ABC transporters: human disease and pharmacotherapeutic potential.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are a 48-member superfamily of membrane proteins that actively transport a variety of biological substrates across lipid membranes. Their functional diversity defines an expansive involvement in myriad aspects of human biology. At least 21 ABC transporters underlie rare monogenic disorders, with even more implicated in the predisposition to and symptomology of common and complex diseases. Such broad (patho)physiological relevance places this class of proteins at the intersection of disease causation and therapeutic potential, underlining them as promising targets for drug discovery, as exemplified by the transformative CFTR (ABCC7) modulator therapies for cystic fibrosis. This review will explore the growing relevance of ABC transporters to human disease and their potential as small-molecule drug targets.

Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules.

The NADase SARM1 (sterile alpha and TIR motif containing 1) is a key executioner of axon degeneration and a therapeutic target for several neurodegenerative conditions. We show that a potent SARM1 inhibitor undergoes base exchange with the nicotinamide moiety of nicotinamide adenine dinucleotide (NAD+) to produce the bona fide inhibitor 1AD. We report structures of SARM1 in complex with 1AD, NAD+ mimetics and the allosteric activator nicotinamide mononucleotide (NMN). NMN binding triggers reorientation of the armadillo repeat (ARM) domains, which disrupts ARM:TIR interactions and leads to formation of a two-stranded TIR domain assembly. The active site spans two molecules in these assemblies, explaining the requirement of TIR domain self-association for NADase activity and axon degeneration. Our results reveal the mechanisms of SARM1 activation and substrate binding, providing rational avenues for the design of new therapeutics targeting SARM1.

Pharmacological SARM1 inhibition protects axon structure and function in paclitaxel-induced peripheral neuropathy.

Axonal degeneration is an early and ongoing event that causes disability and disease progression in many neurodegenerative disorders of the peripheral and central nervous systems. Chemotherapy-induced peripheral neuropathy (CIPN) is a major cause of morbidity and the main cause of dose reductions and discontinuations in cancer treatment. Preclinical evidence indicates that activation of the Wallerian-like degeneration pathway driven by sterile alpha and TIR motif containing 1 (SARM1) is responsible for axonopathy in CIPN. SARM1 is the central driver of an evolutionarily conserved programme of axonal degeneration downstream of chemical, inflammatory, mechanical or metabolic insults to the axon. SARM1 contains an intrinsic NADase enzymatic activity essential for its pro-degenerative functions, making it a compelling therapeutic target to treat neurodegeneration characterized by axonopathies of the peripheral and central nervous systems. Small molecule SARM1 inhibitors have the potential to prevent axonal degeneration in peripheral and central axonopathies and to provide a transformational disease-modifying treatment for these disorders. Using a biochemical assay for SARM1 NADase we identified a novel series of potent and selective irreversible isothiazole inhibitors of SARM1 enzymatic activity that protected rodent and human axons in vitro. In sciatic nerve axotomy, we observed that these irreversible SARM1 inhibitors decreased a rise in nerve cADPR and plasma neurofilament light chain released from injured sciatic nerves in vivo. In a mouse paclitaxel model of CIPN we determined that Sarm1 knockout mice prevented loss of axonal function, assessed by sensory nerve action potential amplitudes of the tail nerve, in a gene-dosage-dependent manner. In that CIPN model, the irreversible SARM1 inhibitors prevented loss of intraepidermal nerve fibres induced by paclitaxel and provided partial protection of axonal function assessed by sensory nerve action potential amplitude and mechanical allodynia.

SARM1 is a metabolic sensor activated by an increased NMN/NAD+ ratio to trigger axon degeneration.

