Analysis of orphan designation status for FDA approved drugs, and case studies in oncology, neuroscience and metabolic diseases.

Many new drugs have been approved over the past decade for rare or orphan diseases. The passage of the Orphan Drug Act (ODA) in 1983 has provided key economic and regulatory incentives to provide medicines for patients who are suffering from rare diseases that may not be commercially attractive for research and development. We have analyzed 497 novel drugs approved from 2010 - June 13, 2022, of which 220 were given orphan designation status. We discuss trends over this time period, potential risks for long development times, and provide example case studies of successful development and launch of novel drugs for rare diseases.

Structural insight into allosteric modulation of protease-activated receptor 2.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

A survey of the clinical pipeline in neuroscience.

Many new first-in-class drugs for neuroscience indications have been introduced in the past decade including new treatments for migraine, amyotrophic lateral sclerosis, depression, and multiple sclerosis. However, significant unmet patient needs remain in areas such as chronic pain, neurodegeneration, psychiatric diseases, and epilepsy. This review summarizes some of the advanced clinical compounds for these indications. Additionally, current opportunities and challenges that remain with respect to genetic validation, biomarkers, and translational models are discussed.

The clinical trial landscape in amyotrophic lateral sclerosis-Past, present, and future.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease marked by progressive loss of muscle function. It is the most common adult-onset form of motor neuron disease, affecting about 16 000 people in the United States alone. The average survival is about 3 years. Only two interventional drugs, the antiglutamatergic small-molecule riluzole and the more recent antioxidant edaravone, have been approved for the treatment of ALS to date. Therapeutic strategies under investigation in clinical trials cover a range of different modalities and targets, and more than 70 different drugs have been tested in the clinic to date. Here, we summarize and classify interventional therapeutic strategies based on their molecular targets and phenotypic effects. We also discuss possible reasons for the failure of clinical trials in ALS and highlight emerging preclinical strategies that could provide a breakthrough in the battle against this relentless disease.

Emerging small-molecule therapeutic approaches for amyotrophic lateral sclerosis and frontotemporal dementia.

Novel treatments are desperately needed for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In this review article, a survey of emerging small-molecule approaches for ALS and FTD therapies is provided. These approaches include targeting aberrant liquid-liquid phase separation and stress granule assembly, modulation of RNA-protein interactions, inhibition of TDP-43 phosphorylation, inhibition of poly(ADP-ribose) polymerases (PARP), RNA-targeting approaches to reduce RAN translation of dipeptide repeat proteins from repeat expansions of C9ORF72, and novel autophagy activation pathways. This review details the emerging small-molecule tools and leads in these areas, along with a critical perspective on the key challenges facing these opportunities.

Tool inhibitors and assays to interrogate the biology of the TRAF2 and NCK interacting kinase.

The TRAF2 and NCK interacting kinase (TNIK) has been proposed to play a role in cytoskeletal organization and synaptic plasticity and has been linked, among others, to neurological disorders. However, target validation efforts for TNIK have been hampered by the limited kinase selectivity of small molecule probes and possible functional compensation in mouse models. Both issues are at least in part due to its close homology to the kinases MINK1 (or MAP4K6) and MAP4K4 (or HGK). As part of our interest in validating TNIK as a therapeutic target for neurological diseases, we set up a panel of biochemical and cellular assays, which are described herein. We then examined the activity of known amino-pyridine-based TNIK inhibitors (1, 3) and prepared structurally very close analogs that lack the ability to inhibit the target. We also developed a structurally orthogonal, naphthyridine-based TNIK inhibitor (9) and an inactive control molecule of the same chemical series. These validated small-molecule probes will enable dissection of the function of TNIK family in the context of human disease biology.

Design and synthesis of peptide-based macrocyclic cyclophilin inhibitors.

