DART.2: bidirectional synaptic pharmacology with thousandfold cellular specificity.

Precision pharmacology aims to manipulate specific cellular interactions within complex tissues. In this pursuit, we introduce DART.2 (drug acutely restricted by tethering), a second-generation cell-specific pharmacology technology. The core advance is optimized cellular specificity-up to 3,000-fold in 15 min-enabling the targeted delivery of even epileptogenic drugs without off-target effects. Additionally, we introduce brain-wide dosing methods as an alternative to local cannulation and tracer reagents for brain-wide dose quantification. We describe four pharmaceuticals-two that antagonize excitatory and inhibitory postsynaptic receptors, and two that allosterically potentiate these receptors. Their versatility is showcased across multiple mouse-brain regions, including cerebellum, striatum, visual cortex and retina. Finally, in the ventral tegmental area, we find that blocking inhibitory inputs to dopamine neurons accelerates locomotion, contrasting with previous optogenetic and pharmacological findings. Beyond enabling the bidirectional perturbation of chemical synapses, these reagents offer intersectional precision-between genetically defined postsynaptic cells and neurotransmitter-defined presynaptic partners.

Amide- and bis-amide-linked highly potent and broadly active antifungal agents for the treatment of invasive fungal infections- towards the discovery of pre-clinical development candidate FC12406.

Most fungal infections are common, localized to skin or mucosal surfaces and can be treated effectively with topical antifungal agents. However, while invasive fungal infections (IFIs) are uncommon, they are very difficult to control medically, and are associated with high mortality rates. We have previously described highly potent bis-guanidine-containing heteroaryl-linked antifungal agents, and were interested in expanding the range of agents to novel series so as to reduce the degree of aromaticity (with a view to making the compounds more drug-like), and provide broadly active high potency derivatives. We have investigated the replacement of the central aryl ring from our original series by both amide and a bis-amide moieties, and have found particular structure-activity relationships (SAR) for both series', resulting in highly active antifungal agents against both mold and yeast pathogens. In particular, we describe the in vitro antifungal activity, absorption, distribution, metabolism and elimination (ADME) properties, and off-target properties of FC12406 (34), which was selected as a pre-clinical development candidate.

Compound FC-10696 Inhibits Egress of Marburg Virus.

Marburg virus (MARV) VP40 protein (mVP40) directs egress and spread of MARV, in part, by recruiting specific host WW domain-containing proteins via its conserved PPxY late (L) domain motif to facilitate efficient virus-cell separation. We reported previously that small-molecule compounds targeting the viral PPxY/host WW domain interaction inhibited VP40-mediated egress and spread. Here, we report on the antiviral potency of novel compound FC-10696, which emerged from extensive structure-activity relationship (SAR) of a previously described series of PPxY inhibitors. We show that FC-10696 inhibits egress of mVP40 virus-like particles (VLPs) and egress of authentic MARV from HeLa cells and primary human macrophages. Moreover, FC-10696 treated-mice displayed delayed onset of weight loss and clinical signs and significantly lower viral loads compared to controls, with 14% of animals surviving 21 days following a lethal MARV challenge. Thus, FC-10696 represents a first-in-class, host-oriented inhibitor effectively targeting late stages of the MARV life cycle.

Synthesis and evaluation of potent novel inhibitors of human sulfide:quinone oxidoreductase.

Here we report the first small-molecule inhibitors of human sulfide:quinone oxidoreductase (SQOR) that decrease the rate of breakdown of hydrogen sulfide (H2S), a potent cardioprotective signaling molecule. SQOR is a mitochondrial membrane-bound protein that catalyzes a two-electron oxidation of H2S to sulfane sulfur (S0), using glutathione (or sulfite) and coenzyme Q (CoQ) as S0 and electron acceptor, respectively. Inhibition of SQOR may constitute a new approach for the treatment of heart failure with reduced ejection fraction. Starting from top hits identified in a high-throughput screen, we conducted SAR development guided by docking of lead candidates into our crystal structure of SQOR. We identified potent SQOR inhibitors such as 19 which has an IC50 of 29 nM for SQOR inhibition and favorable pharmacokinetic and ADME properties required for in vivo efficacy testing.

