Development and validation of PAMPA-BBB QSAR model to predict brain penetration potential of novel drug candidates.

Efficiently circumventing the blood-brain barrier (BBB) poses a major hurdle in the development of drugs that target the central nervous system. Although there are several methods to determine BBB permeability of small molecules, the Parallel Artificial Membrane Permeability Assay (PAMPA) is one of the most common assays in drug discovery due to its robust and high-throughput nature. Drug discovery is a long and costly venture, thus, any advances to streamline this process are beneficial. In this study, ∼2,000 compounds from over 60 NCATS projects were screened in the PAMPA-BBB assay to develop a quantitative structure-activity relationship model to predict BBB permeability of small molecules. After analyzing both state-of-the-art and latest machine learning methods, we found that random forest based on RDKit descriptors as additional features provided the best training balanced accuracy (0.70 ± 0.015) and a message-passing variant of graph convolutional neural network that uses RDKit descriptors provided the highest balanced accuracy (0.72) on a prospective validation set. Finally, we correlated in vitro PAMPA-BBB data with in vivo brain permeation data in rodents to observe a categorical correlation of 77%, suggesting that models developed using data from PAMPA-BBB can forecast in vivo brain permeability. Given that majority of prior research has relied on in vitro or in vivo data for assessing BBB permeability, our model, developed using the largest PAMPA-BBB dataset to date, offers an orthogonal means to estimate BBB permeability of small molecules. We deposited a subset of our data into PubChem bioassay database (AID: 1845228) and deployed the best performing model on the NCATS Open Data ADME portal (https://opendata.ncats.nih.gov/adme/). These initiatives were undertaken with the aim of providing valuable resources for the drug discovery community.

Use of physiological based pharmacokinetic modeling for cross-species prediction of pharmacokinetic and tissue distribution profiles of a novel niclosamide prodrug.

Introduction: Niclosamide (Nc) is an FDA-approved anthelmintic drug that was recently identified in a drug repurposing screening to possess antiviral activity against SARS-CoV-2. However, due to the low solubility and permeability of Nc, its in vivo efficacy was limited by its poor oral absorption. Method: The current study evaluated a novel prodrug of Nc (PDN; NCATS-SM4705) in improving in vivo exposure of Nc and predicted pharmacokinetic profiles of PDN and Nc across different species. ADME properties of the prodrug were determined in humans, hamsters, and mice, while the pharmacokinetics (PK) of PDN were obtained in mice and hamsters. Concentrations of PDN and Nc in plasma and tissue homogenates were measured by UPLC-MS/MS. A physiologically based pharmacokinetic (PBPK) model was developed based on physicochemical properties, pharmacokinetic and tissue distribution data in mice, validated by the PK profiles in hamsters and applied to predict pharmacokinetic profiles in humans. Results: Following intravenous and oral administration of PDN in mice, the total plasma clearance (CLp) and volume of distribution at steady-state (Vdss) were 0.061-0.063 L/h and 0.28-0.31 L, respectively. PDN was converted to Nc in both liver and blood, improving the systemic exposure of Nc in mice and hamsters after oral administration. The PBPK model developed for PDN and in vivo formed Nc could adequately simulate plasma and tissue concentration-time profiles in mice and plasma profiles in hamsters. The predicted human CLp/F and Vdss/F after an oral dose were 2.1 L/h/kg and 15 L/kg for the prodrug respectively. The predicted Nc concentrations in human plasma and lung suggest that a TID dose of 300 mg PDN would provide Nc lung concentrations at 8- to 60-fold higher than in vitro IC50 against SARS-CoV-2 reported in cell assays. Conclusion: In conclusion, the novel prodrug PDN can be efficiently converted to Nc in vivo and improves the systemic exposure of Nc in mice after oral administration. The developed PBPK model adequately depicts the mouse and hamster pharmacokinetic and tissue distribution profiles and highlights its potential application in the prediction of human pharmacokinetic profiles.

Preclinical validation of a novel therapeutic strategy for choroid plexus carcinoma.

Choroid plexus carcinoma (CPC) is a rare infantile brain tumor with an aggressive clinical course that often leaves children with debilitating side effects due to aggressive and toxic chemotherapies. Development of novel therapeutical strategies for this disease have been extremely limited owing to the rarity of the disease and the paucity of biologically relevant substrates. We conducted the first high-throughput screen (HTS) on a human patient-derived CPC cell line (Children Cancer Hospital Egypt, CCHE-45) and identified 427 top hits highlighting key molecular targets in CPC. Furthermore, a combination screen with a wide variety of targets revealed multiple synergistic combinations that may pave the way for novel therapeutical strategies against CPC. Based on in vitro efficiency, central nervous system (CNS) penetrance ability and feasible translational potential, two combinations using a DNA alkylating or topoisomerase inhibitors in combination with an ataxia telangiectasia mutated and rad3 (ATR) inhibitor (topotecan/elimusertib and melphalan/elimusertib respectively) were validated in vitro and in vivo. Pharmacokinetic assays established increased brain penetrance with intra-arterial (IA) delivery over intra-venous (IV) delivery and demonstrated a higher CNS penetrance for the combination melphalan/elimusertib. The mechanisms of synergistic activity for melphalan/elimusertib were assessed through transcriptome analyses and showed dysregulation of key oncogenic pathways (e.g. MYC, mammalian target of rapamycin mTOR, p53) and activation of critical biological processes (e.g. DNA repair, apoptosis, hypoxia, interferon gamma). Importantly, IA administration of melphalan combined with elimusertib led to a significant increase in survival in a CPC genetic mouse model. In conclusion, this study is, to the best of our knowledge, the first that identifies multiple promising combinatorial therapeutics for CPC and emphasizes the potential of IA delivery for the treatment of CPC.

