Structural basis of substrate recognition and catalysis by fucosyltransferase 8.

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80-2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

Intrinsic apoptosis circumvents the functional decline of circulating platelets but does not cause the storage lesion.

The circulating life span of blood platelets is regulated by the prosurvival protein BCL-XL It restrains the activity of BAK and BAX, the essential prodeath mediators of intrinsic apoptosis. Disabling the platelet intrinsic apoptotic pathway in mice by deleting BAK and BAX results in a doubling of platelet life span and concomitant thrombocytosis. Apoptotic platelets expose phosphatidylserine (PS) via a mechanism that is distinct from that driven by classical agonists. Whether there is any role for apoptotic PS in platelet function in vivo, however, is unclear. Apoptosis has also been associated with the platelet storage lesion (PSL), the constellation of biochemical deteriorations that occur during blood bank storage. In this study, we investigated the role of BAK/BAX-mediated apoptosis in hemostasis and thrombosis and in the development of the PSL. We show that although intrinsic apoptosis is rapidly induced during storage at 37°C, it is not detected when platelets are kept at the standard storage temperature of 22°C. Remarkably, loss of BAK and BAX did not prevent the development of the PSL at either temperature. BAK/BAX-deficient mice exhibited increased bleeding times and unstable thrombus formation. This phenotype was not caused by impaired PS exposure, but was associated with a defect in granule release from aged platelets. Strikingly, rejuvenation of BAK/BAX-deficient platelets in vivo completely rescued the observed hemostatic defects. Thus, apoptotic culling of old platelets from the bloodstream is essential to maintain a functional, hemostatically reactive platelet population. Inhibiting intrinsic apoptosis in blood banked platelets is unlikely to yield significant benefit.

Exploration of the P3 region of PEXEL peptidomimetics leads to a potent inhibitor of the Plasmodium protease, plasmepsin V.

The use of arginine isosteres is a known strategy to overcome poor membrane permeability commonly associated with peptides or peptidomimetics that possess this highly polar amino acid. Here, we apply this strategy to peptidomimetics that are potent inhibitors of the malarial protease, plasmepsin V, with the aim of enhancing their activity against Plasmodium parasites, and exploring the structure-activity relationship of the P3 arginine within the S3 pocket of plasmepsin V. Of the arginine isosteres trialled in the P3 position, we discovered that canavanine was the ideal and that this peptidomimetic potently inhibits plasmepsin V, efficiently blocks protein export and inhibits parasite growth. Structure studies of the peptidomimetics bound to plasmepsin V provided insight into the structural basis for the enzyme activity observed in vitro and provides further evidence why plasmepsin V is highly sensitive to substrate modification.

Caspase-9 mediates the apoptotic death of megakaryocytes and platelets, but is dispensable for their generation and function.

Apoptotic caspases, including caspase-9, are thought to facilitate platelet shedding by megakaryocytes. They are known to be activated during platelet apoptosis, and have also been implicated in platelet hemostatic responses. However, the precise requirement for, and the regulation of, apoptotic caspases have never been defined in either megakaryocytes or platelets. To establish the role of caspases in platelet production and function, we generated mice lacking caspase-9 in their hematopoietic system. We demonstrate that both megakaryocytes and platelets possess a functional apoptotic caspase cascade downstream of Bcl-2 family-mediated mitochondrial damage. Caspase-9 is the initiator caspase, and its loss blocks effector caspase activation. Surprisingly, steady-state thrombopoiesis is unperturbed in the absence of caspase-9, indicating that the apoptotic caspase cascade is not required for platelet production. In platelets, loss of caspase-9 confers resistance to the BH3 mimetic ABT-737, blocking phosphatidylserine (PS) exposure and delaying ABT-737-induced thrombocytopenia in vivo. Despite this, steady-state platelet lifespan is normal. Casp9(-/-) platelets are fully capable of physiologic hemostatic responses and functional regulation of adhesive integrins in response to agonist. These studies demonstrate that the apoptotic caspase cascade is required for the efficient death of megakaryocytes and platelets, but is dispensable for their generation and function.