Highly potent, broadly active antifungal agents for the treatment of invasive fungal infections.

Invasive fungal infections have become an important healthcare issue due in large part to high mortality rates under standard of care (SOC) therapies creating an urgent need for new and effective anti-fungal agents. We have developed a series of non-peptide, structurally-constrained analogs of host defence proteins that have distinct advantages over peptides for pharmaceutical uses. Here we report the chemical optimization of bis-guanidine analogs focused on alterations of the central aryl core and the connection of it to the terminal guanidines. This effort resulted in the production of highly potent, broadly active compounds with low mammalian cell cytotoxicity that have comparable or improved antifungal activities over SOC agents. One optimal compound was also found to possess favourable in vitro pharmaceutical and off-target properties suitable for further development.

Biophysical Screens Identify Fragments That Bind to the Viral DNA-Binding Proteins EBNA1 and LANA.

The human gamma-herpesviruses Epstein-Barr virus (EBV) (HHV-4) and Kaposi's sarcoma-associated herpesvirus (KSHV) (HHV-8) are responsible for a number of diseases, including various types of cancer. Epstein-Barr nuclear antigen 1 (EBNA1) from EBV and latency-associated nuclear antigen (LANA) from KSHV are viral-encoded DNA-binding proteins that are essential for the replication and maintenance of their respective viral genomes during latent, oncogenic infection. As such, EBNA1 and LANA are attractive targets for the development of small-molecule inhibitors. To this end, we performed a biophysical screen of EBNA1 and LANA using a fragment library by saturation transfer difference (STD)-NMR spectroscopy and surface plasmon resonance (SPR). We identified and validated a number of unique fragment hits that bind to EBNA1 or LANA. We also determined the high-resolution crystal structure of one fragment bound to EBNA1. Results from this screening cascade provide new chemical starting points for the further development of potent inhibitors for this class of viral proteins.

Small Molecule Inhibitors of the BfrB-Bfd Interaction Decrease Pseudomonas aeruginosa Fitness and Potentiate Fluoroquinolone Activity.

The iron storage protein bacterioferritin (BfrB) is central to bacterial iron homeostasis. The mobilization of iron from BfrB, which requires binding by a cognate ferredoxin (Bfd), is essential to the regulation of cytosolic iron levels in P. aeruginosa. This paper describes the structure-guided development of small molecule inhibitors of the BfrB-Bfd protein-protein interaction. The process was initiated by screening a fragment library and followed by obtaining the structure of a fragment hit bound to BfrB. The structural insights were used to develop a series of 4-(benzylamino)- and 4-((3-phenylpropyl)amino)-isoindoline-1,3-dione analogs that selectively bind BfrB at the Bfd binding site. Challenging P. aeruginosa cells with the 4-substituted isoindoline analogs revealed a dose-dependent growth phenotype. Further investigation determined that the analogs elicit a pyoverdin hyperproduction phenotype that is consistent with blockade of the BfrB-Bfd interaction and ensuing irreversible accumulation of iron in BfrB, with concomitant depletion of iron in the cytosol. The irreversible accumulation of iron in BfrB prompted by the 4-substituted isoindoline analogs was confirmed by visualization of BfrB-iron in P. aeruginosa cell lysates separated on native PAGE gels and stained for iron with Ferene S. Challenging P. aeruginosa cultures with a combination of commercial fluoroquinolone and our isoindoline analogs results in significantly lower cell survival relative to treatment with either antibiotic or analog alone. Collectively, these findings furnish proof of concept for the usefulness of small molecule probes designed to dysregulate bacterial iron homeostasis by targeting a protein-protein interaction pivotal for iron storage in the bacterial cell.

Cypin: A novel target for traumatic brain injury.