Axon degeneration is a central pathological feature of many neurodegenerative diseases. Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) is a nicotinamide adenine dinucleotide (NAD+)-cleaving enzyme whose activation triggers axon destruction. Loss of the biosynthetic enzyme NMNAT2, which converts nicotinamide mononucleotide (NMN) to NAD+, activates SARM1 via an unknown mechanism. Using structural, biochemical, biophysical, and cellular assays, we demonstrate that SARM1 is activated by an increase in the ratio of NMN to NAD+ and show that both metabolites compete for binding to the auto-inhibitory N-terminal armadillo repeat (ARM) domain of SARM1. We report structures of the SARM1 ARM domain bound to NMN and of the homo-octameric SARM1 complex in the absence of ligands. We show that NMN influences the structure of SARM1 and demonstrate via mutagenesis that NMN binding is required for injury-induced SARM1 activation and axon destruction. Hence, SARM1 is a metabolic sensor responding to an increased NMN/NAD+ ratio by cleaving residual NAD+, thereby inducing feedforward metabolic catastrophe and axonal demise.

Small Molecule SARM1 Inhibitors Recapitulate the SARM1-/- Phenotype and Allow Recovery of a Metastable Pool of Axons Fated to Degenerate.

Axonal degeneration is responsible for disease progression and accumulation of disability in many neurodegenerative conditions. The axonal degenerative process can generate a metastable pool of damaged axons that remain structurally and functionally viable but fated to degenerate in the absence of external intervention. SARM1, an NADase that depletes axonal energy stores upon activation, is the central driver of an evolutionarily conserved program of axonal degeneration. We identify a potent and selective small molecule isoquinoline inhibitor of SARM1 NADase that recapitulates the SARM1-/- phenotype and protects axons from degeneration induced by axotomy or mitochondrial dysfunction. SARM1 inhibition post-mitochondrial injury with rotenone allows recovery and rescues axons that already entered the metastable state. We conclude that SARM1 inhibition with small molecules has the potential to treat axonopathies of the central and peripheral nervous systems by preventing axonal degeneration and by allowing functional recovery of a metastable pool of damaged, but viable, axons.

Discovery of a Series of Pyrazinone RORγ Antagonists and Identification of the Clinical Candidate BI 730357.

The interleukin (IL)-23/T helper (Th)17 axis plays a critical role in autoimmune diseases, and there is an increasing number of biologic therapies that target IL-23 and IL-17. The transcription factor retinoic acid receptor-related orphan nuclear receptor γt (RORγt) is important for the activation and differentiation of Th17 cells and thus is an attractive pharmacologic target for the treatment of Th17-mediated diseases. A novel series of pyrazinone RORγ antagonists was discovered through hybridization of two distinct screening hits and scaffold hopping. The series offers attractive potency and selectivity in combination with favorable druglike properties, such as metabolic stability and aqueous solubility. Lead optimization identified a clinical candidate, compound (S)-11 (BI 730357), for the treatment of autoimmune diseases.

Axons Matter: The Promise of Treating Neurodegenerative Disorders by Targeting SARM1-Mediated Axonal Degeneration.

Attempts to develop neuroprotective treatments for neurodegenerative disorders have not yet been clinically successful. Axonal degeneration has been recognized as a predominant driver of disability and disease progression in central nervous system (CNS) diseases such as amyotrophic lateral sclerosis (ALS), multiple sclerosis, and Parkinson's disease, peripheral nervous system (PNS) disorders such as chemotherapy-induced, diabetic, and inherited neuropathies, and ocular disorders, such as glaucoma. In recent years, sterile alpha and TIR motif containing 1 (SARM1) has emerged as the first compelling axonal-specific target for therapeutic intervention. In this review, we discuss the role of axonal degeneration in neurodegenerative disorders, with a focus on SARM1 and the discovery of its intrinsic enzymatic function. Establishment of neurofilament light chain (NfL) as a reliable biomarker of axonal damage, and the availability of an ultrasensitive method for measuring NfL in plasma or serum, provide translational tools to make development of axonal protective, SARM1 inhibitors a viable approach to treat multiple neurodegenerative disorders.

cADPR is a gene dosage-sensitive biomarker of SARM1 activity in healthy, compromised, and degenerating axons.

