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.

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.

Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells.

USP14 is a cysteine protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A showed consistent or significant effects on the level of TDP-43, tau or α-synuclein in HEK293T cells. However, USP14 C114A led to a robust accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated ß-catenin accumulated in the cells expressing USP14 C114A with immunoblotting and immunoprecipitation experiments. The proteasome binding domain of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect ß-catenin, demonstrating USP14 but not UCHL5 has a specific effect on ß-catenin. We used ubiquitin immunoprecipitation and mass spectrometry to identify the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those identified in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Besides ß-catenin, we also verified with immunoblotting that UCHL5 C88A inhibits its own deubiquitination and USP14 C114A inhibits deubiquitination of two proteasomal subunits PSMC1 and PSMD4. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function.

Developmental Regulation of KCC2 Phosphorylation Has Long-Term Impacts on Cognitive Function.

GABAA receptor-mediated currents shift from excitatory to inhibitory during postnatal brain development in rodents. A postnatal increase in KCC2 protein expression is considered to be the sole mechanism controlling the developmental onset of hyperpolarizing synaptic transmission, but here we identify a key role for KCC2 phosphorylation in the developmental EGABA shift. Preventing phosphorylation of KCC2 in vivo at either residue serine 940 (S940), or at residues threonine 906 and threonine 1007 (T906/T1007), delayed or accelerated the postnatal onset of KCC2 function, respectively. Several models of neurodevelopmental disorders including Rett syndrome, Fragile × and Down's syndrome exhibit delayed postnatal onset of hyperpolarizing GABAergic inhibition, but whether the timing of the onset of hyperpolarizing synaptic inhibition during development plays a role in establishing adulthood cognitive function is unknown; we have used the distinct KCC2-S940A and KCC2-T906A/T1007A knock-in mouse models to address this issue. Altering KCC2 function resulted in long-term abnormalities in social behavior and memory retention. Tight regulation of KCC2 phosphorylation is therefore required for the typical timing of the developmental onset of hyperpolarizing synaptic inhibition, and it plays a fundamental role in the regulation of adulthood cognitive function.

Identification of a Core Amino Acid Motif within the α Subunit of GABAARs that Promotes Inhibitory Synaptogenesis and Resilience to Seizures.

The fidelity of inhibitory neurotransmission is dependent on the accumulation of γ-aminobutyric acid type A receptors (GABAARs) at the appropriate synaptic sites. Synaptic GABAARs are constructed from α(1-3), ß(1-3), and γ2 subunits, and neurons can target these subtypes to specific synapses. Here, we identify a 15-amino acid inhibitory synapse targeting motif (ISTM) within the α2 subunit that promotes the association between GABAARs and the inhibitory scaffold proteins collybistin and gephyrin. Using mice in which the ISTM has been introduced into the α1 subunit (Gabra1-2 mice), we show that the ISTM is critical for axo-axonic synapse formation, the efficacy of GABAergic neurotransmission, and seizure sensitivity. The Gabra1-2 mutation rescues seizure-induced lethality in Gabra2-1 mice, which lack axo-axonic synapses due to the deletion of the ISTM from the α2 subunit. Taken together, our data demonstrate that the ISTM plays a critical role in promoting inhibitory synapse formation, both in the axonic and somatodendritic compartments.

Wild-Type, but Not Mutant N296H, Human Tau Restores Aß-Mediated Inhibition of LTP in Tau-/- mice.

Microtubule associated protein tau (MAPT) is involved in the pathogenesis of Alzheimer's disease and many forms of frontotemporal dementia (FTD). We recently reported that Aß-mediated inhibition of hippocampal long-term potentiation (LTP) in mice requires tau. Here, we asked whether expression of human MAPT can restore Aß-mediated inhibition on a mouse Tau-/- background and whether human tau with an FTD-causing mutation (N296H) can interfere with Aß-mediated inhibition of LTP. We used transgenic mouse lines each expressing the full human MAPT locus using bacterial artificial chromosome technology. These lines expressed all six human tau protein isoforms on a Tau-/- background. We found that the human wild-type MAPT H1 locus was able to restore Aß42-mediated impairment of LTP. In contrast, Aß42 did not reduce LTP in slices in two independently generated transgenic lines expressing tau protein with the mutation N296H associated with frontotemporal dementia (FTD). Basal phosphorylation of tau measured as the ratio of AT8/Tau5 immunoreactivity was significantly reduced in N296H mutant hippocampal slices. Our data show that human MAPT is able to restore Aß42-mediated inhibition of LTP in Tau-/- mice. These results provide further evidence that tau protein is central to Aß-induced LTP impairment and provide a valuable tool for further analysis of the links between Aß, human tau and impairment of synaptic function.