The efficient assembly of an 18-membered macrocyclic peptide core was realized by a straightforward and convergent approach utilizing ring-closing metathesis of the corresponding linear tetrapeptides as the key transformation. This approach allowed for the facile preparation of a focused library of novel macrocycles that culminated in the discovery of a cyclophilin A inhibitor with a Kd=5.4µM.

Drug discovery strategies to outer membrane targets in Gram-negative pathogens.

This review will cover selected recent examples of drug discovery strategies which target the outer membrane (OM) of Gram-negative bacteria either by disruption of outer membrane function or by inhibition of essential gene products necessary for outer membrane assembly. Significant advances in pathway elucidation, structural biology and molecular inhibitor designs have created new opportunities for drug discovery within this target-class space.

Novel antibacterial targets and compounds revealed by a high-throughput cell wall reporter assay.

UNLABELLED: A high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter expressed in Escherichia coli was executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action. E. coli mutants resistant to these compounds display no cross-resistance to antibiotics of other classes. Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step in the synthesis of lipid A, the outer membrane anchor of lipopolysaccharide (LPS). To our knowledge, this compound is the first reported inhibitor of LpxH. Resistance to the pyrazole compound mapped to mutations in either LolC or LolE, components of the essential LolCDE transporter complex, which is required for trafficking of lipoproteins to the outer membrane. Biochemical experiments with E. coli spheroplasts showed that the pyrazole compound is capable of inhibiting the release of lipoproteins from the inner membrane. Both of these compounds have significant promise as chemical probes to further interrogate the potential of these novel cell wall components for antimicrobial therapy. IMPORTANCE: The prevalence of antibacterial resistance, particularly among Gram-negative organisms, signals a need for novel antibacterial agents. A phenotypic screen using AmpC as a sensor for compounds that inhibit processes involved in Gram-negative envelope biogenesis led to the identification of two novel inhibitors with unique mechanisms of action targeting Escherichia coli outer membrane biogenesis. One compound inhibits the transport system for lipoprotein transport to the outer membrane, while the other compound inhibits synthesis of lipopolysaccharide. These results indicate that it is still possible to uncover new compounds with intrinsic antibacterial activity that inhibit novel targets related to the cell envelope, suggesting that the Gram-negative cell envelope still has untapped potential for therapeutic intervention.

New natural products as new leads for antibacterial drug discovery.

Natural products have been a rich source of antibacterial drugs for many decades, but investments in this area have declined over the past two decades. The purpose of this review article is to provide a recent survey of new natural product classes and the mechanisms by which they work.

A Drug Discovery Perspective on FDA Expedited Development and Incentive Programs.

Expedited development and approval pathways at the Food and Drug Administration (FDA) such as Priority review, Fast Track Designation, Breakthrough Designation, and Accelerated Approval are programs available to drug sponsors that aim to incentivize and expedite the delivery of drugs to patients in need. In addition, other incentive programs such as Orphan Drug Designation (ODD), Qualified Infectious Disease Product Designation (QIDP), and Rare Pediatric Disease Designation (RPDD) are available to drug sponsors to help motivate development of drugs that may have lower economic incentive for commercialization. These programs have been largely effective, and many new innovative drugs since 2010 have accessed these programs. This Perspective highlights how these programs have been used in recent FDA drug approvals and discusses future ways sponsors and regulatory agencies may further enable development of these innovative drugs in the most expeditious fashion.

Discovery of a potent respiratory syncytial virus RNA polymerase inhibitor.

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.

An Analysis of Successful Hit-to-Clinical Candidate Pairs.

An analysis of 156 published clinical candidates from the Journal of Medicinal Chemistry between 2018 and 2021 was conducted to identify lead generation strategies most frequently employed leading to drug candidates. As in a previous publication, the most frequent lead generation strategies resulting in clinical candidates were from known compounds (59%) followed by random screening approaches (21%). The remainder of the approaches included directed screening, fragment screening, DNA-encoded library screening (DEL), and virtual screening. An analysis of similarity was also conducted based on Tanimoto-MCS and revealed most clinical candidates were distant from their original hits; however, most shared a key pharmacophore that translated from hit-to-clinical candidate. An examination of frequency of oxygen, nitrogen, fluorine, chlorine, and sulfur incorporation in clinical candidates was also conducted. The three most similar and least similar hit-to-clinical pairs from random screening were examined to provide perspective on changes that occur that lead to successful clinical candidates.