Activity refinement of aryl amino acetamides that target the P. falciparum STAR-related lipid transfer 1 protein.

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.

Bcl-xL-inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets.

BH3 mimetics are a new class of proapo-ptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the pro-survival function of one or more Bcl-2-related proteins. Agents that inhibit Bcl-x(L) induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time- and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ibα and glycoprotein VI, and functional down-regulation of integrin α(IIb)ß(3). Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a time-dependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time. Overall, these studies demonstrate that Bcl-x(L)-inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets.

7-N-Substituted-3-oxadiazole Quinolones with Potent Antimalarial Activity Target the Cytochrome bc1 Complex.

The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.

Optimization of 2,3-Dihydroquinazolinone-3-carboxamides as Antimalarials Targeting PfATP4.

There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na+ homeostasis and affect parasite pH, exhibited a fast-to-moderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.

Substrate Peptidomimetic Inhibitors of P. falciparum Plasmepsin X with Potent Antimalarial Activity.

Plasmepsin X (PMX) is an aspartyl protease that processes proteins essential for Plasmodium parasites to invade and egress from host erythrocytes during the symptomatic asexual stage of malaria. PMX substrates possess a conserved cleavage region denoted by the consensus motif, SFhE (h=hydrophobic amino acid). Peptidomimetics reflecting the P3 -P1 positions of the consensus motif were designed and showed potent and selective inhibition of PMX. It was established that PMX prefers Phe in the P1 position, di-substitution at the ß-carbon of the P2 moiety and a hydrophobic P3 group which was supported by modelling of the peptidomimetics in complex with PMX. The peptidomimetics were shown to arrest asexual P. falciparum parasites at the schizont stage by impairing PMX substrate processing. Overall, the peptidomimetics described will assist in further understanding PMX substrate specificity and have the potential to act as a template for future antimalarial design.

The Invention of WM382, a Highly Potent PMIX/X Dual Inhibitor toward the Treatment of Malaria.

Drug resistance to first-line antimalarials-including artemisinin-is increasing, resulting in a critical need for the discovery of new agents with novel mechanisms of action. In collaboration with the Walter and Eliza Hall Institute and with funding from the Wellcome Trust, a phenotypic screen of Merck's aspartyl protease inhibitor library identified a series of plasmepsin X (PMX) hits that were more potent than chloroquine. Inspired by a PMX homology model, efforts to optimize the potency resulted in the discovery of leads that, in addition to potently inhibiting PMX, also inhibit another essential aspartic protease, plasmepsin IX (PMIX). Further potency and pharmacokinetic profile optimization efforts culminated in the discovery of WM382, a very potent dual PMIX/X inhibitor with robust in vivo efficacy at multiple stages of the malaria parasite life cycle and an excellent resistance profile.

Insights Into Drug Repurposing, as Well as Specificity and Compound Properties of Piperidine-Based SARS-CoV-2 PLpro Inhibitors.

The COVID-19 pandemic continues unabated, emphasizing the need for additional antiviral treatment options to prevent hospitalization and death of patients infected with SARS-CoV-2. The papain-like protease (PLpro) domain is part of the SARS-CoV-2 non-structural protein (nsp)-3, and represents an essential protease and validated drug target for preventing viral replication. PLpro moonlights as a deubiquitinating (DUB) and deISGylating enzyme, enabling adaptation of a DUB high throughput (HTS) screen to identify PLpro inhibitors. Drug repurposing has been a major focus through the COVID-19 pandemic as it may provide a fast and efficient route for identifying clinic-ready, safe-in-human antivirals. We here report our effort to identify PLpro inhibitors by screening the ReFRAME library of 11,804 compounds, showing that none inhibit PLpro with any reasonable activity or specificity to justify further progression towards the clinic. We also report our latest efforts to improve piperidine-scaffold inhibitors, 5c and 3k, originally developed for SARS-CoV PLpro. We report molecular details of binding and selectivity, as well as in vitro absorption, distribution, metabolism and excretion (ADME) studies of this scaffold. A co-crystal structure of SARS-CoV-2 PLpro bound to inhibitor 3k guides medicinal chemistry efforts to improve binding and ADME characteristics. We arrive at compounds with improved and favorable solubility and stability characteristics that are tested for inhibiting viral replication. Whilst still requiring significant improvement, our optimized small molecule inhibitors of PLpro display decent antiviral activity in an in vitro SARS-CoV-2 infection model, justifying further optimization.

