Changes in parrot diversity after human arrival to the Caribbean.

Humans did not arrive on most of the world's islands until relatively recently, making islands favorable places for disentangling the timing and magnitude of natural and anthropogenic impacts on species diversity and distributions. Here, we focus on Amazona parrots in the Caribbean, which have close relationships with humans (e.g., as pets as well as sources of meat and colorful feathers). Caribbean parrots also have substantial fossil and archaeological records that span the Holocene. We leverage this exemplary record to showcase how combining ancient and modern DNA, along with radiometric dating, can shed light on diversification and extinction dynamics and answer long-standing questions about the magnitude of human impacts in the region. Our results reveal a striking loss of parrot diversity, much of which took place during human occupation of the islands. The most widespread species, the Cuban Parrot, exhibits interisland divergences throughout the Pleistocene. Within this radiation, we identified an extinct, genetically distinct lineage that survived on the Turks and Caicos until Indigenous human settlement of the islands. We also found that the narrowly distributed Hispaniolan Parrot had a natural range that once included The Bahamas; it thus became "endemic" to Hispaniola during the late Holocene. The Hispaniolan Parrot also likely was introduced by Indigenous people to Grand Turk and Montserrat, two islands where it is now also extirpated. Our research demonstrates that genetic information spanning paleontological, archaeological, and modern contexts is essential to understand the role of humans in altering the diversity and distribution of biota.

Structural Analysis and Development of Notum Fragment Screening Hits.

The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer's disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 µM. Analysis of the fragments' diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 µM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.

Virtual Screening Directly Identifies New Fragment-Sized Inhibitors of Carboxylesterase Notum with Nanomolar Activity.

Notum is a negative regulator of Wnt signaling acting through the hydrolysis of a palmitoleoylate ester, which is required for Wnt activity. Inhibitors of Notum could be of use in diseases where dysfunctional Notum activity is an underlying cause. A docking-based virtual screen (VS) of a large commercial library was used to shortlist 952 compounds for experimental validation as inhibitors of Notum. The VS was successful with 31 compounds having an IC50 < 500 nM. A critical selection process was then applied with two clusters and two singletons (1-4d) selected for hit validation. Optimization of 4d guided by structural biology identified potent inhibitors of Notum activity that restored Wnt/ß-catenin signaling in cell-based models. The [1,2,4]triazolo[4,3-b]pyradizin-3(2H)-one series 4 represent a new chemical class of Notum inhibitors and the first to be discovered by a VS campaign. These results demonstrate the value of VS with well-designed docking models based on X-ray structures.

Structural Insights into Notum Covalent Inhibition.

The carboxylesterase Notum hydrolyzes a palmitoleate moiety from Wingless/Integrated(Wnt) ligands and deactivates Wnt signaling. Notum inhibitors can restore Wnt signaling which may be of therapeutic benefit for pathologies such as osteoporosis and Alzheimer's disease. We report the identification of a novel class of covalent Notum inhibitors, 4-(indolin-1-yl)-4-oxobutanoate esters. High-resolution crystal structures of the Notum inhibitor complexes reveal a common covalent adduct formed between the nucleophile serine-232 and hydrolyzed butyric esters. The covalent interaction in solution was confirmed by mass spectrometry analysis. Inhibitory potencies vary depending on the warheads used. Mechanistically, the resulting acyl-enzyme intermediate carbonyl atom is positioned at an unfavorable angle for the approach of the active site water, which, combined with strong hydrophobic interactions with the enzyme pocket residues, hinders the intermediate from being further processed and results in covalent inhibition. These insights into Notum catalytic inhibition may guide development of more potent Notum inhibitors.

Carboxylesterase Notum Is a Druggable Target to Modulate Wnt Signaling.

Regulation of the Wnt signaling pathway is critically important for a number of cellular processes in both development and adult mammalian biology. This Perspective will provide a summary of current and emerging therapeutic opportunities in modulating Wnt signaling, especially through inhibition of Notum carboxylesterase activity. Notum was recently shown to act as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group. Inhibition of Notum activity may represent a new approach to treat disease where aberrant Notum activity has been identified as the underlying cause. Reliable screening technologies are available to identify inhibitors of Notum, and structural studies are accelerating the discovery of new inhibitors. A selection of these hits have been optimized to give fit-for-purpose small molecule inhibitors of Notum. Three noteworthy examples are LP-922056 (26), ABC99 (27), and ARUK3001185 (28), which are complementary chemical tools for exploring the role of Notum in Wnt signaling.

