NewGen Therapeutics (United States)

Abstract To better understand molecular mechanisms underlying psychiatric disorders, we need improved analytic approaches for integrating large-scale genomic data with brain-based transcriptomics. However, one critical component of individual variation - different levels of gene regulation due to genetic ancestry diversity - has not been traditionally incorporated into such analyses. To address this, we leveraged the ancestral diversity of individuals in adult and developing brain Genotype-Expression (GEx) reference panels of the PsychENCODE Project and psychiatric GWAS of the Psychiatric Genomics Consortium to enhance detection of GReX (Genetically Regulated gene expression) and transcriptome-wide association study (TWAS) signals. To investigate alternative GEx-level choices, we trained GReX models using rigorously constructed subsets of the human postmortem dorsolateral prefrontal cortex GEx panel, generated through downsampling, segregating, and mixing samples of Admixed African (AA) and European (EUR) ancestries, while considering disease status in the subset design. TWAS-based gene trait associations (GTAs) were obtained by integrating these GReX models with ancestry-specific GWASs of bipolar disorder (BIP), major depressive disorder (MDD), post-traumatic stress disorder (PTSD) and schizophrenia (SCZ). Genes with ancestry-specific GReX were enriched in specialized pathways involving mitochondrial functions, organelle structure, and metabolism, while genes with GReX in both ancestries demonstrated high concordance (>95%) in predictor SNP weight directions. Applying GReX from either ancestry to a single ancestry GWAS produced GTAs with concordant effects sizes while each uncovering unique FDR significant trait associated signals at the gene and pathway levels. GTAs based on AA GWAS meta analyzed with GTAs based on EUR GWAS enhanced signals and alleviated noise. EUR-specific GReX produced TWAS pathways that included corticosteroid signaling in PTSD, TGF-beta and neurotrophins in MDD, and inflammation and viral life cycle in SCZ. AA-specific GReX produced TWAS pathways that included glutamine signaling in PTSD, proline-peptide DNA activity in MDD, and immune cytotoxicity, serotoninergic and dopaminergic pathways in SCZ. Genes with ancestry-specific GReX in adult and developing brain were part of similar pathways. Finally, the developing brain TWAS were enriched in specialized developmental and neuronal pathways and produced a higher proportion of shared signals between ancestries. In conclusion, we demonstrate the benefits of leveraging diverse ancestral backgrounds in TWAS analysis, provide insights into which genes and pathways are better captured by ancestry-specific panels, and advocate for genomic region-specific TWAS integration strategies over a uniform genome-wide approach to uncover molecular mechanisms.

The Triggering Receptor Expressed on Myeloid Cells-2 (TREM2), a pivotal innate immune receptor, orchestrates functions such as inflammatory responses, phagocytosis, cell survival, and neuroprotection. TREM2 variants R47H and R62H have been associated with Alzheimer's disease, yet the underlying mechanisms remain elusive. Our previous research established that TREM2 binds to heparan sulfate (HS) and variants R47H and R62H exhibit reduced affinity for HS. Building upon this groundwork, our current study delves into the interplay between TREM2 and HS and its impact on microglial function. We confirm TREM2's binding to cell surface HS and demonstrate that TREM2 interacts with HS, forming HS-TREM2 binary complexes on microglia cell surfaces. Employing various biochemical techniques, including Surface Plasmon Resonance, low molecular weight HS microarray screening, and serial HS mutant cell surface binding assays, we demonstrate TREM2's robust affinity for HS, and the effective binding requires a minimum HS size of approximately 10 saccharide units. Notably, TREM2 selectively binds specific HS structures, with 6-O-sulfation and, to a lesser extent, the iduronic acid residue playing crucial roles. N-sulfation and 2-O-sulfation are dispensable for this interaction. Furthermore, we reveal that 6-O-sulfation is essential for HS-TREM2 ternary complex formation on the microglial cell surface, and HS and its 6-O-sulfation are necessary for TREM2-mediated ApoE3 uptake in microglia. By delineating the interaction between HS and TREM2 on the microglial cell surface and demonstrating its role in facilitating TREM2-mediated ApoE uptake by microglia, our findings provide valuable insights that can inform targeted interventions for modulating microglial functions in Alzheimer's disease.