Cytosolic PSD-95 interactor (cypin), the primary guanine deaminase in the brain, plays key roles in shaping neuronal circuits and regulating neuronal survival. Despite this pervasive role in neuronal function, the ability for cypin activity to affect recovery from acute brain injury is unknown. A key barrier in identifying the role of cypin in neurological recovery is the absence of pharmacological tools to manipulate cypin activity in vivo. Here, we use a small molecule screen to identify two activators and one inhibitor of cypin's guanine deaminase activity. The primary screen identified compounds that change the initial rate of guanine deamination using a colorimetric assay, and secondary screens included the ability of the compounds to protect neurons from NMDA-induced injury and NMDA-induced decreases in frequency and amplitude of miniature excitatory postsynaptic currents. Hippocampal neurons pretreated with activators preserved electrophysiological function and survival after NMDA-induced injury in vitro, while pretreatment with the inhibitor did not. The effects of the activators were abolished when cypin was knocked down. Administering either cypin activator directly into the brain one hour after traumatic brain injury significantly reduced fear conditioning deficits 5 days after injury, while delivering the cypin inhibitor did not improve outcome after TBI. Together, these data demonstrate that cypin activation is a novel approach for improving outcome after TBI and may provide a new pathway for reducing the deficits associated with TBI in patients.

A Method for C2 Acylation of 1,3-Indandiones.

The 1,3-indandione scaffold is an important structural motif used in the preparation of a large number of industrial chemical and pharmaceutical compounds. However, few approaches allow for the direct C2 acylation on these building blocks. A method was developed using DMAP and EDCI, which is mild in reactivity, covers a diverse range of carboxylic acid acylating agents, is compatible with electron releasing and withdrawing substituents on the 1,3-indandione partner, and performs well in a polar aprotic solvent (for solubility reasons) This method cleanly afforded twenty five different products in yields of 32-96%.

Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation.

RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). Here we identify Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. We find that dFMRP overexpression (dFMRP OE) mitigates TDP-43 dependent locomotor defects and reduced lifespan in Drosophila. TDP-43 and FMRP form a complex in flies and human cells. In motor neurons, TDP-43 expression increases the association of dFMRP with stress granules and colocalizes with polyA binding protein in a variant-dependent manner. Furthermore, dFMRP dosage modulates TDP-43 solubility and molecular mobility with overexpression of dFMRP resulting in a significant reduction of TDP-43 in the aggregate fraction. Polysome fractionation experiments indicate that dFMRP OE also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP OE mitigates Futsch-dependent morphological phenotypes at the neuromuscular junction including synaptic size and presence of satellite boutons. Our data suggest a model whereby dFMRP is neuroprotective by remodeling TDP-43 containing RNA granules, reducing aggregation and restoring the translation of specific mRNAs in motor neurons.

Calcium Regulation of Hemorrhagic Fever Virus Budding: Mechanistic Implications for Host-Oriented Therapeutic Intervention.

Hemorrhagic fever viruses, including the filoviruses (Ebola and Marburg) and arenaviruses (Lassa and Junín viruses), are serious human pathogens for which there are currently no FDA approved therapeutics or vaccines. Importantly, transmission of these viruses, and specifically late steps of budding, critically depend upon host cell machinery. Consequently, strategies which target these mechanisms represent potential targets for broad spectrum host oriented therapeutics. An important cellular signal implicated previously in EBOV budding is calcium. Indeed, host cell calcium signals are increasingly being recognized to play a role in steps of entry, replication, and transmission for a range of viruses, but if and how filoviruses and arenaviruses mobilize calcium and the precise stage of virus transmission regulated by calcium have not been defined. Here we demonstrate that expression of matrix proteins from both filoviruses and arenaviruses triggers an increase in host cytoplasmic Ca2+ concentration by a mechanism that requires host Orai1 channels. Furthermore, we demonstrate that Orai1 regulates both VLP and infectious filovirus and arenavirus production and spread. Notably, suppression of the protein that triggers Orai activation (Stromal Interaction Molecule 1, STIM1) and genetic inactivation or pharmacological blockade of Orai1 channels inhibits VLP and infectious virus egress. These findings are highly significant as they expand our understanding of host mechanisms that may broadly control enveloped RNA virus budding, and they establish Orai and STIM1 as novel targets for broad-spectrum host-oriented therapeutics to combat these emerging BSL-4 pathogens and potentially other enveloped RNA viruses that bud via similar mechanisms.