SARM1 is the central executioner of pathological axon degeneration, promoting axonal demise in response to axotomy, traumatic brain injury, and neurotoxic chemotherapeutics that induce peripheral neuropathy. SARM1 is an injury-activated NAD+ cleavage enzyme, and this NADase activity is required for the pro-degenerative function of SARM1. At present, SARM1 function is assayed by either analysis of axonal loss, which is far downstream of SARM1 enzymatic activity, or via NAD+ levels, which are regulated by many competing pathways. Here we explored the utility of measuring cADPR, a product of SARM1-dependent cleavage of NAD+, as an in cell and in vivo biomarker of SARM1 enzymatic activity. We find that SARM1 is a major producer of cADPR in cultured dorsal root ganglion (DRG) neurons, sciatic nerve, and brain, demonstrating that SARM1 has basal activity in the absence of injury. Following injury, there is a dramatic SARM1-dependent increase in the levels of axonal cADPR that precedes morphological axon degeneration. In vivo, there is also a rapid and large injury-stimulated increase in cADPR in sciatic and optic nerves. The increase in cADPR after injury is proportional to SARM1 gene dosage, suggesting that SARM1 activity is the prime regulator of cADPR levels. The role of cADPR as an important calcium mobilizing agent prompted exploration of its functional contribution to axon degeneration. We used multiple bacterial and mammalian engineered enzymes to manipulate cADPR levels in neurons but found no changes in the time course of axonal degeneration, suggesting that cADPR is unlikely to be an important contributor to the degenerative mechanism. Using cADPR as a SARM1 biomarker, we find that SARM1 can be partially activated by a diverse array of mitochondrial toxins administered at doses that do not induce axon degeneration. Hence, the subcritical activation of SARM1 induced by mitochondrial dysfunction may contribute to the axonal vulnerability common to many neurodegenerative diseases. Finally, we assay levels of both nerve cADPR and plasma neurofilament light chain (NfL) following nerve injury in vivo, and demonstrate that both biomarkers are excellent readouts of SARM1 activity, with cADPR reporting the early molecular changes in the nerve and NfL reporting subsequent axonal breakdown. The identification and characterization of cADPR as a SARM1 biomarker will help identify neurodegenerative diseases in which SARM1 contributes to axonal loss and expedite target validation studies of SARM1-directed therapeutics.

NAD+ cleavage activity by animal and plant TIR domains in cell death pathways.

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.

Aminopyrazole Carboxamide Bruton's Tyrosine Kinase Inhibitors. Irreversible to Reversible Covalent Reactive Group Tuning.

Potent covalent inhibitors of Bruton's tyrosine kinase (BTK) based on an aminopyrazole carboxamide scaffold have been identified. Compared to acrylamide-based covalent reactive groups leading to irreversible protein adducts, cyanamide-based reversible-covalent inhibitors provided the highest combined BTK potency and EGFR selectivity. The cyanamide covalent mechanism with BTK was confirmed through enzyme kinetic, NMR, MS, and X-ray crystallographic studies. The lead cyanamide-based inhibitors demonstrated excellent kinome selectivity and rat pharmacokinetic properties.

Automatically updating predictive modeling workflows support decision-making in drug design.

Using predictive models for early decision-making in drug discovery has become standard practice. We suggest that model building needs to be automated with minimum input and low technical maintenance requirements. Models perform best when tailored to answering specific compound optimization related questions. If qualitative answers are required, 2-bin classification models are preferred. Integrating predictive modeling results with structural information stimulates better decision making. For in silico models supporting rapid structure-activity relationship cycles the performance deteriorates within weeks. Frequent automated updates of predictive models ensure best predictions. Consensus between multiple modeling approaches increases the prediction confidence. Combining qualified and nonqualified data optimally uses all available information. Dose predictions provide a holistic alternative to multiple individual property predictions for reaching complex decisions.

Investigation of the pyrazinones as PDE5 inhibitors: evaluation of regioisomeric projections into the solvent region.

We describe the design, synthesis and profiling of a novel series of PDE5 inhibitors. We take advantage of an alternate projection into the solvent region to identify compounds with excellent potency, selectivity and pharmacokinetic profiles.

Discovery of INCB10820/PF-4178903, a potent, selective, and orally bioavailable dual CCR2 and CCR5 antagonist.

We report the discovery of a potent, selective, and orally bioavailable dual CCR2 and CCR5 antagonist (3S,4S)-N-[(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine (19). After evaluation in 28-day toxicology studies, compound 19 (INCB10820/PF-4178903) was selected as a clinical candidate.