Preclinical Pharmacokinetics and In Vitro Properties of GS-441524, a Potential Oral Drug Candidate for COVID-19 Treatment.

Preclinical pharmacokinetics (PK) and In Vitro ADME properties of GS-441524, a potential oral agent for the treatment of Covid-19, were studied. GS-441524 was stable in vitro in liver microsomes, cytosols, and hepatocytes of mice, rats, monkeys, dogs, and humans. The plasma free fractions of GS-441524 were 62-78% across all studied species. The in vitro transporter study results showed that GS-441524 was a substrate of MDR1, BCRP, CNT3, ENT1, and ENT2; but not a substrate of CNT1, CNT2, and ENT4. GS-441524 had a low to moderate plasma clearance (CLp), ranging from 4.1 mL/min/kg in dogs to 26 mL/min/kg in mice; the steady state volume distribution (Vdss) ranged from 0.9 L/kg in dogs to 2.4 L/kg in mice after IV administration. Urinary excretion appeared to be the major elimination process for GS-441524. Following oral administration, the oral bioavailability was 8.3% in monkeys, 33% in rats, 39% in mice, and 85% in dogs. The PK and ADME properties of GS-441524 support its further development as an oral drug candidate.

Hydroxynorketamine Pharmacokinetics and Antidepressant Behavioral Effects of (2,6)- and (5R)-Methyl-(2R,6R)-hydroxynorketamines.

(R,S)-Ketamine is rapidly metabolized to form a range of metabolites in vivo, including 12 unique hydroxynorketamines (HNKs) that are distinguished by a cyclohexyl ring hydroxylation at the 4, 5, or 6 position. While both (2R,6R)- and (2S,6S)-HNK readily penetrate the brain and exert rapid antidepressant-like actions in preclinical tests following peripheral administration, the pharmacokinetic profiles and pharmacodynamic actions of 10 other HNKs have not been examined. We assessed the pharmacokinetic profiles of all 12 HNKs in the plasma and brains of male and female mice and compared the relative potencies of four (2,6)-HNKs to induce antidepressant-relevant behavioral effects in the forced swim test in male mice. While all HNKs were readily brain-penetrable following intraperitoneal injection, there were robust differences in peak plasma and brain concentrations and exposures. Forced swim test immobility rank order of potency, from most to least potent, was (2R,6S)-, (2S,6R)-, (2R,6R)-, and (2S,6S)-HNK. We hypothesized that distinct structure-activity relationships and the resulting potency of each metabolite are linked to unique substitution patterns and resultant conformation of the six-membered cyclohexanone ring system. To explore this, we synthesized (5R)-methyl-(2R,6R)-HNK, which incorporates a methyl substitution on the cyclohexanone ring. (5R)-Methyl-(2R,6R)-HNK exhibited similar antidepressant-like potency to (2R,6S)-HNK. These results suggest that conformation of the cyclohexanone ring system in the (2,6)-HNKs is an important factor underlying potency and that additional engineering of this structural feature may improve the development of a new generation of HNKs. Such HNKs may represent novel drug candidates for the treatment of depression.

Pentostatin antagonizes the antiviral activity of MBX-2168 by inhibiting the biosynthesis of the active compound.

MBX-2168 has a mechanism of action similar to that of acyclovir (ACV) and ganciclovir (GCV), but two unique steps differentiate this drug from ACV/GCV. First, MBX-2168 is, at least partially, phosphorylated by the endogenous cellular kinase TAOK3 to a monophosphate. The second involves the removal of a moiety at the 6-position of MBX-2168-MP by adenosine deaminase like protein-1 (ADAL-1). It has been previously demonstrated that co-incubation with pentostatin (dCF), an ADAL-1 inhibitor, antagonizes the anti-viral activity of MBX-2168. We therefore hypothesize that inhibiting ADAL-1 results in a reduction of active compound produced in virus-infected cells. To test this, we examined the effect dCF has on the conversion of MBX-2168 to a triphosphate in HSV-1 and HCMV-infected cells. Our results demonstrate incubation of MBX-2168 alone or with dCF in HCMV-infected cells resulted in 53.1 ± 0.7 and 39.4 ± 1.5 pmol triphosphate/106 cells at 120 h, respectively. Incubation of MBX-2168 alone or with dCF in Vero cells resulted in 12.8 ± 0.1 and 6.7 ± 0.7 pmol triphosphate/106 cells at 24 h, respectively. HSV-1-infected Vero cells demonstrated no statistical difference in triphosphate accumulation at 24 h (13.1 ± 0.3 pmol triphosphate/106 cells). As expected, incubation with dCF resulted in the accumulation of MBX-2168-MP in both HFF (9.8 ± 0.9 pmol MBX-2168-MP/106 cells at 120 h) and Vero cells (4.7 ± 0.3 pmol MBX-2168-MP/106 cells at 24 h) while no detectable levels of monophosphate were observed in cultures not incubated with dCF. We conclude that dCF antagonizes the anti-viral effect of MBX-2168 by inhibiting the production of triphosphate, the active compound.

Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro.

The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-Cov and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, and facilitates the attachment of Spike-bearing viral particles to the cell surface to promote viral entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry and reveals drugs capable of targeting this important step in the viral life cycle.

Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro.

The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-CoV and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, facilitates the attachment of viral particles to the cell surface to promote cell entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry, and reveals drugs capable of targeting this important step in the viral life cycle.