A Second-Generation Oral SARS-CoV-2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19.

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.

Design of Next-Generation DGAT2 Inhibitor PF-07202954 with Longer Predicted Half-Life.

Diacylglycerol O-acyltransferase 2 (DGAT2) inhibitors have been shown to lower liver triglyceride content and are being explored clinically as a treatment for non-alcoholic steatohepatitis (NASH). This work details efforts to find an extended-half-life DGAT2 inhibitor. A basic moiety was added to a known inhibitor template, and the basicity and lipophilicity were fine-tuned by the addition of electrophilic fluorines. A weakly basic profile was required to find an appropriate balance of potency, clearance, and permeability. This work culminated in the discovery of PF-07202954 (12), a weakly basic DGAT2 inhibitor that has advanced to clinical studies. This molecule displays a higher volume of distribution and longer half-life in preclinical species, in keeping with its physicochemical profile, and lowers liver triglyceride content in a Western-diet-fed rat model.

Discovery of the Potent and Selective MC4R Antagonist PF-07258669 for the Potential Treatment of Appetite Loss.

The melanocortin-4 receptor (MC4R) is a centrally expressed, class A GPCR that plays a key role in the regulation of appetite and food intake. Deficiencies in MC4R signaling result in hyperphagia and increased body mass in humans. Antagonism of MC4R signaling has the potential to mitigate decreased appetite and body weight loss in the setting of anorexia or cachexia due to underlying disease. Herein, we report on the identification of a series of orally bioavailable, small-molecule MC4R antagonists using a focused hit identification effort and the optimization of these antagonists to provide clinical candidate 23. Introduction of a spirocyclic conformational constraint allowed for simultaneous optimization of MC4R potency and ADME attributes while avoiding the production of hERG active metabolites observed in early series leads. Compound 23 is a potent and selective MC4R antagonist with robust efficacy in an aged rat model of cachexia and has progressed into clinical trials.

Imaging Leucine-Rich Repeat Kinase 2 In Vivo with 18F-Labeled Positron Emission Tomography Ligand.

Leucine-rich repeat kinase 2 (LRRK2) has been demonstrated to be closely involved in the pathogenesis of Parkinson's disease (PD), and pharmacological blockade of LRRK2 represents a new opportunity for therapeutical treatment of PD and other related neurodegenerative conditions. The development of an LRRK2-specific positron emission tomography (PET) ligand would enable a target occupancy study in vivo and greatly facilitate LRRK2 drug discovery and clinical translation as well as provide a molecular imaging tool for studying physiopathological changes in neurodegenerative diseases. In this work, we present the design and development of compound 8 (PF-06455943) as a promising PET radioligand through a PET-specific structure-activity relationship optimization, followed by comprehensive pharmacology and ADME/neuroPK characterization. Following an efficient 18F-labeling method, we have confirmed high brain penetration of [18F]8 in nonhuman primates (NHPs) and validated its specific binding in vitro by autoradiography in postmortem NHP brain tissues and in vivo by PET imaging studies.

Anti-PD-L1 and anti-CD73 combination therapy promotes T cell response to EGFR-mutated NSCLC.

Treatment with anti-PD-1 and anti-PD-L1 therapies has shown durable clinical benefit in non-small cell lung cancer (NSCLC). However, patients with NSCLC with epidermal growth factor receptor (EGFR) mutations do not respond as well to treatment as patients without an EGFR mutation. We show that EGFR-mutated NSCLC expressed higher levels of CD73 compared with EGFR WT tumors and that CD73 expression was regulated by EGFR signaling. EGFR-mutated cell lines were significantly more resistant to T cell killing compared with WT cell lines through suppression of T cell proliferation and function. In a xenograft mouse model of EGFR-mutated NSCLC, neither anti-PD-L1 nor anti-CD73 antibody alone inhibited tumor growth compared with the isotype control. In contrast, the combination of both antibodies significantly inhibited tumor growth, increased the number of tumor-infiltrating CD8+ T cells, and enhanced IFN-γ and TNF-α production of these T cells. Consistently, there were increases in gene expression that corresponded to inflammation and T cell function in tumors treated with the combination of anti-PD-L1 and anti-CD73. Together, these results further support the combination of anti-CD73 and anti-PD-L1 therapies in treating EGFR-mutated NSCLC, while suggesting that increased T cell activity may play a role in response to therapy.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Discovery and Characterization of (R)-6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates.

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance.

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties.

By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.

Discovery and preclinical profiling of 3-[4-(morpholin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]benzonitrile (PF-06447475), a highly potent, selective, brain penetrant, and in vivo active LRRK2 kinase inhibitor.

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of 14 (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies.

Discovery and preclinical characterization of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo-[3,4-b]pyrazine (PF470): a highly potent, selective, and efficacious metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulator.

A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (1), a systematic structure-activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (PF470, 14) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound 14 demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of 14 to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-mediated type IV hypersensitivity in a 90-day NHP regulatory toxicology study.