Translocation of sphingosine kinase 1 to the plasma membrane is mediated by calcium- and integrin-binding protein 1.

SK1 (sphingosine kinase 1) plays an important role in many aspects of cellular regulation. Most notably, elevated cellular SK1 activity leads to increased cell proliferation, protection from apoptosis, and induction of neoplastic transformation. We have previously shown that translocation of SK1 from the cytoplasm to the plasma membrane is integral for oncogenesis mediated by this enzyme. The molecular mechanism mediating this translocation of SK1 has remained undefined. Here, we demonstrate a direct role for CIB1 (calcium and integrin-binding protein 1) in this process. We show that CIB1 interacts with SK1 in a Ca(2+)-dependent manner at the previously identified "calmodulin-binding site" of SK1. We also demonstrate that CIB1 functions as a Ca(2+)-myristoyl switch, providing a mechanism whereby it translocates SK1 to the plasma membrane. Both small interfering RNA knockdown of CIB1 and the use of a dominant-negative CIB1 we have generated prevent the agonist-dependent translocation of SK1. Furthermore, we demonstrate the requirement of CIB1-mediated translocation of SK1 in controlling cellular sphingosine 1-phosphate generation and associated anti-apoptotic signaling.

Optimization of Benzothiazole and Thiazole Hydrazones as Inhibitors of Schistosome BCL-2.

Limited therapeutic options are available for the treatment of human schistosomiasis caused by the parasitic Schistosoma flatworm. The B cell lymphoma-2 (BCL-2)-regulated apoptotic cell death pathway in schistosomes was recently characterized and shown to share similarities with the intrinsic apoptosis pathway in humans. Here, we exploit structural differences in the human and schistosome BCL-2 (sBCL-2) pro-survival proteins toward a novel treatment strategy for schistosomiasis. The benzothiazole hydrazone scaffold previously employed to target human BCL-XL was repurposed as a starting point to target sBCL-2. We utilized X-ray structural data to inform optimization and then applied a scaffold-hop strategy to identify the 5-carboxamide thiazole hydrazone scaffold (43) with potent sBCL-2 activity (IC50 30 nM). Human BCL-XL potency (IC50 13 nM) was inadvertently preserved during the optimization process. The lead analogues from this study exhibit on-target activity in model fibroblast cell lines dependent on either sBCL-2 or human BCL-XL for survival. Further optimization of the thiazole hydrazone class is required to exhibit activity in schistosomes and enhance the potential of this strategy for treating schistosomiasis.

Development and application of a high-throughput screening assay for identification of small molecule inhibitors of the P. falciparum reticulocyte binding-like homologue 5 protein.

