Identification of cell division cycle protein 20 in various forms of acute and chronic kidney injury in mice.

Tubular epithelial cell fate following exposure to various types of injurious stimuli can be decided at distinct cell cycle checkpoints. One such checkpoint occurs during mitosis, known as the spindle assembly checkpoint, and is tightly regulated through the actions of cell division cycle protein 20 (CDC20). Due to our paucity of knowledge about the role of CDC20 in the kidney, the present study was designed to investigate the expression levels and distribution of CDC20 within the kidney and how pharmacological inhibition of CDC20 function affects kidney recovery using various rodent models of kidney injury. CDC20 is normally detected in distal tubules, but upon injury by either cisplatin administration or ureter obstruction, CDC20 accumulation is considerably elevated. Blockade of CDC20 activity using a selective pharmacological inhibitor, Apcin, lowered serum creatinine, tubular damage, and DNA injury following acute kidney injury compared with vehicle-treated mice. In unilateral ureteral obstruction, Apcin reduced tissue kidney injury molecule-1 levels, sirius red staining, and tubulointerstitial α-smooth muscle actin staining in the tissue. The findings in the present study demonstrated that elevations in CDC20 levels in the kidney are associated with kidney injury and that inhibition of CDC20 can alleviate and reverse some of the pathological effects on the architecture and function of kidney.NEW & NOTEWORTHY To our knowledge, this is the first study to characterize the expression and localization of cell division cycle 20 protein (CDC20) in normal and acute, and chronically injured kidneys. Tubular epithelial cell damage was markedly reduced through the administration of a selective inhibitor of CDC20, Apcin. This study provides new evidence that CDC20 can be induced in damaged kidney cells and negatively impact the recovery of the kidney following acute kidney injury.

The similarity of class II HLA genotypes defines patterns of autoreactivity in idiopathic bone marrow failure disorders.

Idiopathic aplastic anemia (IAA) is a rare autoimmune bone marrow failure (BMF) disorder initiated by a human leukocyte antigen (HLA)-restricted T-cell response to unknown antigens. As in other autoimmune disorders, the predilection for certain HLA profiles seems to represent an etiologic factor; however, the structure-function patterns involved in the self-presentation in this disease remain unclear. Herein, we analyzed the molecular landscape of HLA complexes of a cohort of 300 IAA patients and almost 3000 healthy and disease controls by deeply dissecting their genotypic configurations, functional divergence, self-antigen binding capabilities, and T-cell receptor (TCR) repertoire specificities. Specifically, analysis of the evolutionary divergence of HLA genotypes (HED) showed that IAA patients carried class II HLA molecules whose antigen-binding sites were characterized by a high level of structural homology, only partially explained by specific risk allele profiles. This pattern implies reduced HLA binding capabilities, confirmed by binding analysis of hematopoietic stem cell (HSC)-derived self-peptides. IAA phenotype was associated with the enrichment in a few amino acids at specific positions within the peptide-binding groove of DRB1 molecules, affecting the interface HLA-antigen-TCR ß and potentially constituting the basis of T-cell dysfunction and autoreactivity. When analyzing associations with clinical outcomes, low HED was associated with risk of malignant progression and worse survival, underlying reduced tumor surveillance in clearing potential neoantigens derived from mechanisms of clonal hematopoiesis. Our data shed light on the immunogenetic risk associated with IAA etiology and clonal evolution and on general pathophysiological mechanisms potentially involved in other autoimmune disorders.

Structure-Activity relationship of 1-(Furan-2ylmethyl)Pyrrolidine-Based Stimulation-2 (ST2) inhibitors for treating graft versus host disease.

An elevated plasma level of soluble ST2 (sST2) is a risk biomarker for graft-versus-host disease (GVHD) and death in patients receiving hematopoietic cell transplantation (HCT). sST2 functions as a trap for IL-33 and amplifies the pro-inflammatory type 1 and 17 response while suppressing the tolerogenic type 2 and regulatory T cells activation during GVHD development. We previously identified small-molecule ST2 inhibitors particularly iST2-1 that reduces plasma sST2 levels and improved survival in two animal models. Here, we reported the structure-activity relationship of the furanylmethylpyrrolidine-based ST2 inhibitors based on iST2-1. Based on the biochemical AlphaLISA assay, we improved the activity of iST2-1 by 6-fold (∼6 µM in IC50 values) in the inhibition of ST2/IL-33 and confirmed the activities of the compounds in a cellular reporter assay. To determine the inhibition of the alloreactivity in vitro, we used the mixed lymphocyte reaction assay to demonstrate that our ST2 inhibitors decreased CD4+ and CD8+ T cells proliferation and increased Treg population. The data presented in this work are critical to the development of ST2 inhibitors in future.

