Joseph M. Ready

Discovery of a Proneurogenic, Neuroprotective Chemical

An in vivo screen was performed in search of chemicals capable of enhancing neuron formation in the hippocampus of adult mice. Eight of 1000 small molecules tested enhanced neuron formation in the subgranular zone of the dentate gyrus. Among these was an aminopropyl carbazole, designated P7C3, endowed with favorable pharmacological properties. In vivo studies gave evidence that P7C3 exerts its proneurogenic activity by protecting newborn neurons from apoptosis. Mice missing the gene encoding neuronal PAS domain protein 3 (NPAS3) are devoid of hippocampal neurogenesis and display malformation and electrophysiological dysfunction of the dentate gyrus. Prolonged administration of P7C3 to npas3(-/-) mice corrected these deficits by normalizing levels of apoptosis of newborn hippocampal neurons. Prolonged administration of P7C3 to aged rats also enhanced neurogenesis in the dentate gyrus, impeded neuron death, and preserved cognitive capacity as a function of terminal aging. PAPERCLIP:

Development of Proneurogenic, Neuroprotective Small Molecules

Degeneration of the hippocampus is associated with Alzheimers disease and occurs very early in the progression of the disease. Current options for treating the cognitive symptoms associated with Alzheimers are inadequate, giving urgency to the search for novel therapeutic strategies. Pharmacologic agents that safely enhance hippocampal neurogenesis may provide new therapeutic approaches. We discovered the first synthetic molecule, named P7C3, which protects newborn neurons from apoptotic cell death, and thus promotes neurogenesis in mice and rats in the subgranular zone of the hippocampal dentate gyrus, the site of normal neurogenesis in adult mammals. We describe the results of a medicinal chemistry campaign to optimize the potency, toxicity profile, and stability of P7C3. Systematic variation of nearly every position of the lead compound revealed elements conducive toward increases in activity and regions subject to modification. We have discovered compounds that are orally available, nontoxic, stable in mice, rats, and cell culture, and capable of penetrating the blood-brain barrier. The most potent compounds are active at nanomolar concentrations. Finally, we have identified derivatives that may facilitate mode-of-action studies through affinity chromatography or photo-cross-linking.

Evolution of a Synthetic Strategy : Total Synthesis of (±)-Welwitindolinone A Isonitrile

An efficient and highly stereoselective total synthesis of the natural product (+/-)-welwitindolinone A isonitrile (1) is described. The bicyclo[4.2.0]octane core of 1 was established by a regio- and diastereoselective [2+2] ketene cycloaddition. The C12 quaternary center and vicinal stereogenic chlorine were installed in a single operation with excellent stereocontrol via a chloronium ion mediated semipinacol rearrangement. Described strategies for construction of the spiro-oxinole include a SmI2-LiCl mediated reductive cyclization and a novel anionic cyclization that simultaneously constructs the spiro-oxindole and vinyl isonitrile moieties.

P7C3 Neuroprotective Chemicals Function by Activating the Rate-Limiting Enzyme in NAD Salvage

The P7C3 class of aminopropyl carbazole chemicals fosters the survival of neurons in a variety of rodent models of neurodegeneration or nerve cell injury. To uncover its mechanism of action, an active derivative of P7C3 was modified to contain both a benzophenone for photocrosslinking and an alkyne for CLICK chemistry. This derivative was found to bind nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme involved in the conversion of nicotinamide into nicotinamide adenine dinucleotide (NAD). Administration of active P7C3 chemicals to cells treated with doxorubicin, which induces NAD depletion, led to a rebound in intracellular levels of NAD and concomitant protection from doxorubicin-mediated toxicity. Active P7C3 variants likewise enhanced the activity of the purified NAMPT enzyme, providing further evidence that they act by increasing NAD levels through its NAMPT-mediated salvage.

Neuroprotective efficacy of aminopropyl carbazoles in a mouse model of Parkinson disease

We previously reported the discovery of P7C3, an aminopropyl carbazole having proneurogenic and neuroprotective properties in newborn neural precursor cells of the dentate gyrus. Here, we provide evidence that P7C3 also protects mature neurons in brain regions outside of the hippocampus. P7C3 blocks 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated cell death of dopaminergic neurons in the substantia nigra of adult mice, a model of Parkinson disease (PD). Dose–response studies show that the P7C3 analog P7C3A20 blocks cell death with even greater potency and efficacy, which parallels the relative potency and efficacy of these agents in blocking apoptosis of newborn neural precursor cells of the dentate gyrus. P7C3 and P7C3A20 display similar relative effects in blocking 1-methyl-4-phenylpyridinium (MPP+)-mediated death of dopaminergic neurons in Caenorhabditis elegans, as well as in preserving C. elegans mobility following MPP+ exposure. Dimebon, an antihistaminergic drug that is weakly proneurogenic and neuroprotective in the dentate gyrus, confers no protection in either the mouse or the worm models of PD. We further demonstrate that the hippocampal proneurogenic efficacy of eight additional analogs of P7C3 correlates with their protective efficacy in MPTP-mediated neurotoxicity. In vivo screening of P7C3 analogs for proneurogenic efficacy in the hippocampus may thus provide a reliable means of predicting neuroprotective efficacy. We propose that the chemical scaffold represented by P7C3 and P7C3A20 provides a basis for optimizing and advancing pharmacologic agents for the treatment of patients with PD.