Ancient DNA from the extinct Haitian cave-rail (Nesotrochis steganinos) suggests a biogeographic connection between the Caribbean and Old World.

Worldwide decline in biodiversity during the Holocene has impeded a comprehensive understanding of pre-human biodiversity and biogeography. This is especially true on islands, because many recently extinct island taxa were morphologically unique, complicating assessment of their evolutionary relationships using morphology alone. The Caribbean remains an avian hotspot but was more diverse before human arrival in the Holocene. Among the recently extinct lineages is the enigmatic genus Nesotrochis, comprising three flightless species. Based on morphology, Nesotrochis has been considered an aberrant rail (Rallidae) or related to flufftails (Sarothruridae). We recovered a nearly complete mitochondrial genome of Nesotrochis steganinos from fossils, discovering that it is not a rallid but instead is sister to Sarothruridae, volant birds now restricted to Africa and New Guinea, and the recently extinct, flightless Aptornithidae of New Zealand. This result suggests a widespread or highly dispersive most recent common ancestor of the group. Prior to human settlement, the Caribbean avifauna had a far more cosmopolitan origin than is evident from extant species.

Ancient mitogenomics elucidates diversity of extinct West Indian tortoises.

We present 10 nearly complete mitochondrial genomes of the extinct tortoise Chelonoidis alburyorum from the Bahamas. While our samples represent morphologically distinct populations from six islands, their genetic divergences were shallow and resembled those among Galápagos tortoises. Our molecular clock estimates revealed that divergence among Bahamian tortoises began ~ 1.5 mya, whereas divergence among the Galápagos tortoises (C. niger complex) began ~ 2 mya. The inter-island divergences of tortoises from within the Bahamas and within the Galápagos Islands are much younger (0.09-0.59 mya, and 0.08-1.43 mya, respectively) than the genetic differentiation between any other congeneric pair of tortoise species. The shallow mitochondrial divergences of the two radiations on the Bahamas and the Galápagos Islands suggest that each archipelago sustained only one species of tortoise, and that the taxa currently regarded as distinct species in the Galápagos should be returned to subspecies status. The extinct tortoises from the Bahamas have two well-supported clades: the first includes one sample from Great Abaco and two from Crooked Island; the second clade includes tortoises from Great Abaco, Eleuthera, Crooked Island, Mayaguana, Middle Caicos, and Grand Turk. Tortoises belonging to both clades on Great Abaco and Crooked Island suggest late Holocene inter-island transport by prehistoric humans.

Bird populations and species lost to Late Quaternary environmental change and human impact in the Bahamas.

Comparing distributional information derived from fossils with the modern distribution of species, we summarize the changing bird communities of the Bahamian Archipelago across deep ecological time. While our entire dataset consists of 7,600+ identified fossils from 32 sites on 15 islands (recording 137 species of resident and migratory birds), we focus on the landbirds from four islands with the best fossil records, three from the Late Pleistocene (∼25 to 10 ka [1,000 y ago]) and one from the Holocene (∼10 to 0 ka). The Late Pleistocene sites feature 51 resident species that have lost one or more Bahamian populations; 29 of these species do not occur in any of the younger Holocene sites (or in the Bahamas today). Of these 29 species, 17 have their closest affinities to species now or formerly living in Cuba and/or North America. A set of 27 species of landbirds, most of them extant somewhere today, was more widespread in the Bahamas in the prehistoric Holocene (∼10 to 0.5 ka) than they are today; 16 of these 27 species were recorded as Pleistocene fossils as well. No single site adequately captures the entire landbird fauna of the combined focal islands. Information from all sites is required to assess changes in Bahamian biodiversity (including endemism) since the Late Pleistocene. The Bahamian islands are smaller, flatter, lower, and more biotically depauperate than the Greater Antilles, resulting in more vulnerable bird communities.

A new genus and species of pigeon (Aves, Columbidae) from the Kingdom of Tonga, with an evaluation of hindlimb osteology of columbids from Oceania.