Truncation of the TAR DNA-binding protein 43 is not a prerequisite for cytoplasmic relocalization, and is suppressed by caspase inhibition and by introduction of the A90V sequence variant.

The RNA-binding and -processing protein TAR DNA-binding protein 43 (TDP-43) is heavily linked to the underlying causes and pathology of neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In these diseases, TDP-43 is mislocalized, hyperphosphorylated, ubiquitinated, aggregated and cleaved. The importance of TDP-43 cleavage in the disease pathogenesis is still poorly understood. Here we detail the use of D-sorbitol as an exogenous stressor that causes TDP-43 cleavage in HeLa cells, resulting in a 35 kDa truncated product that accumulates in the cytoplasm within one hour of treatment. We confirm that the formation of this 35 kDa cleavage product is mediated by the activation of caspases. Inhibition of caspases blocks the cleavage of TDP-43, but does not prevent the accumulation of full-length protein in the cytoplasm. Using D-sorbitol as a stressor and caspase activator, we also demonstrate that the A90V variant of TDP-43, which lies adjacent to the caspase cleavage site within the nuclear localization sequence of TDP-43, confers partial resistance against caspase-mediated generation of the 35 kDa cleavage product.

Cytoplasmic Relocalization of TAR DNA-Binding Protein 43 Is Not Sufficient to Reproduce Cellular Pathologies Associated with ALS In vitro.

Mutations in the gene TARDBP, which encodes TAR DNA-binding protein 43 (TDP-43), are a rare cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While the majority of mutations are found in the C-terminal glycine-rich domain, an alanine to valine amino acid change at position 90 (A90V) in the bipartite nuclear localization signal (NLS) of TDP-43 has been described. This sequence variant has previously been shown to cause cytoplasmic mislocalization of TDP-43 and decrease protein solubility, leading to the formation of insoluble aggregates. Since the A90V mutation has been described both in patients as well as healthy controls, its pathogenic potential in ALS and FTD remains unclear. Here we compare properties of overexpressed A90V to the highly pathogenic M337V mutation. Though both mutations drive mislocalization of the protein to the cytoplasm to the same extent, M337V produces more significant damage in terms of protein solubility, levels of pathogenic phosphorylation, and formation of C-terminal truncated protein species. Furthermore, the M337V, but not the A90V mutant, leads to a downregulation of histone deacetylase 6 and Ras GTPase-activating protein-binding protein. We conclude that in the absence of another genetic or environmental 'hit' the A90V variant is not sufficient to cause the deleterious phenotypes associated with ALS and FTD, despite prominent cytoplasmic protein relocalization of TDP-43.

Increased 4R tau expression and behavioural changes in a novel MAPT-N296H genomic mouse model of tauopathy.

The microtubule-associated protein tau is implicated in various neurodegenerative diseases including Alzheimer's disease, progressive supranuclear palsy and corticobasal degeneration, which are characterized by intracellular accumulation of hyperphosphorylated tau. Mutations in the tau gene MAPT cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). In the human central nervous system, six tau isoforms are expressed, and imbalances in tau isoform ratios are associated with pathology. To date, few animal models of tauopathy allow for the potential influence of these protein isoforms, relying instead on cDNA-based transgene expression. Using the P1-derived artificial chromosome (PAC) technology, we created mouse lines expressing all six tau isoforms from the human MAPT locus, harbouring either the wild-type sequence or the disease-associated N296H mutation on an endogenous Mapt-/- background. Animals expressing N296H mutant tau recapitulated early key features of tauopathic disease, including a tau isoform imbalance and tau hyperphosphorylation in the absence of somatodendritic tau inclusions. Furthermore, N296H animals displayed behavioural anomalies such as hyperactivity, increased time in the open arms of the elevated plus maze and increased immobility during the tail suspension test. The mouse models described provide an excellent model to study the function of wild-type or mutant tau in a highly physiological setting.

The green tea polyphenol (-)-epigallocatechin gallate prevents the aggregation of tau protein into toxic oligomers at substoichiometric ratios.

The accumulation of amyloid-beta (Aß) and tau aggregates is a pathological hallmark of Alzheimer's disease. Both polypeptides form fibrillar deposits, but several lines of evidence indicate that Aß and tau form toxic oligomeric aggregation intermediates. Depleting such structures could thus be a powerful therapeutic strategy. We generated a fragment of tau (His-K18ΔK280) that forms stable, toxic, oligomeric tau aggregates in vitro. We show that (-)-epigallocatechin gallate (EGCG), a green tea polyphenol that was previously found to reduce Aß aggregation, inhibits the aggregation of tau K18ΔK280 into toxic oligomers at ten- to hundred-fold substoichiometric concentrations, thereby rescuing toxicity in neuronal model cells.