Neuroprotective efficacy of aminopropyl carbazoles in a mouse model of amyotrophic lateral sclerosis

We previously reported the discovery of P7C3, an aminopropyl carbazole having proneurogenic and neuroprotective properties in newborn neural precursor cells of the hippocampal dentate gyrus. We have further found that chemicals having efficacy in this in vivo screening assay also protect dopaminergic neurons of the substantia nigra following exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a mouse model of Parkinson disease. Here, we provide evidence that an active analog of P7C3, known as P7C3A20, protects ventral horn spinal cord motor neurons from cell death in the G93A-SOD1 mutant mouse model of amyotrophic lateral sclerosis (ALS). P7C3A20 is efficacious in this model when administered at disease onset, and protection from cell death correlates with preservation of motor function in assays of walking gait and in the accelerating rotarod test. The prototypical member of this series, P7C3, delays disease progression in G93A-SOD1 mice when administration is initiated substantially earlier than the expected time of symptom onset. Dimebon, an antihistaminergic drug with significantly weaker proneurogenic and neuroprotective efficacy than P7C3, confers no protection in this ALS model. We propose that the chemical scaffold represented by P7C3 and P7C3A20 may provide a basis for the discovery and optimization of pharmacologic agents for the treatment of ALS.

The P7C3 class of neuroprotective compounds exerts antidepressant efficacy in mice by increasing hippocampal neurogenesis

Augmenting hippocampal neurogenesis represents a potential new strategy for treating depression. Here we test this possibility by comparing hippocampal neurogenesis in depression-prone ghrelin receptor (Ghsr)-null mice to that in wild-type littermates and by determining the antidepressant efficacy of the P7C3 class of neuroprotective compounds. Exposure of Ghsr-null mice to chronic social defeat stress (CSDS) elicits more severe depressive-like behavior than in CSDS-exposed wild-type littermates, and exposure of Ghsr-null mice to 60% caloric restriction fails to elicit antidepressant-like behavior. CSDS resulted in more severely reduced cell proliferation and survival in the ventral dentate gyrus (DG) subgranular zone of Ghsr-null mice than in that of wild-type littermates. Also, caloric restriction increased apoptosis of DG subgranular zone cells in Ghsr-null mice, although it had the opposite effect in wild-type littermates. Systemic treatment with P7C3 during CSDS increased survival of proliferating DG cells, which ultimately developed into mature (NeuN+) neurons. Notably, P7C3 exerted a potent antidepressant-like effect in Ghsr-null mice exposed to either CSDS or caloric restriction, while the more highly active analog P7C3-A20 also exerted an antidepressant-like effect in wild-type littermates. Focal ablation of hippocampal stem cells with radiation eliminated this antidepressant effect, further attributing the P7C3 class antidepressant effect to its neuroprotective properties and resultant augmentation of hippocampal neurogenesis. Finally, P7C3-A20 demonstrated greater proneurogenic efficacy than a wide spectrum of currently marketed antidepressant drugs. Taken together, our data confirm the role of aberrant hippocampal neurogenesis in the etiology of depression and suggest that the neuroprotective P7C3-compounds represent a novel strategy for treating patients with this disease.

Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration

A shot in the arm for damaged tissue Tissue damage can be caused by injury, disease, and even certain medical treatments. There is great interest in identifying drugs that accelerate tissue regeneration and recovery, especially drugs that might benefit multiple organ systems. Zhang et al. describe a compound with this desired activity, at least in mice (see the Perspective by FitzGerald). SW033291 promotes recovery of the hematopoietic system after bone marrow transplantation, prevents the development of ulcerative colitis in the intestine, and accelerates liver regeneration after hepatic surgery. It acts by inhibiting an enzyme that degrades prostaglandins, lipid signaling molecules that have been implicated in tissue stem cell maintenance. Science, this issue 10.1126/science.aaa2340; see also p. 1208 A compound that inhibits prostaglandin degradation enhances tissue regeneration in multiple organs in mice. [Also see Perspective by FitzGerald] INTRODUCTION Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. To date, therapeutic interventions have largely focused on targeting two PGE2 biosynthetic enzymes, cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2), with the aim of reducing PGE2 production. In this study, we take the converse approach: We examine the role of a prostaglandin-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), as a negative regulator of tissue repair, and we explore whether inhibition of this enzyme can potentiate tissue regeneration in mouse models. RATIONALE We used 15-PGDH knockout mice to elucidate the role of 15-PGDH in regulating tissue levels of PGE2 and tissue repair capacity in multiple organs. We then developed SW033291, a potent small-molecule inhibitor of 15-PGDH with activity in vivo. We used SW033291 to investigate the therapeutic potential of 15-PGDH inhibitors in tissue regeneration and to identify a 15-PGDH–regulated hematopoietic pathway within the bone marrow niche. RESULTS We found that in comparison with wild-type mice, 15-PGDH–deficient mice display a twofold increase in PGE2 levels across multiple tissues—including bone marrow, colon, and liver—and that they show increased fitness of these tissues in response to damage. The mutant mice also show enhanced hematopoietic capacity, with increased neutrophils, increased bone marrow SKL (Sca-1+ C-kit+ Lin−) cells (enriched for stem cells), and greater capacity to generate erythroid and myeloid colonies in cell culture. The 15-PGDH–deficient mice respond to colon injury from dextran sulfate sodium (DSS) with a twofold increase in cell proliferation in colon crypts, which confers resistance to DSS-induced colitis. The mutant mice also respond to partial hepatectomy with a greater than twofold increase in hepatocyte proliferation, which leads to accelerated and more extensive liver regeneration. SW033291, a potent small-molecule inhibitor of 15-PGDH (inhibitor dissociation constant Ki ~0.1 nM), recapitulates in mice the phenotypes of 15-PGDH gene knockout, inducing increased hematopoiesis, resistance to DSS colitis, and more rapid liver regeneration after partial hepatectomy. Moreover, SW033291-treated mice show a 6-day-faster reconstitution of hematopoiesis after bone marrow transplantation, with accelerated recovery of neutrophils, platelets, and erythrocytes, and greater recovery of bone marrow SKL cells. This effect is mediated by bone marrow CD45– cells, which respond to increased PGE2 with a fourfold increase in production of CXCL12 and SCF, two cytokines that play key roles in hematopoietic stem cell homing and maintenance. CONCLUSIONS Studying mouse models, we have shown that 15-PGDH negatively regulates tissue regeneration and repair in the bone marrow, colon, and liver. Of most direct utility, our observations identify 15-PGDH as a therapeutic target and provide a chemical formulation, SW033291, that is an active 15-PGDH inhibitor in vivo and that potentiates repair in multiple tissues. SW033291 or related compounds may merit clinical investigation as a strategy to accelerate recovery after bone marrow transplantation and other tissue injuries. Inhibiting 15-PGDH accelerates tissue repair. (A) The enzyme 15-PGDH degrades and negatively regulates PGE2. (B) SW033291 inhibits 15-PGDH, increases tissue levels of PGE2, and induces CXCL12 and SCF expression from CD45– bone marrow cells. This in turn accelerates homing of transplanted hematopoietic stem cells (HSC), generation of mature blood elements, and post-transplant recovery of normal blood counts. Inhibiting 15-PGDH similarly stimulates cell proliferation after injury to colon or liver, accelerating repair of these tissues. Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. Here, we show that inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme, potentiates tissue regeneration in multiple organs in mice. In a chemical screen, we identify a small-molecule inhibitor of 15-PGDH (SW033291) that increases prostaglandin PGE2 levels in bone marrow and other tissues. SW033291 accelerates hematopoietic recovery in mice receiving a bone marrow transplant. The same compound also promotes tissue regeneration in mouse models of colon and liver injury. Tissues from 15-PGDH knockout mice demonstrate similar increased regenerative capacity. Thus, 15-PGDH inhibition may be a valuable therapeutic strategy for tissue regeneration in diverse clinical contexts.

Chiral Allene-Containing Phosphines in Asymmetric Catalysis

We demonstrate that allenes, chiral 1,2-dienes, appended with basic functionality can serve as ligands for transition metals. We describe an allene-containing bisphosphine that, when coordinated to Rh(I), promotes the asymmetric addition of arylboronic acids to α-keto esters with high enantioselectivity. Solution and solid-state structural analysis reveals that one olefin of the allene can coordinate to transition metals, generating bi- and tridentate ligands.

Allenes in Asymmetric Catalysis. Asymmetric Ring-Opening of Meso-Epoxides Catalyzed by Allene-Containing Phosphine Oxides

Unsymmetrically substituted allenes (1,2-dienes) are inherently chiral and can be prepared in optically pure form. Nonetheless, to date the allene framework has not been incorporated into ligands for asymmetric catalysis. Since allenes project functionality differently than either tetrahedral carbon or chiral biaryls, they may create complementary chiral environments. This study demonstrates that optically active, C(2)-symmetric allene-containing bisphosphine oxides can catalyze the addition of SiCl(4) to meso-epoxides with high enantioselectivity. The epoxide opening likely involves generation of a Lewis acidic, cationic (bisphosphine oxide)SiCl(3) complex. The fact that high asymmetric induction is observed suggests that allenes may represent a new platform for the development of ligands and catalysts for asymmetric synthesis.