The region from New Guinea through Oceania sustains the world's most diverse set of columbids. We describe osteological characters of the hindlimb (femur, tibiotarsus, tarsometatarsus) that divide the Papuan-Oceanic pigeons and doves into three groups based on functional morphology: "arboreal" (Hemiphaga, Ducula, Ptilinopus, Drepanoptila, Gymnophaps), "intermediate" (Columba, Macropygia, Reinwardtoena), and "terrestrial" (Gallicolumba [includes Alopecoenas], Trugon, Microgoura, Goura, Chalcophaps, Geopelia, Henicophaps, Caloenas, Didunculus, Otidiphaps). The arboreal and terrestrial groups are each distinctive osteologically, especially in the tibiotarsus and tarsometatarsus, which are short relative to the femur in the arboreal group, and long relative to the femur in the terrestrial group. The intermediate pigeons are more similar to arboreal than to terrestrial pigeons, but nonetheless fit in neither group. To estimate the phylogenetic relationships among or within these three groups is somewhat tentative using hindlimb osteology alone, although all five genera of arboreal pigeons have independent molecular evidence of relatedness, as do most of the genera of terrestrial pigeons.                Using the hindlimb and other osteological data as a framework, we describe a new extinct genus and species of pigeon, Tongoenas burleyi, from Holocene archaeological and Pleistocene paleontological sites on six islands (Foa, Lifuka, `Uiha, Ha`afeva, Tongatapu, and `Eua) in the Kingdom of Tonga. Tongoenas was a large-sized member of the "arboreal" pigeon group, with osteological characters that relate it to Ducula, Gymnophaps, and Hemiphaga (generally canopy frugivores) rather than with the "terrestrial" pigeons (more ground-dwelling and granivorous) such as Gallicolumba, Trugon, Microgoura, Goura, etc. (others listed above). Among volant columbids, living or extinct, only the species of Goura (from New Guinea) are larger than Tongoenas. From most of the same prehistoric sites, we also report new material of the nearly as large, extinct pigeon Ducula shutleri Worthy Burley, recently described from islands in the Vava`u Group of Tonga. Thus, D. shutleri also was widespread in Tonga before human impact. The prehistoric anthropogenic loss in Tonga of Tongoenas burleyi, Ducula shutleri, and other columbids undoubtedly had a negative impact on the dispersal regimes of Tongan forest trees. At first human contact about 2850 years ago, at least nine species of columbids in six genera inhabited the Tongan islands, where only four species in three genera exist today.

Screening of a Custom-Designed Acid Fragment Library Identifies 1-Phenylpyrroles and 1-Phenylpyrrolidines as Inhibitors of Notum Carboxylesterase Activity.

The Wnt family of proteins are secreted signaling proteins that play key roles in regulating cellular functions. Recently, carboxylesterase Notum was shown to act as a negative regulator of Wnt signaling by mediating the removal of an essential palmitoleate. Here we disclose two new chemical scaffolds that inhibit Notum enzymatic activity. Our approach was to create a fragment library of 250 acids for screening against Notum in a biochemical assay followed by structure determination by X-ray crystallography. Twenty fragments were identified as hits for Notum inhibition, and 14 of these fragments were shown to bind in the palmitoleate pocket of Notum. Optimization of 1-phenylpyrrole 20, guided by structure-based drug design, identified 20z as the most potent compound from this series. Similarly, the optimization of 1-phenylpyrrolidine 8 gave acid 26. This work demonstrates that inhibition of Notum activity can be achieved by small, drug-like molecules possessing favorable in vitro ADME profiles.

Ancient DNA and high-resolution chronometry reveal a long-term human role in the historical diversity and biogeography of the Bahamian hutia.

Quaternary paleontological and archaeological evidence often is crucial for uncovering the historical mechanisms shaping modern diversity and distributions. We take an interdisciplinary approach using multiple lines of evidence to understand how past human activity has shaped long-term animal diversity in an island system. Islands afford unique opportunities for such studies given their robust fossil and archaeological records. Herein, we examine the only non-volant terrestrial mammal endemic to the Bahamian Archipelago, the hutia Geocapromys ingrahami. This capromyine rodent once inhabited many islands but is now restricted to several small cays. Radiocarbon dated fossils indicate that hutias were present on the Great Bahama Bank islands before humans arrived at AD ~800-1000; all dates from other islands post-date human arrival. Using ancient DNA from a subset of these fossils, along with modern representatives of Bahamian hutia and related taxa, we develop a fossil-calibrated phylogeny. We found little genetic divergence among individuals from within either the northern or southern Bahamas but discovered a relatively deep North-South divergence (~750 ka). This result, combined with radiocarbon dating and archaeological evidence, reveals a pre-human biogeographic divergence, and an unexpected human role in shaping Bahamian hutia diversity and biogeography across islands.

Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α-hydroxyketones.

A narrow substrate range is a major limitation in exploiting enzymes more widely as catalysts in synthetic organic chemistry. For enzymes using two substrates, the simultaneous optimisation of both substrate specificities is also required for the rapid expansion of accepted substrates. Transketolase (TK) catalyses the reversible transfer of a C2 -ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for industrial applications. Herein, TK from Escherichia coli was engineered to accept both pyruvate, as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3-formylbenzoic acid (FBA). Twenty single-mutant variants were first designed and characterised experimentally. Beneficial mutations were then recombined to construct a small library. Screening of this library identified the best variant with a 9.2-fold improvement in the yield towards pyruvate and propionaldehyde, relative to wild-type (WT). Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% enantiomeric excess (ee) for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3-FBA. The best variant was able to convert 47% of substrate into product within 24 h, whereas no conversion was observed for WT. Docking experiments suggested a cooperation between the mutations responsible for donor and acceptor recognition, which would promote the activity towards both the acceptor and donor. The variants obtained have the potential to be used for developing catalytic pathways to a diverse range of high-value products.

Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors.

The carboxylesterase Notum is a key negative regulator of the Wnt signaling pathway by mediating the depalmitoleoylation of Wnt proteins. Our objective was to discover potent small molecule inhibitors of Notum suitable for exploring the regulation of Wnt signaling in the central nervous system. Scaffold-hopping from thienopyrimidine acids 1 and 2, supported by X-ray structure determination, identified 3-methylimidazolin-4-one amides 20-24 as potent inhibitors of Notum with activity across three orthogonal assay formats (biochemical, extra-cellular, occupancy). A preferred example 24 demonstrated good stability in mouse microsomes and plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated efflux. Pharmacokinetic studies with 24 were performed in vivo in mouse with single oral administration of 24 showing good plasma exposure and reasonable CNS penetration. We propose that 24 is a new chemical tool suitable for cellular studies to explore the fundamental biology of Notum.

An improved, scalable synthesis of Notum inhibitor LP-922056 using 1-chloro-1,2-benziodoxol-3-one as a superior electrophilic chlorinating agent.

Background: The carboxylesterase Notum has been shown to act as a key negative regulator of the Wnt signalling pathway by mediating the depalmitoleoylation of Wnt proteins. LP-922056 (1) is an orally active inhibitor of Notum. We are investigating the role of Notum in modulating Wnt signalling in the central nervous system and wished to establish if 1 would serve as a peripherally restricted control. An accessible and improved synthetic route would allow 1 to become more readily available as a chemical tool to explore the fundamental biology of Notum and build target validation to underpin new drug discovery programs. Results: An improved, scalable synthesis of 1 is reported. Key modifications include: (1) the introduction of the C7-cyclopropyl group was most effectively achieved with a Suzuki-Miyaura cross-coupling reaction with MIDA-boronate 11 (5 → 6), and (2) C6 chlorination was performed with 1-chloro-1,2-benziodoxol-3-one (12) (6 → 7) as a mild and selective electrophilic chlorination agent. This 7-step route from 16 has been reliably performed on large scale to produce multigram quantities of 1 in good efficiency and high purity. Pharmacokinetic studies in mouse showed CNS penetration of 1 is very low with a brain/plasma concentration ratio of just 0.01. A small library of amides 17 were prepared from acid 1 to explore if 1 could be modified to deliver a CNS penetrant tool by capping off the acid as an amide. Although significant Notum inhibition activity could be achieved, none of these amides demonstrated the required combination of metabolic stability along with cell permeability without evidence of P-gp mediated efflux. Conclusion: Mouse pharmacokinetic studies demonstrate that 1 is unsuitable for use in models of disease where brain penetration is an essential requirement of the compound but would be an ideal peripherally restricted control. These data will contribute to the understanding of drug levels of 1 to overlay with appropriate in vivo efficacy endpoints, i.e., the PK-PD relationship. The identification of a suitable analogue of 1 (or 17) which combines Notum inhibition with CNS penetration would be a valuable chemical probe for investigating the role of Notum in disease models.