The P. falciparum parasite, responsible for the disease in humans known as malaria, must invade erythrocytes to provide an environment for self-replication and survival. For invasion to occur, the parasite must engage several ligands on the host erythrocyte surface to enable adhesion, tight junction formation and entry. Critical interactions include binding of erythrocyte binding-like ligands and reticulocyte binding-like homologues (Rhs) to the surface of the host erythrocyte. The reticulocyte binding-like homologue 5 (Rh5) is the only member of this family that is essential for invasion and it binds to the basigin host receptor. The essential nature of Rh5 makes it an important vaccine target, however to date, Rh5 has not been targeted by small molecule intervention. Here, we describe the development of a high-throughput screening assay to identify small molecules which interfere with the Rh5-basigin interaction. To validate the utility of this assay we screened a known drug library and the Medicines for Malaria Box and demonstrated the reproducibility and robustness of the assay for high-throughput screening purposes. The screen of the known drug library identified the known leukotriene antagonist, pranlukast. We used pranlukast as a model inhibitor in a post screening evaluation cascade. We procured and synthesised analogues of pranlukast to assist in the hit confirmation process and show which structural moieties of pranlukast attenuate the Rh5 - basigin interaction. Evaluation of pranlukast analogues against P. falciparum in a viability assay and a schizont rupture assay show the parasite activity was not consistent with the biochemical inhibition of Rh5, questioning the developability of pranlukast as an antimalarial. The high-throughput assay developed from this work has the capacity to screen large collections of small molecules to discover inhibitors of P. falciparum Rh5 for future development of invasion inhibitory antimalarials.

Optimization of 5-substituted thiazolyl ureas and 6-substituted imidazopyridines as potential HIV-1 latency reversing agents.

A persistent latent reservoir of virus in CD4+ T cells is a major barrier to cure HIV. Activating viral transcription in latently infected cells using small molecules is one strategy being explored to eliminate latency. We previously described the use of a FlpIn.FM HEK293 cellular assay to identify and then optimize the 2-acylaminothiazole class to exhibit modest activation of HIV gene expression. Here, we implement two strategies to further improve the activation of viral gene expression and physicochemical properties of this class. Firstly, we explored rigidification of the central oxy-carbon linker with a variety of saturated heterocycles, and secondly, investigated bioisosteric replacement of the 2-acylaminothiazole moiety. The optimization process afforded lead compounds (74 and 91) from the 2-piperazinyl thiazolyl urea and the imidazopyridine class. The lead compounds from each class demonstrate potent activation of HIV gene expression in the FlpIn.FM HEK293 cellular assay (both with LTR EC50s of 80 nM) and in the Jurkat Latency 10.6 cell model (LTR EC50 220 and 320 nM respectively), but consequently activate gene expression non-specifically in the FlpIn.FM HEK293 cellular assay (CMV EC50 70 and 270 nM respectively) manifesting in cellular cytotoxicity. The lead compounds have potential for further development as novel latency reversing agents.

Clinical MDR1 inhibitors enhance Smac-mimetic bioavailability to kill murine LSCs and improve survival in AML models.

The specific targeting of inhibitor of apoptosis (IAP) proteins by Smac-mimetic (SM) drugs, such as birinapant, has been tested in clinical trials of acute myeloid leukemia (AML) and certain solid cancers. Despite their promising safety profile, SMs have had variable and limited success. Using a library of more than 5700 bioactive compounds, we screened for approaches that could sensitize AML cells to birinapant and identified multidrug resistance protein 1 inhibitors (MDR1i) as a class of clinically approved drugs that can enhance the efficacy of SM therapy. Genetic or pharmacological inhibition of MDR1 increased intracellular levels of birinapant and sensitized AML cells from leukemia murine models, human leukemia cell lines, and primary AML samples to killing by birinapant. The combination of clinical MDR1 and IAP inhibitors was well tolerated in vivo and more effective against leukemic cells, compared with normal hematopoietic progenitors. Importantly, birinapant combined with third-generation MDR1i effectively killed murine leukemic stem cells (LSCs) and prolonged survival of AML-burdened mice, suggesting a therapeutic opportunity for AML. This study identified a drug combination strategy that, by efficiently killing LSCs, may have the potential to improve outcomes in patients with AML.

Discovery of Acylsulfonohydrazide-Derived Inhibitors of the Lysine Acetyltransferase, KAT6A, as Potent Senescence-Inducing Anti-Cancer Agents.

A high-throughput screen designed to discover new inhibitors of histone acetyltransferase KAT6A uncovered CTX-0124143 (1), a unique aryl acylsulfonohydrazide with an IC50 of 1.0 µM. Using this acylsulfonohydrazide as a template, we herein disclose the results of our extensive structure-activity relationship investigations, which resulted in the discovery of advanced compounds such as 55 and 80. These two compounds represent significant improvements on our recently reported prototypical lead WM-8014 (3) as they are not only equivalently potent as inhibitors of KAT6A but are less lipophilic and significantly more stable to microsomal degradation. Furthermore, during this process, we discovered a distinct structural subclass that contains key 2-fluorobenzenesulfonyl and phenylpyridine motifs, culminating in the discovery of WM-1119 (4). This compound is a highly potent KAT6A inhibitor (IC50 = 6.3 nM; KD = 0.002 µM), competes with Ac-CoA by binding to the Ac-CoA binding site, and has an oral bioavailability of 56% in rats.

A high-throughput screen to identify novel synthetic lethal compounds for the treatment of E-cadherin-deficient cells.

The cell-cell adhesion protein E-cadherin (CDH1) is a tumor suppressor that is required to maintain cell adhesion, cell polarity and cell survival signalling. Somatic mutations in CDH1 are common in diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In addition, germline mutations in CDH1 predispose to the autosomal dominant cancer syndrome Hereditary Diffuse Gastric Cancer (HDGC). One approach to target cells with mutations in specific tumor suppressor genes is synthetic lethality. To identify novel synthetic lethal compounds for the treatment of cancers associated with E-cadherin loss, we have undertaken a high-throughput screening campaign of ~114,000 lead-like compounds on an isogenic pair of human mammary epithelial cell lines - with and without CDH1 expression. This unbiased approach identified 12 novel compounds that preferentially harmed E-cadherin-deficient cells. Validation of these compounds using both real-time and end-point viability assays identified two novel compounds with significant synthetic lethal activity, thereby demonstrating that E-cadherin loss creates druggable vulnerabilities within tumor cells. In summary, we have identified novel synthetic lethal compounds that may provide a new strategy for the prevention and treatment of both sporadic and hereditary LBC and DGC.

Identification of 5-Substituted 2-Acylaminothiazoles That Activate Tat-Mediated Transcription in HIV-1 Latency Models.

The persistent reservoir of cells latently infected with human immunodeficiency virus (HIV)-integrated proviral DNA necessitates lifelong suppressive antiretroviral therapy (ART). Epigenetic targeted compounds have shown promise as potential latency-reversing agents; however, these drugs have undesirable toxicity and lack specificity for HIV. We utilized a novel HEK293-derived FlpIn dual-reporter cell line, which quantifies specific HIV provirus reactivation (LTR promoter) relative to nonspecific host cell gene expression (CMV promoter), to identify the 5-substituted 2-acylaminothiazole hit class. Here, we describe the optimization of the hit class, defining the functionality necessary for HIV gene activation and for improving in vitro metabolism and solubility. The optimized compounds displayed enhanced HIV gene expression in HEK293 and Jurkat 10.6 latency cellular models and increased unspliced HIV RNA in resting CD4+ T cells isolated from HIV-infected individuals on ART, demonstrating the potential of the 2-acylaminothiazole class as latency-reversing agents.

Discovery of Benzoylsulfonohydrazides as Potent Inhibitors of the Histone Acetyltransferase KAT6A.

A high-throughput screen for inhibitors of the histone acetyltransferase, KAT6A, led to identification of an aryl sulfonohydrazide derivative (CTX-0124143) that inhibited KAT6A with an IC50 of 1.0 µM. Elaboration of the structure-activity relationship and medicinal chemistry optimization led to the discovery of WM-8014 (97), a highly potent inhibitor of KAT6A (IC50 = 0.008 µM). WM-8014 competes with acetyl-CoA (Ac-CoA), and X-ray crystallographic analysis demonstrated binding to the Ac-CoA binding site. Through inhibition of KAT6A activity, WM-8014 induces cellular senescence and represents a unique pharmacological tool.