Discovery of novel positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu5).

Novel in vitro mGlu(5) positive allosteric modulators with good potency, solubility, and low lipophilicity are described. Compounds were identified which did not rely on the phenylacetylene and carbonyl functionalities previously observed to be required for in vitro activity. Investigation of the allosteric binding requirements of a series of dihydroquinolinone analogs led to phenylacetylene azachromanone 4 (EC(50) 11.5 nM). Because of risks associated with potential metabolic and toxicological liabilities of the phenylacetylene, this moiety was successfully replaced with a phenoxymethyl group (27; EC(50) 156.3 nM). Derivation of a second-generation of mGlu(5) PAMs lacking a ketone carbonyl resulted in azaindoline (33), azabenzimidazole (36), and N-methyl 8-azaoxazine (39) phenylacetylenes. By scoping nitrogen substituents and phenylacetylene replacements in 39, we identified phenoxymethyl 8-azaoxazine 47 (EC(50) 50.1 nM) as a potent and soluble mGlu(5) PAM devoid of both undesirable phenylacetylene and carbonyl functionalities.

2,6-Disubstituted pyrazines and related analogs as NR2B site antagonists of the NMDA receptor with anti-depressant activity.

Herein we describe the discovery of compounds that are competitive antagonists of the CP101-606 binding site within the NR2B subtype of the NMDA receptor. The compounds identified do not possess phenolic functional groups such as those in ifenprodil and related analogs. Initial identification of hits in this series focused on a basic, secondary amine side chain which led to good potency, but also presented a hERG liability. Further modifications led to examples of non-basic replacements which demonstrated much less liability in this regard. Finally, one compound in the series, 6a, was tested in the mouse forced swim depression assay and found to show activity (s.c. 60 mg/kg).

A Decade of FDA-Approved Drugs (2010-2019): Trends and Future Directions.

A total of 378 novel drugs and 27 biosimilars approved by the U.S. Food and Drug Administration (FDA) between 2010 and 2019 were evaluated according to approval numbers by year, therapeutic areas, modalities, route of administration, first-in-class designation, approval times, and expedited review categories. From this review, oncology remains the top therapy area (25%), followed by infection (15%) and central nervous system disorders (11%). Regulatory incentives have been effective as evidenced by an increase in orphan drugs as well as antibacterial drugs approved under the GAIN act. Clinical development times may be increasing, perhaps as a result of the increase in orphan drug indications. Small molecules continue to mostly adhere to "Rule of 5" (Ro5) parameters, but innovation in new modalities is rapidly progressing with approvals for antisense oligonucleotides (ASO), small-interfering RNA (siRNAs), and antibody-directed conjugates (ADCs). Finally, novel targets and scientific breakthroughs that address areas of unmet clinical need are discussed in detail.

In vitro metabolism studies of nomifensine monooxygenation pathways: metabolite identification, reaction phenotyping, and bioactivation mechanism.