Cathepsin K (CtsK) is a cysteine protease with potent collagenase activity. CtsK is highly expressed by bone-resorbing osteoclasts and plays an essential role in bone remodeling. Although CtsK is known to bind heparan sulfate (HS), the structural details of the interaction, and how HS ultimately regulates the biological functions of CtsK, remains largely unknown. In this report, we determined that CtsK preferably binds to larger HS oligosaccharides, such as dodecasaccharides (12mer), and that the12mer can induce monomeric CtsK to form a stable dimer in solution. Interestingly, while HS has no effect on the peptidase activity of CtsK, it greatly inhibits the collagenase activity of CtsK in a manner dependent on sulfation level. By forming a complex with CtsK, HS was able to preserve the full peptidase activity of CtsK for prolonged periods, likely by stabilizing its active conformation. Crystal structures of Ctsk with a bound 12mer, alone and in the presence of the endogenous inhibitor cystatin-C reveal the location of HS binding is remote from the active site. Mutagenesis based on these complex structures identified 6 basic residues of Ctsk that play essential roles in mediating HS-binding. At last, we show that HS 12mers can effectively block osteoclast resorption of bone in vitro. Combined, we have shown that HS can function as a multifaceted regulator of CtsK and that HS-based oligosaccharide might be explored as a new class of selective CtsK inhibitor in many diseases that involve exaggerated bone resorption.

Burkholderia pseudomallei is the causative agent of melioidosis, which is increasingly being reported worldwide. Mortality rates as high as 40% have been reported based on clinical patient outcomes in the endemic areas of Australia and Thailand. Novel therapies are needed to reduce treatment duration and adverse effects and improve treatment outcomes. Epetraborole, a novel antibiotic, targets leucyl-tRNA synthetase (LeuRS), an essential enzyme that catalyzes the attachment of leucine to transfer RNA. Epetraborole was evaluated for in vitro activity and efficacy in a murine model to assess clinical relevance against Burkholderia pseudomallei infections for possible treatment of melioidosis. Epetraborole was tested against 13 clinically derived and three reference B. pseudomallei strains that have a broad spectrum of susceptibilities to the standard-of-care (SoC) drugs for melioidosis, which showed that epetraborole exhibited minimal inhibitory concentrations of 0.25–4 μg/mL. Ex vivo studies using THP-1 macrophages confirmed the potency of epetraborole and demonstrated synergy between epetraborole and ceftazidime. In the acute pulmonary murine infection model of melioidosis, epetraborole demonstrated equivalent efficacy when delivered orally or subcutaneously, which compared well with the standard-of-care drug ceftazidime. In addition, adding epetraborole to ceftazidime significantly improved antimicrobial activity in this animal model. This work warrants further exploration of epetraborole as a candidate for treating melioidosis and substantiates LeuRS as a clinically relevant drug target in B. pseudomallei.

The efficacy and safety of chemotherapy are two major challenges when it comes to treating ovarian cancer. The associated undesirable side effects of chemotherapy agents jeopardize the clinical intent and the efficiency of the therapy. Multiple studies have been published describing new developments and novel strategies utilizing the latest therapeutic and drug delivery technologies to address the efficacy and safety of chemotherapeutics in ovarian cancers. We have identified five novel technologies that are available and, if used, have the potential to mitigate the above-mentioned challenges. Nanocarriers in different forms (Nano-gel, Aptamer, peptide medicated formulations, Antibody-drug conjugation, surface charge, and nanovesicle technologies) are developed and available to be employed to target the cancerous tissue. These strategies are promising to improve clinical efficacy and reduce side effects. We have systematically searched and analyzed published data, as well as the authors intent for the described technology on each publication. We narrowed to 81 key articles and extracted their data to be discussed in this review. In summary, the selected articles investigated the pharmacokinetic properties of drugs combined with nanocarriers and found significant improvement in efficacy and safety by reducing the IC50 values and drug doses. These key papers described promising novel technologies in anti-cancer therapeutic approaches to enable sustained drug release and achieve prolonged drug performance near the tumor site or target tissue.