Quinoxaline-based inhibitors of Ebola and Marburg VP40 egress.

We prepared a series of quinoxalin-2-mercapto-acetyl-urea analogs and evaluated them for their ability to inhibit viral egress in our Marburg and Ebola VP40 VLP budding assays in HEK293T cells. We also evaluated selected compounds in our bimolecular complementation assay (BiMC) to detect and visualize a Marburg mVP40-Nedd4 interaction in live mammalian cells. Antiviral activity was assessed for selected compounds using a live recombinant vesicular stomatitis virus (VSV) (M40 virus) that expresses the EBOV VP40 PPxY L-domain. Finally selected compounds were evaluated in several ADME assays to have an early assessment of their drug properties. Our compounds had low nM potency in these assays (e.g., compounds 21, 24, 26, 39), and had good human liver microsome stability, as well as little or no inhibition of P450 3A4.

Synthesis and evaluation of orally active small molecule HIV-1 Nef antagonists.

The HIV-1 Nef accessory factor enhances viral replication and promotes immune system evasion of HIV-infected cells, making it an attractive target for drug discovery. Recently we described a novel class of diphenylpyrazolodiazene compounds that bind directly to Nef in vitro and inhibit Nef-dependent HIV-1 infectivity and replication in cell culture. However, these first-generation Nef antagonists have several structural liabilities, including an azo linkage that led to poor oral bioavailability. The azo group was therefore replaced with either a one- or two-carbon linker. The resulting set of non-azo analogs retained nanomolar binding affinity for Nef by surface plasmon resonance, while inhibiting HIV-1 replication with micromolar potency in cell-based assays without cytotoxicity. Computational docking studies show that these non-azo analogs occupy the same predicted binding site within the HIV-1 Nef dimer interface as the original azo compound. Computational methods also identified a hot spot for inhibitor binding within this site that is defined by conserved HIV-1 Nef residues Asp108, Leu112, and Pro122. Pharmacokinetic evaluation of the non-azo B9 analogs in mice showed that replacement of the azo linkage dramatically enhanced oral bioavailability without substantially affecting plasma half-life or clearance. The improved oral bioavailability of non-azo diphenylpyrazolo Nef antagonists provides a starting point for further drug lead optimization in support of future efficacy testing in animal models of HIV/AIDS.

Small-molecule probes targeting the viral PPxY-host Nedd4 interface block egress of a broad range of RNA viruses.

UNLABELLED: Budding of filoviruses, arenaviruses, and rhabdoviruses is facilitated by subversion of host proteins, such as Nedd4 E3 ubiquitin ligase, by viral PPxY late (L) budding domains expressed within the matrix proteins of these RNA viruses. As L domains are important for budding and are highly conserved in a wide array of RNA viruses, they represent potential broad-spectrum targets for the development of antiviral drugs. To identify potential competitive blockers, we used the known Nedd4 WW domain-PPxY interaction interface as the basis of an in silico screen. Using PPxY-dependent budding of Marburg (MARV) VP40 virus-like particles (VLPs) as our model system, we identified small-molecule hit 1 that inhibited Nedd4-PPxY interaction and PPxY-dependent budding. This lead candidate was subsequently improved with additional structure-activity relationship (SAR) analog testing which enhanced antibudding activity into the nanomolar range. Current lead compounds 4 and 5 exhibit on-target effects by specifically blocking the MARV VP40 PPxY-host Nedd4 interaction and subsequent PPxY-dependent egress of MARV VP40 VLPs. In addition, lead compounds 4 and 5 exhibited antibudding activity against Ebola and Lassa fever VLPs, as well as vesicular stomatitis and rabies viruses (VSV and RABV, respectively). These data provide target validation and suggest that inhibition of the PPxY-Nedd4 interaction can serve as the basis for the development of a novel class of broad-spectrum, host-oriented antivirals targeting viruses that depend on a functional PPxY L domain for efficient egress. IMPORTANCE: There is an urgent and unmet need for the development of safe and effective therapeutics against biodefense and high-priority pathogens, including filoviruses (Ebola and Marburg) and arenaviruses (e.g., Lassa and Junin) which cause severe hemorrhagic fever syndromes with high mortality rates. We along with others have established that efficient budding of filoviruses, arenaviruses, and other viruses is critically dependent on the subversion of host proteins. As disruption of virus budding would prevent virus dissemination, identification of small-molecule compounds that block these critical viral-host interactions should effectively block disease progression and transmission. Our findings provide validation for targeting these virus-host interactions as we have identified lead inhibitors with broad-spectrum antiviral activity. In addition, such inhibitors might prove useful for newly emerging RNA viruses for which no therapeutics would be available.

Acinetobacter baumannii OxPhos inhibitors as selective anti-infective agents.

The Gram-negative bacterium Acinetobacter baumannii is an opportunistic pathogen in humans and infections are poorly treated by current therapy. Recent emergence of multi-drug resistant strains and the lack of new antibiotics demand an immediate action for development of new anti-Acinetobacter agents. To this end, oxidative phosphorylation (OxPhos) was identified as a novel target for drug discovery research. Consequently, a library of ∼10,000 compounds was screened using a membrane-based ATP synthesis assay. One hit identified was the 2-iminobenzimidazole 1 that inhibited the OxPhos of A. baumannii with a modestly high selectivity against mitochondrial OxPhos, and displayed an MIC of 25µM (17µg/mL) against the pathogen. The 2-iminobenzimidazole 1 was found to inhibit the type 1 NADH-quinone oxidoreductase (NDH-1) of A. baumannii OxPhos by a biochemical approach. Among various derivatives that were synthesized to date, des-hydroxy analog 5 is among the most active with a relatively tight SAR requirement for the N'-aminoalkyl side chain. Analog 5 also showed less cytotoxicity against NIH3T3 and HepG2 mammalian cell lines, demonstrating the potential for this series of compounds as anti-Acinetobacter agents. Additional SAR development and target validation is underway.

A selective small molecule NOP (ORL-1 receptor) partial agonist for the treatment of anxiety.

Small molecule (1) has been identified as a selective partial agonist of Opioid Receptor Like-1 (ORL-1) with potential utility for the treatment of anxiety and other disorders. Nociceptin (orphanin FQ) is an endogenous peptide ligand that binds to ORL-1, however it does not bind the classical δ, µ and κ opioid receptors with high affinity. The synthesis of 1 involved using a molecular diversity approach, to rapidly advance a library of compounds for biological testing. A lead selective potent partial agonist (35-fold ORL-1/Mu) progressed to ORL-1 (NOP or OP4) proof of concept testing in advanced studies. The synthetic approach and biological data for the related chemical series will be presented.

Ubiquilin-2 (UBQLN2) binds with high affinity to the C-terminal region of TDP-43 and modulates TDP-43 levels in H4 cells: characterization of inhibition by nucleic acids and 4-aminoquinolines.