Discovery of 2-phenoxyacetamides as inhibitors of the Wnt-depalmitoleating enzyme NOTUM from an X-ray fragment screen.

NOTUM is a carboxylesterase that has been shown to act by mediating the O-depalmitoleoylation of Wnt proteins resulting in suppression of Wnt signaling. Here, we describe the development of NOTUM inhibitors that restore Wnt signaling for use in in vitro disease models where NOTUM over activity is an underlying cause. A crystallographic fragment screen with NOTUM identified 2-phenoxyacetamide 3 as binding in the palmitoleate pocket with modest inhibition activity (IC50 33 µM). Optimization of hit 3 by SAR studies guided by SBDD identified indazole 38 (IC50 0.032 µM) and isoquinoline 45 (IC50 0.085 µM) as potent inhibitors of NOTUM. The binding of 45 to NOTUM was rationalized through an X-ray co-crystal structure determination which showed a flipped binding orientation compared to 3. However, it was not possible to combine NOTUM inhibition activity with metabolic stability as the majority of the compounds tested were rapidly metabolized in an NADPH-independent manner.

Ancient DNA from a 2,500-year-old Caribbean fossil places an extinct bird (Caracara creightoni) in a phylogenetic context.

Since the late Pleistocene humans have caused the extinction of species across our planet. Placing these extinct species in the tree of life with genetic data is essential to understanding the ecological and evolutionary implications of these losses. While ancient DNA (aDNA) techniques have advanced rapidly in recent decades, aDNA from tropical species, especially birds, has been historically difficult to obtain, leaving a gap in our knowledge of the extinction processes that have influenced current distributions and biodiversity. Here we report the recovery of a nearly complete mitochondrial genome from a 2,500 year old (late Holocene) bone of an extinct species of bird, Caracara creightoni, recovered from the anoxic saltwater environment of a blue hole in the Bahamas. Our results suggest that this extinct species is sister (1.6% sequence divergence) to a clade containing the extant C. cheriway and C. plancus. Caracara creightoni shared a common ancestor with these extant species during the Pleistocene (1.2-0.4 MYA) and presumably survived on Cuba when the Bahamas was mostly underwater during Quaternary interglacial intervals (periods of high sea levels). Tropical blue holes have been collecting animals for thousands of years and will continue to improve our understanding of faunal extinctions and distributions. In particular, new aDNA techniques combined with radiocarbon dating from Holocene Bahamian fossils will allow us to place other extinct (species-level loss) and extirpated (population-level loss) vertebrate taxa in improved phylogenetic, evolutionary, biogeographic, and temporal contexts.

New approaches for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent chronic neurodegenerative disease. Current approved therapies are symptomatic treatments having some effect on cognitive function. Therapies that target ß-amyloid (Aß) have been the focus of efforts to develop a disease modification treatment for AD but these approaches have failed to show any clinical benefit so far. Beyond the 'Aß hypothesis', there are a number of newer approaches to treat AD with neuroinflammation emerging as a very active area of research based on risk gene analysis. This short review will summarize approved drug therapies, recent clinical trials and new approaches for the treatment of AD.

Another new species of flightless Rail (Aves: Rallidae: Rallus) from Abaco, The Bahamas.

We describe a late Pleistocene species of extinct rail, Rallus gracilipes n. sp., from Sawmill Sink blue hole on Abaco Island, Little Bahama Bank, The Bahamas. The only other extinct rail known from any Bahamian island is the smaller Rallus cyanocavi, also from late Pleistocene contexts at Sawmill Sink. No fossils of R. gracilipes or R. cyanocavi have been found in Holocene sites on Abaco; the loss of both of these species is likely to be due to changes in climate, habitat, and island area during the Pleistocene-Holocene Transition.

Small Molecule Neuropilin-1 Antagonists Combine Antiangiogenic and Antitumor Activity with Immune Modulation through Reduction of Transforming Growth Factor Beta (TGFß) Production in Regulatory T-Cells.

We report the design, synthesis, and biological evaluation of some potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumors, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229. The design of these molecules was informed and supported by X-ray crystal structures. Compound 1 (EG01377) was identified as having properties suitable for further investigation. Compound 1 was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, 1 was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1+, FoxP3+, and CD25+ populations of Tregs from mice, 1 was able to block a glioma-conditioned medium-induced increase in TGFß production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies.