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.

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.

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.

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.

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.

Protease-activated receptor-2 ligands reveal orthosteric and allosteric mechanisms of receptor inhibition.

Protease-activated receptor-2 (PAR2) has been implicated in multiple pathophysiologies but drug discovery is challenging due to low small molecule tractability and a complex activation mechanism. Here we report the pharmacological profiling of a potent new agonist, suggested by molecular modelling to bind in the putative orthosteric site, and two novel PAR2 antagonists with distinctly different mechanisms of inhibition. We identify coupling between different PAR2 binding sites. One antagonist is a competitive inhibitor that binds to the orthosteric site, while a second antagonist is a negative allosteric modulator that binds at a remote site. The allosteric modulator shows probe dependence, more effectively inhibiting peptide than protease activation of PAR2 signalling. Importantly, both antagonists are active in vivo, inhibiting PAR2 agonist-induced acute paw inflammation in rats and preventing activation of mast cells and neutrophils. These results highlight two distinct mechanisms of inhibition that potentially could be targeted for future development of drugs that modulate PAR2.

Prolonged tau clearance and stress vulnerability rescue by pharmacological activation of autophagy in tauopathy neurons.

Tauopathies are neurodegenerative diseases associated with accumulation of abnormal tau protein in the brain. Patient iPSC-derived neuronal cell models replicate disease-relevant phenotypes ex vivo that can be pharmacologically targeted for drug discovery. Here, we explored autophagy as a mechanism to reduce tau burden in human neurons and, from a small-molecule screen, identify the mTOR inhibitors OSI-027, AZD2014 and AZD8055. These compounds are more potent than rapamycin, and robustly downregulate phosphorylated and insoluble tau, consequently reducing tau-mediated neuronal stress vulnerability. MTORC1 inhibition and autophagy activity are directly linked to tau clearance. Notably, single-dose treatment followed by washout leads to a prolonged reduction of tau levels and toxicity for 12 days, which is mirrored by a sustained effect on mTORC1 inhibition and autophagy. This new insight into the pharmacodynamics of mTOR inhibitors in regulation of neuronal autophagy may contribute to development of therapies for tauopathies.

Axl receptor tyrosine kinase is a regulator of apolipoprotein E.

Alzheimer's disease (AD), the leading cause of dementia, is a chronic neurodegenerative disease. Apolipoprotein E (apoE), which carries lipids in the brain in the form of lipoproteins, plays an undisputed role in AD pathophysiology. A high-throughput phenotypic screen was conducted using a CCF-STTG1 human astrocytoma cell line to identify small molecules that could upregulate apoE secretion. AZ7235, a previously discovered Axl kinase inhibitor, was identified to have robust apoE activity in brain microglia, astrocytes and pericytes. AZ7235 also increased expression of ATP-binding cassette protein A1 (ABCA1), which is involved in the lipidation and secretion of apoE. Moreover, AZ7235 did not exhibit Liver-X-Receptor (LXR) activity and stimulated apoE and ABCA1 expression in the absence of LXR. Target validation studies using AXL-/- CCF-STTG1 cells showed that Axl is required to mediate AZ7235 upregulation of apoE and ABCA1. Intriguingly, apoE expression and secretion was significantly attenuated in AXL-deficient CCF-STTG1 cells and reconstitution of Axl or kinase-dead Axl significantly restored apoE baseline levels, demonstrating that Axl also plays a role in maintaining apoE homeostasis in astrocytes independent of its kinase activity. Lastly, these effects may require human apoE regulatory sequences, as AZ7235 exhibited little stimulatory activity toward apoE and ABCA1 in primary murine glia derived from neonatal human APOE3 targeted-replacement mice. Collectively, we identified a small molecule that exhibits robust apoE and ABCA1 activity independent of the LXR pathway in human cells and elucidated a novel relationship between Axl and apoE homeostasis in human astrocytes.

A High-Throughput Cellular Screening Assay for Small-Molecule Inhibitors and Activators of Cytoplasmic Dynein-1-Based Cargo Transport.

Cytoplasmic dynein-1 (hereafter dynein) is a six-subunit motor complex that transports a variety of cellular components and pathogens along microtubules. Dynein's cellular functions are only partially understood, and potent and specific small-molecule inhibitors and activators of this motor would be valuable for addressing this issue. It has also been hypothesized that an inhibitor of dynein-based transport could be used in antiviral or antimitotic therapy, whereas an activator could alleviate age-related neurodegenerative diseases by enhancing microtubule-based transport in axons. Here, we present the first high-throughput screening (HTS) assay capable of identifying both activators and inhibitors of dynein-based transport. This project is also the first collaborative screening report from the Medical Research Council and AstraZeneca agreement to form the UK Centre for Lead Discovery. A cellular imaging assay was used, involving chemically controlled recruitment of activated dynein complexes to peroxisomes. Such a system has the potential to identify molecules that affect multiple aspects of dynein biology in vivo. Following optimization of key parameters, the assay was developed in a 384-well format with semiautomated liquid handling and image acquisition. Testing of more than 500,000 compounds identified both inhibitors and activators of dynein-based transport in multiple chemical series. Additional analysis indicated that many of the identified compounds do not affect the integrity of the microtubule cytoskeleton and are therefore candidates to directly target the transport machinery.