Recently, it was reported that mutations in the ubiquitin-like protein ubiquilin-2 (UBQLN2) are associated with X-linked amyotrophic lateral sclerosis (ALS), and that both wild-type and mutant UBQLN2 can co-localize with aggregates of C-terminal fragments of TAR DNA binding protein (TDP-43). Here, we describe a high affinity interaction between UBQLN2 and TDP-43 and demonstrate that overexpression of both UBQLN2 and TDP-43 reduces levels of both exogenous and endogenous TDP-43 in human H4 cells. UBQLN2 bound with high affinity to both full length TDP-43 and a C-terminal TDP-43 fragment (261-414 aa) with KD values of 6.2nM and 8.7nM, respectively. Both DNA oligonucleotides and 4-aminoquinolines, which bind to TDP-43, also inhibited UBQLN2 binding to TDP-43 with similar rank order affinities compared to inhibition of oligonucleotide binding to TDP-43. Inhibitor characterization experiments demonstrated that the DNA oligonucleotides noncompetitively inhibited UBQLN2 binding to TDP-43, which is consistent with UBQLN2 binding to the C-terminal region of TDP-43. Interestingly, the 4-aminoquinolines were competitive inhibitors of UBQLN2 binding to TDP-43, suggesting that these compounds also bind to the C-terminal region of TDP-43. In support of the biochemical data, co-immunoprecipitation experiments demonstrated that both TDP-43 and UBQLN2 interact in human neuroglioma H4 cells. Finally, overexpression of UBQLN2 in the presence of overexpressed full length TDP-43 or C-terminal TDP-43 (170-414) dramatically lowered levels of both full length TDP-43 and C-terminal TDP-43 fragments (CTFs). Consequently, these data suggest that UBQLN2 enhances the clearance of TDP-43 and TDP-43 CTFs and therefore may play a role in the development of TDP-43 associated neurotoxicity.

Anilino-monoindolylmaleimides as potent and selective JAK3 inhibitors.

We designed a series of anilino-indoylmaleimides based on structural elements from literature JAK3 inhibitors 3 and 4, and our lead 5. These new compounds were tested as inhibitors of JAKs 1, 2 and 3 and TYK2 for therapeutic intervention in rheumatoid arthritis (RA). Our requirements, based on current scientific rationale for optimum efficacy against RA with reduced side effects, was for potent, mixed JAK1 and 3 inhibition, and selectivity over JAK2. Our efforts yielded a potent JAK3 inhibitor 11d and its eutomer 11e. These compounds were highly selective for inhibition of JAK3 over JAK2 and TYK. The compounds displayed only modest JAK1 inhibition.

Intramolecular Rearrangement of α-Amino Acid Amide Derivatives of 2-Aminobenzothiazoles.

We have found that α-amino acid amide derivatives of 2-aminobenzothiazoles undergo a time-dependent, thermal rearrangement in which the amine group attacks the 2-position carbon of the thiazole ring to form a 5,5-spiro ring system. This is followed by sulfur leaving and air oxidation to the corresponding symmetrical disulfide. The isolated yields of such products are quite high (>70%) if there is conformational bias to further promote the intramolecular reaction such as for the 2-aminobenzothiazole amides derived from proline or 4-aminopiperidine-4-carboxylic acid. This rearrangement has not been described previously for α-amino acid amide derivatives of 2-aminobenzothiazoles. However, a related reaction involving 2-semicarbazido benzothiazoles has been recently reported.

PROTAC-mediated degradation of HIV-1 Nef efficiently restores cell-surface CD4 and MHC-I expression and blocks HIV-1 replication.

The HIV-1 Nef accessory factor enhances the viral life cycle in vivo, promotes immune escape of HIV-infected cells, and represents an attractive antiretroviral drug target. However, Nef lacks enzymatic activity and an active site, complicating traditional occupancy-based drug development. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef. Nef-binding compounds, based on an existing hydroxypyrazole core, were coupled to ligands for ubiquitin E3 ligases via flexible linkers. The resulting bivalent PROTACs induced formation of a ternary complex between Nef and the cereblon E3 ubiquitin ligase thalidomide-binding domain in vitro and triggered Nef degradation in a T cell expression system. Nef-directed PROTACs efficiently rescued Nef-mediated MHC-I and CD4 downregulation in T cells and suppressed HIV-1 replication in donor PBMCs. Targeted degradation is anticipated to reverse all HIV-1 Nef functions and may help restore adaptive immune responses against HIV-1 reservoir cells in vivo.