Pharmacological expansion of type 2 alveolar epithelial cells promotes regenerative lower airway repair.

Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to lower airway repair. Agents that promote the selective expansion of these cells might stimulate regeneration of the compromised alveolar epithelium, an etiology-defining event in several pulmonary diseases. From a high-content imaging screen of the drug repurposing library ReFRAME, we identified that dipeptidyl peptidase 4 (DPP4) inhibitors, widely used type 2 diabetes medications, selectively expand AEC2s and are broadly efficacious in several mouse models of lung damage. Mechanism of action studies revealed that the protease DPP4, in addition to processing incretin hormones, degrades IGF-1 and IL-6, essential regulators of AEC2 expansion whose levels are increased in the luminal compartment of the lung in response to drug treatment. To selectively target DPP4 in the lung with sufficient drug exposure, we developed NZ-97, a locally delivered, lung persistent DPP4 inhibitor that broadly promotes efficacy in mouse lung damage models with minimal peripheral exposure and good tolerability. This work reveals DPP4 as a central regulator of AEC2 expansion and affords a promising therapeutic approach to broadly stimulate regenerative repair in pulmonary disease.

Bicyclic pyrrolidine inhibitors of Toxoplasma gondii phenylalanine t-RNA synthetase with antiparasitic potency in vitro and brain exposure.

Previous studies have shown that bicyclic azetidines are potent and selective inhibitors of apicomplexan phenylalanine tRNA synthetase (PheRS), leading to parasite growth inhibition in vitro and in vivo, including in models of Toxoplasma infection. Despite these useful properties, additional optimization is required for the development of efficacious treatments of toxoplasmosis from this inhibitor series, in particular to achieve sufficient exposure in the brain. Here, we describe a series of PheRS inhibitors built on a new bicyclic pyrrolidine core scaffold designed to retain the exit-vector geometry of the isomeric bicyclic azetidine core scaffold while offering avenues to sample diverse chemical space. Relative to the parent series, bicyclic pyrrolidines retain reasonable potency and target selectivity for parasite PheRS vs. host. Further structure-activity relationship studies revealed that the introduction of aliphatic groups improved potency, ADME and PK properties, including brain exposure. The identification of this new scaffold provides potential opportunities to extend the analog series to further improve selectivity and potency and ultimately deliver a novel, efficacious treatment of toxoplasmosis.

Drug repurposing screen identifies lonafarnib as respiratory syncytial virus fusion protein inhibitor.

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC50: 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.

Discovery of CMX990: A Potent SARS-CoV-2 3CL Protease Inhibitor Bearing a Novel Warhead.

There remains a need to develop novel SARS-CoV-2 therapeutic options that improve upon existing therapies by an increased robustness of response, fewer safety liabilities, and global-ready accessibility. Functionally critical viral main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target due to its homology within the coronaviral family, and lack thereof toward human proteases. In this disclosure, we outline the advent of a novel SARS-CoV-2 3CLpro inhibitor, CMX990, bearing an unprecedented trifluoromethoxymethyl ketone warhead. Compared with the marketed drug nirmatrelvir (combination with ritonavir = Paxlovid), CMX990 has distinctly differentiated potency (∼5× more potent in primary cells) and human in vitro clearance (>4× better microsomal clearance and >10× better hepatocyte clearance), with good in vitro-to-in vivo correlation. Based on its compelling preclinical profile and projected once or twice a day dosing supporting unboosted oral therapy in humans, CMX990 advanced to a Phase 1 clinical trial as an oral drug candidate for SARS-CoV-2.

Synthesis and structure-activity relationships of aryl fluorosulfate-based inhibitors as novel antitubercular agents.

To identify new compounds that can effectively inhibit Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), we screened, synthesized, and evaluated a series of novel aryl fluorosulfate derivatives for their in vitro inhibitory activity against Mtb. Compound 21b exhibited an in vitro minimum inhibitory concentration (MIC) of 0.06 µM against Mtb, no cytotoxicity against both HEK293T and HepG2 mammalian cell lines, and had good in vivo mouse plasma exposure and lung concentration with a 20 mg/kg oral dose, which supports advanced development as a new chemical entity for TB treatment.

A covalent inhibitor of the YAP-TEAD transcriptional complex identified by high-throughput screening.

Yes-associated protein (YAP), the master transcriptional effector downstream of the Hippo pathway, regulates essential cell growth and regenerative processes in animals. However, the activation of YAP observed in cancers drives cellular proliferation, metastasis, chemoresistance, and immune suppression, making it of key interest in developing precision therapeutics for oncology. As such, pharmacological inhibition of YAP by targeting its essential co-regulators, TEA domain transcription factors (TEADs) would likely promote tumor clearance in sensitive tumor types. From a fluorescence polarization-based high throughput screen of over 800 000 diverse small molecules, here we report the identification of a pyrazolopyrimidine-based scaffold that inhibits association of YAP and TEADs. Medicinal chemistry-based optimization identified mCMY020, a potent, covalent inhibitor of TEAD transcriptional activity that occupies a conserved, central palmitoylation site on TEADs.

Pharmacological YAP activation promotes regenerative repair of cutaneous wounds.

Chronic cutaneous wounds remain a persistent unmet medical need that decreases life expectancy and quality of life. Here, we report that topical application of PY-60, a small-molecule activator of the transcriptional coactivator Yes-associated protein (YAP), promotes regenerative repair of cutaneous wounds in pig and human models. Pharmacological YAP activation enacts a reversible pro-proliferative transcriptional program in keratinocytes and dermal cells that results in accelerated re-epithelization and regranulation of the wound bed. These results demonstrate that transient topical administration of a YAP activating agent may represent a generalizable therapeutic approach to treating cutaneous wounds.

Multistep Synthesis of Analogues of Remdesivir: Incorporating Heterocycles at the C-1' Position.

Studies suggest that the 1'ß-CN moiety in remdesivir sterically clashes with the Ser861 residue of the RNA-dependent-RNA polymerase (RdRp), causing a delayed chain termination in the RNA replication process. Replacing C1'ß-CN with 5-membered heterocycles such as tetrazoles, oxadiazoles, and triazoles can augment the inhibitory activity and pharmacokinetic profile of C-nucleotides. Synthesis of tetrazole-, triazole-, and oxadiazole-integrated C1' analogues of remdesivir was attempted using general synthetic routes. The final compounds 26, 28, and 29 did not inhibit viral replication; however, the synthetic intermediates, i.e., 27 and 50, exhibited an IC90 = 14.1 µM each. The trifluoromethyl-substituted 1,2,4-oxadiazole 59 showed an IC90 of 33.5 µM. This work adds to the growing evidence of the beneficial medicinal impact of C1,1'-disubstituted C-nucleotides.

S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling.

KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.

Structure-activity relationship of BMS906024 derivatives for Cryptosporidium parvum growth inhibition.

BMS906024, a γ-secretase inhibitor that blocks Notch signaling, was previously shown to inhibit Cryptosporidium parvum growth in vitro. A structure-activity relationship (SAR) analysis of BMS906024 reported herein demonstrates the importance of the stereochemistry of the C-3 benzodiazepine and the succinyl ß-substituent. However, concomitant removal of the succinyl α-substituent and switching the primary amide with secondary amides was tolerated. For example, 32 (SH287) inhibited C. parvum growth in HCT-8 host cells with an EC50 = 6.4 nM and an EC90 = 16 nM; however, blocking C. parvum growth with BMS906024 derivatives was correlative with inhibition of Notch signaling, highlighting that additional SAR analysis will be needed to separate these two activities.

Optimization of 3-aminotetrahydrothiophene 1,1-dioxides with improved potency and efficacy as non-electrophilic antioxidant response element (ARE) activators.

Activating NRF2-driven transcription with non-electrophilic small molecules represents an attractive strategy to therapeutically target disease states associated with oxidative stress and inflammation. In this study, we describe a campaign to optimize the potency and efficacy of a previously identified bis-sulfone based non-electrophilic ARE activator 2. This work identifies the efficacious analog 17, a compound with a non-cytotoxic profile in IMR32 cells, as well as ARE activators 18 and 22, analogs with improved cellular potency. In silico drug-likeness prediction suggested the optimized bis-sulfones 17, 18, and 22 will likely be of pharmacological utility.