Multiple GSH adducts of the oxidative products of nomifensine (M1-M9) in human hepatocytes and liver microsomes have been identified recently. The current study reports three new types of monooxygenated metabolites of nomifensine identified in human liver microsomes: C-linked hydroxylated metabolites with modifications at the A ring (H1 and H4), an N-hydroxylamine (H6), and nomifensine N-oxides (H7 and H8). GSH conjugate formation in incubates containing cDNA-expressed P450s and GSH suggests that nomifensine GSH-sulfinamides (M1 and M2) are formed through the reaction between GSH and the oxidative product of H6. C-linked GSH conjugates M3, M4, M5, and M6 are probably formed via nomifensine benzoquinone imine intermediates via H4 and/or nomifensine epoxides. C-linked GSH conjugates M7, M8, and M9 are probably formed through similar mechanisms via H1. Nomifensine N-oxides do not form reactive metabolites that react with GSH. In vitro metabolism studies using a panel of cDNA-expressed human P450 and flavin monooxygenase (FMO) isoforms (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, FMO1, FMO3, and FMO5) indicated that CYP3A4, CYP2C19, and CYP2B6 generate the largest quantities of H1, H4, and H6, respectively. H7 and H8 are formed almost exclusively by FMOs. The contribution of the individual P450s involved in the formation of H1, H4, and H6 in human liver microsomes was confirmed by the inhibition of product formation by monoclonal anti-cytochrome 450 antibodies. These results showed that CYP3A4 and CYP2B6 contributed primarily to the formation of H1 and H6, respectively. CYP2C19 and CYP1A2 seemed to contribute significantly to the formation of H4.

Identification of multiple glutathione conjugates of 8-amino- 2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline maleate (nomifensine) in liver microsomes and hepatocyte preparations: evidence of the bioactivation of nomifensine.

8-Amino-2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline maleate (nomifensine), an antidepressant drug, was withdrawn from the market because of increased incidence of hemolytic anemia, as well as kidney and liver toxicity. Although the nature of the potentially reactive metabolites formed after nomifensine metabolism remains unknown and no glutathione (GSH) adducts of these nomifensine reactive metabolites have been reported, bioactivation has been postulated as a potential mechanism for the toxicity of nomifensine. This study was conducted to probe the potential bioactivation pathways of nomifensine in human and animal hepatocytes and in liver microsomes using GSH as a trapping agent. Two types of GSH conjugates were characterized by liquid chromatography/tandem mass spectrometry: 1) aniline oxidation followed by GSH conjugation leading to the formation of nomifensine-GSH sulfinamides (M1 and M2); and 2) arene oxidation followed by GSH conjugation yielding a range of arene C-linked GSH adducts (M3-M9). Nine GSH adducts (M1-M9) were identified in liver microsomes of humans, dogs, monkeys, and rats and in human and rat hepatocytes. In dog hepatocyte preparations, six GSH adducts (M1-M6) were identified. The GSH adducts in dog and rat liver microsomes were formed primarily through aniline and arene oxidation, respectively. Both pathways contributed significantly to the formation of the GSH adducts in human and monkey liver microsomes. The bioactivation pathways proposed here account for the formation of the observed GSH conjugates. These investigations have confirmed the aniline and the arene groups in nomifensine as potential toxicophores capable of generating reactive intermediates.

SAR development of a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides as kappa opioid receptor antagonists. Part 2.

Further structure activity relationship studies on a previously reported 8-azabicyclo[3.2.1]octan-3-yloxy-benzamide series of potent and selective kappa opioid receptor antagonists is discussed. Modification of the pendant N-substitution to include a cyclohexylurea moiety produced analogs with greater in vitro opioid and hERG selectivity such as 12 (kappa IC50=172 nM, mu:kappa ratio=93, delta:kappa ratio=>174, hERG IC50=>33 microM). Changes to the linker conformation and identity as well as to the benzamide ring moiety were also investigated.

Discovery of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides as selective antagonists of the kappa opioid receptor. Part 1.

Initial high throughput screening efforts identified highly potent and selective kappa opioid receptor antagonist 3 (κ IC(50)=77 nM; µ:κ and δ:κ IC(50) ratios>400) which lacked CNS exposure in vivo. Modification of this scaffold resulted in development of a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides showing potent and selectivity κ antagonism as well as good brain exposure. Analog 6c (κ IC(50)=20 nM; µ:κ=36, δ:κ=415) was also shown to reverse κ-agonist induced rat diuresis in vivo.