Metabolism of a serotonin-4 receptor partial agonist 4-{4-[4-tetrahydrofuran-3-yloxy)-benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol (TBPT): identification of an unusual pharmacologically active cyclized oxazolidine metabolite in human.

4-{4-[4-Tetrahydrofuran-3-yloxy)-benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol (PF-4995274, TBPT) is a new agent that is a partial agonist of the human serotonin-4 (5-HT4) receptor and is under investigation for neurological disorders. Metabolism of TBPT was examined in vitro in human liver microsomes and human hepatocytes. Metabolites were also identified in the plasma of healthy human subjects in a phase 1 clinical study. Human-derived metabolite profiles were compared with corresponding profiles obtained in laboratory animal species. There were two major routes of metabolism in vitro: N-dealkylation of the methyltetrahydropyran moiety (M1) and hydroxylation at the seven position of the benzisoxazole moiety (M4). These were also observed in human plasma; however, in that matrix, the major metabolite was an unusual cyclized oxazolidine entity (M2). M2 was proposed to be formed via generation of an intermediate 4° iminium ion on the piperidine ring followed by spontaneous cyclization by attack of the ß-hydroxyl substituent of the tetrahydropyran ring to form a cyclized oxazolidine product. An authentic standard of the metabolite was generated using a methylene-blue-sensitized photochemical oxidation reaction as well as microbial transformation. Further investigation of this metabolite showed that it also possessed 5-HT4 agonism activity similar to the parent. The metabolite was 150-fold more highly protein bound in human plasma than TBPT, which is consistent with its presence as a major circulating metabolite while being only a minor metabolite in in vitro systems. Overall, this illustrates the importance of understanding the complex dispositional properties of a pharmacologically active metabolite.

Identification of multiple 5-HT4 partial agonist clinical candidates for the treatment of Alzheimer's disease.

The cognitive impairments observed in Alzheimer's disease (AD) are in part a consequence of reduced acetylcholine (ACh) levels resulting from a loss of cholinergic neurons. Preclinically, serotonin 4 receptor (5-HT(4)) agonists are reported to modulate cholinergic function and therefore may provide a new mechanistic approach for treating cognitive deficits associated with AD. Herein we communicate the design and synthesis of potent, selective, and brain penetrant 5-HT(4) agonists. The overall goal of the medicinal chemistry strategy was identification of structurally diverse clinical candidates with varying intrinsic activities. The exposure-response relationships between binding affinity, intrinsic activity, receptor occupancy, drug exposure, and pharmacodynamic activity in relevant preclinical models of AD were utilized as key selection criteria for advancing compounds. On the basis of their excellent balance of pharmacokinetic attributes and safety, two lead 5-HT(4) partial agonist candidates 2d and 3 were chosen for clinical development.

Design and discovery of 6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943), a selective brain penetrant PDE9A inhibitor for the treatment of cognitive disorders.

6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943) is a novel PDE9A inhibitor identified using parallel synthetic chemistry and structure-based drug design (SBDD) and has advanced into clinical trials. Selectivity for PDE9A over other PDE family members was achieved by targeting key residue differences between the PDE9A and PDE1C catalytic site. The physicochemical properties of the series were optimized to provide excellent in vitro and in vivo pharmacokinetics properties in multiple species including humans. It has been reported to elevate central cGMP levels in the brain and CSF of rodents. In addition, it exhibits procognitive activity in several rodent models and synaptic stabilization in an amyloid precursor protein (APP) transgenic mouse model. Recent disclosures from clinical trials confirm that it is well tolerated in humans and elevates cGMP in cerebral spinal fluid of healthy volunteers, confirming that it is a quality pharmacological tool for testing clinical hypotheses in disease states associated with impairment of cGMP signaling or cognition.

MEDI3617, a human anti-angiopoietin 2 monoclonal antibody, inhibits angiogenesis and tumor growth in human tumor xenograft models.

Angiopoietin 2 (Ang2) is an important regulator of angiogenesis, blood vessel maturation and integrity of the vascular endothelium. The correlation between the dynamic expression of Ang2 in tumors with regions of high angiogenic activity and a poor prognosis in many tumor types makes Ang2 an ideal drug target. We have generated MEDI3617, a human anti-Ang2 monoclonal antibody that neutralizes Ang2 by preventing its binding to the Tie2 receptor in vitro, and inhibits angiogenesis and tumor growth in vivo. Treatment of mice with MEDI3617 resulted in inhibition of angiogenesis in several mouse models including: FGF2-induced angiogenesis in a basement extract plug model, tumor and retinal angiogenesis. In xenograft tumor models, treatment with MEDI3617 resulted in a reduction in tumor angiogenesis and an increase in tumor hypoxia. The administration of MEDI3617 as a single agent to mice bearing human tumor xenografts resulted in tumor growth inhibition against a broad spectrum of tumor types. Combining MEDI3617 with chemotherapy or bevacizumab resulted in a delay in tumor growth and no body weight loss was observed in the combination groups. These results, combined with pharmacodynamic studies, demonstrate that treatment of tumor-bearing mice with MEDI3617 significantly inhibited tumor growth as a single agent by blocking tumor angiogenesis. Together, these data show that MEDI3617 is a robust antiangiogenic agent and support the clinical evaluation and biomarker development of MEDI3617 in cancer patients.