Targeted protein S-nitrosylation of ACE2 inhibits SARS-CoV-2 infection.

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.

Phenotypic screening of the ReFRAME drug repurposing library to discover new drugs for treating sickle cell disease.

Stem cell transplantation and genetic therapies offer potential cures for patients with sickle cell disease (SCD), but these options require advanced medical facilities and are expensive. Consequently, these treatments will not be available for many years to the majority of patients suffering from this disease. What is urgently needed now is an inexpensive oral drug in addition to hydroxyurea, the only drug approved by the FDA that inhibits sickle-hemoglobin polymerization. Here, we report the results of the first phase of our phenotypic screen of the 12,657 compounds of the Scripps ReFRAME drug repurposing library using a recently developed high-throughput assay to measure sickling times following deoxygenation to 0% oxygen of red cells from sickle trait individuals. The ReFRAME library is a very important collection because the compounds are either FDA-approved drugs or have been tested in clinical trials. From dose-response measurements, 106 of the 12,657 compounds exhibit statistically significant antisickling at concentrations ranging from 31 nM to 10 µM. Compounds that inhibit sickling of trait cells are also effective with SCD cells. As many as 21 of the 106 antisickling compounds emerge as potential drugs. This estimate is based on a comparison of inhibitory concentrations with free concentrations of oral drugs in human serum. Moreover, the expected therapeutic potential for each level of inhibition can be predicted from measurements of sickling times for cells from individuals with sickle syndromes of varying severity. Our results should motivate others to develop one or more of these 106 compounds into drugs for treating SCD.

Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection.

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

HIV protease inhibitors Nelfinavir and Lopinavir/Ritonavir markedly improve lung pathology in SARS-CoV-2-infected Syrian hamsters despite lack of an antiviral effect.

Nelfinavir is an HIV protease inhibitor that has been widely prescribed as a component of highly active antiretroviral therapy, and has been reported to exert in vitro antiviral activity against SARS-CoV-2. We here assessed the effect of Nelfinavir in a SARS-CoV-2 infection model in hamsters. Despite the fact that Nelfinavir, [50 mg/kg twice daily (BID) for four consecutive days], did not reduce viral RNA load and infectious virus titres in the lung of infected animals, treatment resulted in a substantial improvement of SARS-CoV-2-induced lung pathology. This was accompanied by a dense infiltration of neutrophils in the lung interstitium which was similarly observed in non-infected hamsters. Nelfinavir resulted also in a marked increase in activated neutrophils in the blood, as observed in non-infected animals. Although Nelfinavir treatment did not alter the expression of chemoattractant receptors or adhesion molecules on human neutrophils, in vitro migration of human neutrophils to the major human neutrophil attractant CXCL8 was augmented by this protease inhibitor. Nelfinavir appears to induce an immunomodulatory effect associated with increasing neutrophil number and functionality, which may be linked to the marked improvement in SARS-CoV-2 lung pathology independent of its lack of antiviral activity. Since Nelfinavir is no longer used for the treatment of HIV, we studied the effect of two other HIV protease inhibitors, namely the combination Lopinavir/Ritonavir (Kaletra™) in this model. This combination resulted in a similar protective effect as Nelfinavir against SARS-CoV2 induced lung pathology in hamsters.

Bicyclic azetidines target acute and chronic stages of Toxoplasma gondii by inhibiting parasite phenylalanyl t-RNA synthetase.

Toxoplasma gondii commonly infects humans and while most infections are controlled by the immune response, currently approved drugs are not capable of clearing chronic infection in humans. Hence, approximately one third of the world's human population is at risk of reactivation, potentially leading to severe sequelae. To identify new candidates for treating chronic infection, we investigated a series of compounds derived from diversity-oriented synthesis. Bicyclic azetidines are potent low nanomolar inhibitors of phenylalanine tRNA synthetase (PheRS) in T. gondii, with excellent selectivity. Biochemical and genetic studies validate PheRS as the primary target of bicyclic azetidines in T. gondii, providing a structural basis for rational design of improved analogs. Favorable pharmacokinetic properties of a lead compound provide excellent protection from acute infection and partial protection from chronic infection in an immunocompromised mouse model of toxoplasmosis. Collectively, PheRS inhibitors of the bicyclic azetidine series offer promise for treatment of chronic toxoplasmosis.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

The combined treatment of Molnupiravir and Favipiravir results in a potentiation of antiviral efficacy in a SARS-CoV-2 hamster infection model.