Design and synthesis of novel CCR2 antagonists: investigation of non-aryl/heteroaryl binding motifs.

This report describes the design and synthesis of a series of CCR2 antagonists incorporating novel non-aryl/heteroaryl RHS (right hand side) motifs. Previous SAR in the area has suggested an aryl/heteroaryl substituent as a necessary structural feature for binding to the CCR2 receptor. Herein we describe the SAR with regards to potency (binding to hCCR2), dofetilide activity and metabolic stability (in vitro HLM) for this series. The resulting outcome was the identification of compounds with excellent properties for the investigation of the role of CCR2 in disease.

Discovery of ((1S,3R)-1-isopropyl-3-((3S,4S)-3-methoxy-tetrahydro-2H-pyran-4-ylamino)cyclopentyl)(4-(5-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)methanone, PF-4254196, a CCR2 antagonist with an improved cardiovascular profile.

We describe the systematic optimization, focused on the improvement of CV-TI, of a series of CCR2 antagonists. This work resulted in the identification of 10 (((1S,3R)-1-isopropyl-3-((3S,4S)-3-methoxy-tetrahydro-2H-pyran-4-ylamino)cyclopentyl)(4-(5-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)methanone) which possessed a low projected human dose 35-45mg BID and a CV-TI=3800-fold.

Discovery of INCB8761/PF-4136309, a Potent, Selective, and Orally Bioavailable CCR2 Antagonist.

We report the discovery of a new (S)-3-aminopyrrolidine series of CCR2 antagonists. Structure-activity relationship studies on this new series led to the identification of 17 (INCB8761/PF-4136309) that exhibited potent CCR2 antagonistic activity, high selectivity, weak hERG activity, and an excellent in vitro and in vivo ADMET profile. INCB8761/PF-4136309 has entered human clinical trials.

1-(2-Ethoxyethyl)-1H-pyrazolo[4,3-d]pyrimidines as potent phosphodiesterase 5 (PDE5) inhibitors.

1H-Pyrazolo[4,3-d]pyrimidines are a class of potent and selective second generation phosphodiesterase 5 (PDE5) inhibitors. This work explores the potency, selectivity and efficacy of 1-(2-ethoxyethyl)-1H-pyrazolo[4,5-d]pyrimidines as PDE5 inhibitors resulting in the advancement of a clinical candidate.

1-(2-(2,2,2-trifluoroethoxy)ethyl-1H-pyrazolo[4,3-d]pyrimidines as potent phosphodiesterase 5 (PDE5) inhibitors.

1H-Pyrazolo[4,3-d]pyrimidines were previously disclosed as a potent second generation class of phosphodiesterase 5 (PDE5) inhibitors. This work explores the advancement of more selective and potent PDE5 inhibitors resulting from the substitution of 2-(2,2,2-trifluoroethoxy)ethyl at the 1 position in the so-called alkoxy pocket.

Design, synthesis, and biological evaluation of 3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one, a potent, orally active, brain penetrant inhibitor of phosphodiesterase 5 (PDE5).

We recently described a novel series of aminopyridopyrazinones as PDE5 inhibitors. Efforts toward optimization of this series culminated in the identification of 3-[4-(2-hydroxyethyl)piperazin-1-yl]-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one, which possessed an excellent potency and selectivity profile and demonstrated robust in vivo blood pressure lowering in a spontaneously hypertensive rat (SHR) model. Furthermore, this compound is brain penetrant and will be a useful agent for evaluating the therapeutic potential of central inhibition of PDE5. This compound has recently entered clinical trials.

Optimization of the aminopyridopyrazinones class of PDE5 inhibitors: discovery of 3-[(trans-4-hydroxycyclohexyl)amino]-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one.

We describe efforts to improve the pharmacokinetic profile of the aminopyridopyrazinone class of PDE5 inhibitors. These efforts led to the discovery of 3-[(trans-4-hydroxycyclohexyl)amino]-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one, a potent and selective inhibitor of PDE5 with an excellent PK profile.