase inhibitor which targets the vascular endothelial growth factor receptor (VEGFR), the epidermal growth factor receptor (EGFR), as well as the RET-tyrosine kinase. In the current study, it was tested in different cell lines and showed promising results on inhibition versus the toxic effect on A549-hACE2 cells (IC50 0.79 µM) while also showing a reduction of >3 log TCID50/mL for HCoV-229E. The in vivo efficacy of vandetanib was assessed in a mouse model of SARS-CoV-2 infection and statistically significantly reduced the levels of IL-6, IL-10, and TNF-α and mitigated inflammatory cell infiltrates in the lungs of infected animals but did not reduce viral load. Vandetanib also decreased CCL2, CCL3, and CCL4 compared to the infected animals. Vandetanib additionally rescued the decreased IFN-1ß caused by SARS-CoV-2 infection in mice to levels similar to that in uninfected animals. Our results indicate that the FDA-approved anticancer drug vandetanib is worthy of further assessment as a potential therapeutic candidate to block the COVID-19 cytokine storm.

Cross-species efficacy of enzyme replacement therapy for CLN1 disease in mice and sheep.

CLN1 disease, also called infantile neuronal ceroid lipofuscinosis (NCL) or infantile Batten disease, is a fatal neurodegenerative lysosomal storage disorder resulting from mutations in the CLN1 gene encoding the soluble lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1). Therapies for CLN1 disease have proven challenging because of the aggressive disease course and the need to treat widespread areas of the brain and spinal cord. Indeed, gene therapy has proven less effective for CLN1 disease than for other similar lysosomal enzyme deficiencies. We therefore tested the efficacy of enzyme replacement therapy (ERT) by administering monthly infusions of recombinant human PPT1 (rhPPT1) to PPT1-deficient mice (Cln1-/-) and CLN1R151X sheep to assess how to potentially scale up for translation. In Cln1-/- mice, intracerebrovascular (i.c.v.) rhPPT1 delivery was the most effective route of administration, resulting in therapeutically relevant CNS levels of PPT1 activity. rhPPT1-treated mice had improved motor function, reduced disease-associated pathology, and diminished neuronal loss. In CLN1R151X sheep, i.c.v. infusions resulted in widespread rhPPT1 distribution and positive treatment effects measured by quantitative structural MRI and neuropathology. This study demonstrates the feasibility and therapeutic efficacy of i.c.v. rhPPT1 ERT. These findings represent a key step toward clinical testing of ERT in children with CLN1 disease and highlight the importance of a cross-species approach to developing a successful treatment strategy.

Computational and Experimental Approaches Identify Beta-Blockers as Potential SARS-CoV-2 Spike Inhibitors.

Finding antivirals for SARS-CoV-2 is still a major challenge, and many computational and experimental approaches have been employed to find a solution to this problem. While the global vaccination campaigns are the primary driver of controlling the current pandemic, orally bioavailable small-molecule drugs and biologics are critical to overcome this global issue. Improved therapeutics and prophylactics are required to treat people with circulating and emerging new variants, addressing severe infection, and people with underlying or immunocompromised conditions. The SARS-CoV-2 envelope spike is a challenging target for viral entry inhibitors. Pindolol presented a good docking score in a previous virtual screening using computational docking calculations after screening a Food and Drug Administration (FDA)-approved drug library of 2400 molecules as potential candidates to block the SARS-CoV-2 spike protein interaction with the angiotensin-converting enzyme 2 (ACE-2). Here, we expanded the computational evaluation to identify five beta-blockers against SARS-CoV-2 using several techniques, such as microscale thermophoresis, NanoDSF, and in vitro assays in different cell lines. These data identified carvedilol with a K d of 364 ± 22 nM for the SARS-CoV-2 spike and in vitro activity (EC50 of 7.57 µM, CC50 of 18.07 µM) against SARS-CoV-2 in Calu-3 cells. We have shown how we can apply multiple computational and experimental approaches to find molecules that can be further optimized to improve anti-SARS-CoV-2 activity.

Machine Learning for Discovery of New ADORA Modulators.

Adenosine (ADO) is an extracellular signaling molecule generated locally under conditions that produce ischemia, hypoxia, or inflammation. It is involved in modulating a range of physiological functions throughout the brain and periphery through the membrane-bound G protein-coupled receptors, called adenosine receptors (ARs) A1AR, A2AAR, A2BAR, and A3AR. These are therefore important targets for neurological, cardiovascular, inflammatory, and autoimmune diseases and are the subject of drug development directed toward the cyclic adenosine monophosphate and other signaling pathways. Initially using public data for A1AR agonists we generated and validated a Bayesian machine learning model (Receiver Operator Characteristic of 0.87) that we used to identify molecules for testing. Three selected molecules, crisaborole, febuxostat and paroxetine, showed initial activity in vitro using the HEK293 A1AR Nomad cell line. However, radioligand binding, ß-arrestin assay and calcium influx assay did not confirm this A1AR activity. Nevertheless, several other AR activities were identified. Febuxostat and paroxetine both inhibited orthosteric radioligand binding in the µM range for A2AAR and A3AR. In HEK293 cells expressing the human A2AAR, stimulation of cAMP was observed for crisaborole (EC50 2.8 µM) and paroxetine (EC50 14 µM), but not for febuxostat. Crisaborole also increased cAMP accumulation in A2BAR-expressing HEK293 cells, but it was weaker than at the A2AAR. At the human A3AR, paroxetine did not show any agonist activity at 100 µM, although it displayed binding with a Ki value of 14.5 µM, suggesting antagonist activity. We have now identified novel modulators of A2AAR, A2BAR and A3AR subtypes that are clinically used for other therapeutic indications, and which are structurally distinct from previously reported tool compounds or drugs.

Advancing the Research and Development of Enzyme Replacement Therapies for Lysosomal Storage Diseases.

With the increasing interest in developing gene therapies for rare diseases, it is easy to overlook that there are numerous rare lysosomal storage diseases (LSD) with treatments that have been approved by regulatory agencies in the United States and Europe. These primarily consist of enzyme replacement therapies (ERT), which are recombinant human proteins that are delivered for the life of the patient via different routes and may have distinct safety and distribution advantages over gene therapies. The research and development of ERT is a lengthy and expensive process, which is usually performed in academic laboratories before transfer to pharmaceutical companies and is hence a process ripe for disruption. There may still be considerable scientific and investment potential for ERT, however we need to develop a pipeline of proteins analogous to what has been created in some open science efforts as well as apply technologies to decrease manufacturing costs. In this Perspective, we illustrate the opportunity to fill the rare LSD treatment gap with ERTs while gene therapies are in development for these life-shortening diseases.