BACKGROUND: Favipiravir and Molnupiravir, orally available antivirals, have been reported to exert antiviral activity against SARS-CoV-2. First efficacy data have been recently reported in COVID-19 patients. METHODS: We here report on the combined antiviral effect of both drugs in a SARS-CoV-2 Syrian hamster infection model. The infected hamsters were treated twice daily with the vehicle (the control group) or a suboptimal dose of each compound or a combination of both compounds. FINDINGS: When animals were treated with a combination of suboptimal doses of Molnupiravir and Favipiravir at the time of infection, a marked combined potency at endpoint is observed. Infectious virus titers in the lungs of animals treated with the combination are reduced by ∼5 log10 and infectious virus are no longer detected in the lungs of >60% of treated animals. When start of treatment was delayed with one day a reduction of titers in the lungs of 2.4 log10 was achieved. Moreover, treatment of infected animals nearly completely prevented transmission to co-housed untreated sentinels. Both drugs result in an increased mutation frequency of the remaining viral RNA recovered from the lungs of treated animals. In the combo-treated hamsters, an increased frequency of C-to-T mutations in the viral RNA is observed as compared to the single treatment groups which may explain the pronounced antiviral potency of the combination. INTERPRETATION: Our findings may lay the basis for the design of clinical studies to test the efficacy of the combination of Molnupiravir/Favipiravir in the treatment of COVID-19. FUNDING: stated in the acknowledgment.

Reprogramming of Protein-Targeted Small-Molecule Medicines to RNA by Ribonuclease Recruitment.

Reprogramming known medicines for a novel target with activity and selectivity over the canonical target is challenging. By studying the binding interactions between RNA folds and known small-molecule medicines and mining the resultant dataset across human RNAs, we identified that Dovitinib, a receptor tyrosine kinase (RTK) inhibitor, binds the precursor to microRNA-21 (pre-miR-21). Dovitinib was rationally reprogrammed for pre-miR-21 by using it as an RNA recognition element in a chimeric compound that also recruits RNase L to induce the RNA's catalytic degradation. By enhancing the inherent RNA-targeting activity and decreasing potency against canonical RTK protein targets in cells, the chimera shifted selectivity for pre-miR-21 by 2500-fold, alleviating disease progression in mouse models of triple-negative breast cancer and Alport Syndrome, both caused by miR-21 overexpression. Thus, targeted degradation can dramatically improve selectivity even across different biomolecules, i.e., protein versus RNA.

Chemical Inhibition of ENL/AF9 YEATS Domains in Acute Leukemia.

Transcriptional coregulators, which mediate chromatin-dependent transcriptional signaling, represent tractable targets to modulate tumorigenic gene expression programs with small molecules. Genetic loss-of-function studies have recently implicated the transcriptional coactivator, ENL, as a selective requirement for the survival of acute leukemia and highlighted an essential role for its chromatin reader YEATS domain. Motivated by these discoveries, we executed a screen of nearly 300,000 small molecules and identified an amido-imidazopyridine inhibitor of the ENL YEATS domain (IC50 = 7 µM). Improvements to the initial screening hit were enabled by adopting and expanding upon a SuFEx-based approach to high-throughput medicinal chemistry, ultimately demonstrating that it is compatible with cell-based drug discovery. Through these efforts, we discovered SR-0813, a potent and selective ENL/AF9 YEATS domain inhibitor (IC50 = 25 nM). Armed with this tool and a first-in-class ENL PROTAC, SR-1114, we detailed the biological response of AML cells to pharmacological ENL disruption for the first time. Most notably, we discovered that ENL YEATS inhibition is sufficient to selectively suppress ENL target genes, including HOXA9/10, MYB, MYC, and a number of other leukemia proto-oncogenes. Cumulatively, our study establishes YEATS domain inhibition as a viable approach to disrupt the pathogenic function of ENL in acute leukemia and provides the first thoroughly characterized chemical probe for the ENL YEATS domain.