Phenotypic high-throughput screening platform identifies novel chemotypes for necroptosis inhibition.

Regulated necrosis or necroptosis, mediated by receptor-interacting kinase 1 (RIPK1), RIPK3 and pseudokinase mixed lineage kinase domain-like protein (MLKL), contributes to the pathogenesis of inflammatory, infectious and degenerative diseases. Recently identified necroptosis inhibitors display moderate specificity, suboptimal pharmacokinetics, off-target effects and toxicity, preventing these molecules from reaching the clinic. Here, we developed a cell-based high-throughput screening (HTS) cascade for the identification of small-molecule inhibitors of necroptosis. From the initial library of over 250,000 compounds, the primary screening phase identified 356 compounds that strongly inhibited TNF-α-induced necroptosis, but not apoptosis, in human and murine cell systems, with EC50 < 6.7 µM. From these, 251 compounds were tested for RIPK1 and/or RIPK3 kinase inhibitory activity; some were active and several have novel mechanisms of action. Based on specific chemical descriptors, 110 compounds proceeded into the secondary screening cascade, which then identified seven compounds with maximum ability to reduce MLKL activation, IC50 >100 µM, EC50 2.5-11.5 µM under long-term necroptosis execution in murine fibroblast L929 cells, and full protection from ATP depletion and membrane leakage in human and murine cells. As a proof of concept, compound SN-6109, with binding mode to RIPK1 similar to that of necrostatin-1, confirmed RIPK1 inhibitory activity and appropriate pharmacokinetic properties. SN-6109 was further tested in mice, showing efficacy against TNF-α-induced systemic inflammatory response syndrome. In conclusion, a phenotypic-driven HTS cascade promptly identified robust necroptosis inhibitors with in vivo activity, currently undergoing further medicinal chemistry optimization. Notably, the novel hits highlight the opportunity to identify new molecular mechanisms of action in necroptosis.

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.

Opportunities and Challenges in Phenotypic Screening for Neurodegenerative Disease Research.

Toxic misfolded proteins potentially underly many neurodegenerative diseases, but individual targets which regulate these proteins and their downstream detrimental effects are often unknown. Phenotypic screening is an unbiased method to screen for novel targets and therapeutic molecules and span the range from primitive model organisms such as Sacchaomyces cerevisiae, which allow for high-throughput screening to patient-derived cell-lines that have a close connection to the disease biology but are limited in screening capacity. This perspective will review current phenotypic models, as well as the chemical screening strategies most often employed. Advances in in 3D cell cultures, high-content screens, robotic microscopy, CRISPR screening, and use of machine learning methods to process the enormous amount of data generated by these screens are certain to change the paradigm for phenotypic screening and will be discussed.

Promiscuity of in Vitro Secondary Pharmacology Assays and Implications for Lead Optimization Strategies.

We conducted an analysis on screening data generated from 1445 compounds against a panel of 130 enzymes, ion channels, and receptors to assess secondary pharmacological risks. Hit rates of these targets as well as physicochemical properties for those hits were evaluated. A majority of targets yielded hits with higher clogP, molecular weight, and more basic character than inactive compounds. Although most targets favored lipophilic hits, the average clogP of hits at a given target did not correlate with its hit rate. Furthermore, a matched pair analysis was completed to determine structural changes that impacted off-target activities. A correlation of binding assays used in this analysis illustrated that some pharmacologically related binding assays are highly correlative and may be substituted for a smaller set of surrogate assays.

Discovery of Ligand-Efficient Scaffolds for the Design of Novel Trichomonas vaginalis Uridine Nucleoside Ribohydrolase Inhibitors Using Fragment Screening.

Trichomoniasis is caused by the parasitic protozoan Trichomonas vaginalis. The increasing prevalence of strains resistant to the current 5-nitroimidazole treatments creates the need for novel therapies. T. vaginalis cannot synthesize purine and pyrimidine rings and requires salvage pathway enzymes to obtain them from host nucleosides. The uridine nucleoside ribohydrolase was screened using an 19F NMR-based activity assay against a 2000-compound fragment diversity library. Several series of inhibitors were identified including scaffolds based on acetamides, cyclic ureas or ureas, pyridines, and pyrrolidines. A number of potent singleton compounds were identified, as well. Eighteen compounds with IC50 values of 20 µM or lower were identified, including some with ligand efficiency values of 0.5 or greater. Detergent and jump-dilution counter screens validated all scaffold classes as target-specific, reversible inhibitors. Identified scaffolds differ substantially from 5-nitroimidazoles. Medicinal chemistry using the structure-activity relationship emerging from the fragment hits is being pursued to discover nanomolar inhibitors.

CETSA beyond Soluble Targets: a Broad Application to Multipass Transmembrane Proteins.

Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane bound targets.