e13132 Background: Luminal B breast cancer makes up between 10-20% of cases and is among the most lethal forms of breast cancer. Prognosis is little better than that of the notorious Triple Negative disease. There is currently no effective treatment for Luminal B breast cancer. Recently it has been found that, although mutations in the RAS oncogene are rare in Luminal B, hyper activation of the wild type RAS protein is common due to inactivation of key negative regulators of RAS such as DAB2IP and RASAL. Therefore, targeting RAS is a logical novel approach to developing enhanced therapy for this disease. There are three main isoforms of RAS and there are no currently approved pan-RAS inhibitors. We have developed a small molecule pan-RAS inhibitor which binds directly to all forms of RAS. It acts to block the ability of RAS and to bind and signal through its effectors by disrupting the RAS effector domain. It is active in vitro and in vivo against models of Luminal B breast cancer. Methods: In silico screening of a virtual compound library was performed to identify an initial candidate inhibitor. Iterative rounds of Medicinal Chemistry informed by structural modeling and bioassay in 3D vs 2D growth assays were performed to identify an enhanced activity derivative. Target binding was confirmed by Microscale Thermophoresis and NMR. RAS/RAF complex status was measured by co-immunoprecipitation of the endogenous proteins. Ras signaling was assayed by Western analysis of Phospho-ERK and RAL-GTP pull down assays. In vitro activity was measured using 3D soft agar assays. In vivo activity was measured by ip or po administration against a Luminal B breast cancer cell line. Results: The current lead, designated RAS-F binds all three main isoforms of RAS at low uM kd and modulates the RAS effector domain in NMR studies. It suppresses RAS signaling pathways in transient assays and suppresses Luminal B tumor cell tumor cell growth in soft agar and in vivo. Conclusions: We have developed a pre-clinical pan-RAS inhibitor that is active in vitro and in vivo against Luminal B breast cancer models. The agent also has potential uses against other tumor types partially driven by deregulated wild type RAS such as triple negative breast cancer.

Literature has well-established the importance of 3-O-sulfation of neuronal cell surface glycan heparan sulfate (HS) to its interaction with herpes simplex virus type 1 glycoprotein D (gD). Previous investigations of gD to its viral receptors HVEM and nectin-1 also highlighted the conformational dynamics of gD's N- and C-termini, necessary for viral membrane fusion. However, little is known on the structural interactions of gD with HS. Here, we present our findings on this interface from both the glycan and the protein perspective. We used C-terminal and N-terminal gD variants to probe the role of their respective regions in gD/HS binding. The N-terminal truncation mutants (with Δ1-22) demonstrate equivalent or stronger binding to heparin than their intact glycoproteins, indicating that the first 22 amino acids are disposable for heparin binding. Characterization of the conformational differences between C-terminal truncated mutants by sedimentation velocity analytical ultracentrifugation distinguished between the "open" and "closed" conformations of the glycoprotein D, highlighting the region's modulation of receptor binding. From the glycan perspective, we investigated gD interacting with heparin, heparan sulfate, and other de-sulfated and chemically defined oligosaccharides using surface plasmon resonance and glycan microarray. The results show a strong preference of gD for 6-O-sulfate, with 2-O-sulfation becoming more important in the presence of 6-O-S. Additionally, 3-O-sulfation shifted the chain length preference of gD from longer chain to mid-chain length, reaffirming the sulfation site's importance to the gD/HS interface. Our results shed new light on the molecular details of one of seven known protein-glycan interactions with 3-O-sulfated heparan sulfate.

Abstract Amyotrophic lateral sclerosis (ALS) is an inexorably progressive and degenerative disorder of motor neurons with no currently-known cure. Studies to determine the mechanism of neurotoxicity and the impact of ALS-linked mutations (SOD1, FUS, TARDP, C9ORF72, PFN1, TUBA4A and others) have greatly expanded our knowledge of ALS disease mechanisms and have helped to identify potential targets for ALS therapy. Cellular pathologies (e.g., aggregation of mutant forms of SOD1, TDP43, FUS, Ubiqulin2, PFN1, and C9ORF72), mitochondrial dysfunction, neuro­inflammation, and oxidative damage are major pathways implicated in ALS. Nevertheless, the selective vulnerability of motor neurons remains unexplained. The importance of tubulins for long-axon infrastructure, and the special morphology and function of motor neurons, underscore the central role of the cytoskeleton. The recent linkage of mutations to the tubulin α chain, TUBA4A, to familial and sporadic cases of ALS provides a new investigative opportunity to shed light on both mechanisms of ALS and the vulnerability of motor neurons. In the current study we investigate TUBA4A, a structural microtubule protein with mutations causal to familial ALS, using molecular-dynamic (MD) modeling of protein structure to predict the effects of each mutation and its overall impact on GTP binding, chain stability, tubulin assembly, and aggregation propensity. These studies predict that each of the reported mutations will cause notable structural changes to the TUBA4A (α chain) tertiary protein structure, adversely affecting its physical properties and function. Molecular docking and MD simulations indicate certain α chain mutations (e.g. K430N, R215C, and W407X) will cause structural deviations that impair GTP binding, and may prevent tubulin polymerization. Furthermore, several mutations (including R320C and K430N) confer a significant increase in predicted aggregation propensity of TUBA4A mutants relative to wild-type. Taken together, these in silico modeling studies revealed structural perturbations and disruption of GTP binding, culminating in failure to form the tubulin heterocomplex, which may account for an important mechanism and a trigger in initiation of motor neuron degeneration in ALS.