6-amino-4-(pyrimidin-4-yl)pyridones: novel glycogen synthase kinase-3ß inhibitors.

The synthesis and structure-activity relationships for a novel series of 6-amino-4-(pyrimidin-4-yl)pyridones derived from a high throughput screening hit are discussed. Optimization of lead matter afforded compounds with good potency, selectivity and central nervous system (CNS) exposure.

Diamide amino-imidazoles: a novel series of γ-secretase inhibitors for the treatment of Alzheimer's disease.

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a ß-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aß in guinea pigs. The therapeutic index between Aß reductions and changes in B-cell populations were studied for compound 10 h.

Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: discovery of PF-3084014.

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aß in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aß EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.

Clinical and biological impact of EphA2 overexpression and angiogenesis in endometrial cancer.

OBJECTIVE: EphA2 overexpression predicts poor prognosis in endometrial cancer. To explore mechanisms for this association and assess its potential as therapeutic target, the relationship of EphA2 expression to markers of angiogenesis was examined using patient samples and an orthotopic mouse model of uterine cancer. EXPERIMENTAL DESIGN: Expression of EphA2, estrogen receptor (ER), progesterone receptor (PR), Ki-67, vascular endothelial growth factor (VEGF) and microvessel density (MVD) was evaluated using immunohistochemistry in 85 endometrioid endometrial adenocarcinomas (EEC) by two independent investigators. Results were correlated with clinicopathological characteristics. The effect of EphA2- agonist monoclonal antibody EA5, alone or in combination with docetaxel was studied in vitro and in vivo. Samples were analyzed for markers of angiogenesis, proliferation and apoptosis. RESULTS: Of 85 EEC samples, EphA2 was overexpressed in 47% of tumors and was significantly associated with high VEGF expression (p=0.001) and high MVD counts (p=0.02). High EphA2 expression, high VEGF expression and high MVD counts were significantly associated with shorter disease-specific survival. EA5 led to decrease in EphA2 expression and phosphorylation in vitro. In the murine model, while EA5 (33-88%) and docetaxel (23-55%) individually led to tumor inhibition over controls, combination therapy had the greatest efficacy (78-92%, p.

Pharmacodynamics and pharmacokinetics of the gamma-secretase inhibitor PF-3084014.

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel gamma-secretase inhibitor that reduces amyloid-beta (Abeta) production with an in vitro IC(50) of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC(50) of 2.1 microM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Abeta in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Abeta. To further characterize Abeta dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Abeta, and the magnitude and duration of Abeta lowering exceeded those of the reductions in B-cell endpoints. Other gamma-secretase inhibitors have shown high potency at elevating Abeta in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Abeta11-40 and Abeta1-43 at doses that potently inhibited Abeta1-40 and Abeta1-42. PF-3084014, like previously described gamma-secretase inhibitors, preferentially reduced Abeta1-40 relative to Abeta1-42. Potency at Abeta relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.

Antitumor efficacy of IPI-504, a selective heat shock protein 90 inhibitor against human epidermal growth factor receptor 2-positive human xenograft models as a single agent and in combination with trastuzumab or lapatinib.

IPI-504 is a novel, highly soluble small-molecule inhibitor of heat shock protein 90 (Hsp90), a protein chaperone essential for regulating homeostasis of oncoproteins and cell signaling proteins. Human epidermal growth factor receptor 2 (HER2; ErbB2) oncoprotein, expressed in a subset of metastatic breast cancers, is a Hsp90 client protein. In this study, we investigated the antitumor activity and the mechanism of action of IPI-504 in HER2(+), trastuzumab-sensitive and trastuzumab-refractory cell lines in vitro and in vivo. IPI-504 exhibited potent antiproliferative activities (range of IC(50), 10-40 nmol/L) against several tumor cell lines examined, whereby mechanism of action was mediated through HER2 and Akt degradation. Both intravenous and oral administration of IPI-504 assessed in multiple schedules showed potent tumor growth inhibition in vivo with corresponding degradation of HER2. The tolerability and efficacy of IPI-504 combined with either trastuzumab or lapatinib were also investigated in HER2(+) tumor xenograft models. Combination of IPI-504 with trastuzumab significantly enhanced tumor growth delay and induced greater responses when compared with either agent alone. Although, as expected, trastuzumab alone did not exhibit any significant antitumor activity in the trastuzumab-resistant JIMT-1 model, IPI-504 administered in combination with trastuzumab yielded greater antitumor efficacy than either agent alone. Finally, combination of IPI-504 and lapatinib was well tolerated up to 50 mg/kg IPI-504 and 100 mg/kg lapatinib and resulted in significant delay in tumor growth, including partial and complete tumor responses. These lines of evidence support the development of IPI-504 in HER2-positive breast cancers as a single agent and in combination with either trastuzumab or lapatinib