RNF111-dependent neddylation activates DNA damage-induced ubiquitination.

Ubiquitin-like proteins have been shown to be covalently conjugated to targets. However, the functions of these ubiquitin-like proteins are largely unknown. Here, we have screened most known ubiquitin-like proteins after DNA damage and found that NEDD8 is involved in the DNA damage response. Following various DNA damage stimuli, NEDD8 accumulated at DNA damage sites; this accumulation was dependent on an E2 enzyme (UBE2M) and an E3 ubiquitin ligase (RNF111). We further found that histone H4 was polyneddylated in response to DNA damage, and NEDD8 was conjugated to the N-terminal lysine residues of H4. Interestingly, the DNA damage-induced polyneddylation chain could be recognized by the MIU (motif interacting with ubiquitin) domain of RNF168. Loss of DNA damage-induced neddylation negatively regulated DNA damage-induced foci formation of RNF168 and its downstream functional partners, such as 53BP1 and BRCA1, thus affecting the normal DNA damage repair process.

The FHA and BRCT domains recognize ADP-ribosylation during DNA damage response.

Poly-ADP-ribosylation is a unique post-translational modification participating in many biological processes, such as DNA damage response. Here, we demonstrate that a set of Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains recognizes poly(ADP-ribose) (PAR) both in vitro and in vivo. Among these FHA and BRCT domains, the FHA domains of APTX and PNKP interact with iso-ADP-ribose, the linkage of PAR, whereas the BRCT domains of Ligase4, XRCC1, and NBS1 recognize ADP-ribose, the basic unit of PAR. The interactions between PAR and the FHA or BRCT domains mediate the relocation of these domain-containing proteins to DNA damage sites and facilitate the DNA damage response. Moreover, the interaction between PAR and the NBS1 BRCT domain is important for the early activation of ATM during DNA damage response and ATM-dependent cell cycle checkpoint activation. Taken together, our results demonstrate two novel PAR-binding modules that play important roles in DNA damage response.

N-terminal modified cyclopeptidic mimetics of ApolloTBM as inhibitors of TRF2.

Telomeric repeat binding factor 2 (TRF2) plays an important role in protecting telomeres from being recognized as DNA breaks. TRF2 performs its telomere protecting functions partially by recruiting a number of accessory proteins to telomeres through its TRF homology (TFRH) domain. Identification of small molecular compounds which can bind to the TRFH domain of TRF2 and block the interactions between TRF2 and its associated proteins is crucial for elucidating the molecular mechanisms of these protein-protein interactions. Using a previously identified peptidic mimetic of ApolloTBM as a lead compound, we designed and synthesized a series of novel TRF2 inhibitors by non-peptidic modifications of the N-terminal residues. These compounds can maintain the binding affinities to TRF2 but have much reduced peptidic characteristics compared to the lead compound.

Targeting DCN1-UBC12 Protein-Protein Interaction for Regulation of Neddylation Pathway.

Protein neddylation is one type of posttranslational modifications that regulates the activity of the substrate proteins. Neddylation modification is catalyzed by NEDD8-activating enzyme (NAE, E1), NEDD8-conjugating enzyme (E2), and NEDD8 ligase (E3) to attach NEDD8, an ubiquitin-like molecule, to a lysine residue of a substrate protein. The best known neddylation substrates are cullin family members, which are scaffold components of cullin-RING ligases (CRLs), and cullin neddylation is required for activation of CRLs. In mammalian cells, there are one E1, two E2s (UBC12/UBE2M and UBE2F), and over a dozen E3s. MLN4924, the first-in-class small-molecule inhibitor of NAE, blocks the entire neddylation modification to inactivate activity of all CRLs. MLN4924 is currently in the Phase I/II clinical trials for anticancer application.In the last few years, targeting protein-protein interactions of the neddylation complexes has been pursued as a potential strategy to selectively inhibit the activity of individual CRL. Analysis of the co-crystal structures of DCN1, a co-E3 for neddylation, and its binding partners UBC12 (a neddylation E2) suggested that it may be amenable for the design of potent, small-molecule inhibitors. In this chapter, we will review the discovery of small-molecule inhibitors that block the interactions of DCN1 with UBC12 (hereafter called DCN1 inhibitors) from a number of laboratories, including ours, leading to selective inactivation of CRL-1 and/or CRL-3. We will also discuss potential therapeutic applications of these small-molecule inhibitors.

Small-molecule PROTAC degraders of the Bromodomain and Extra Terminal (BET) proteins - A review.

The PROteolysis TArgeting Chimeric (PROTAC) concept has provided an opportunity for the discovery and development of a completely new type of therapy involving induction of protein degradation. The BET proteins, comprised of BRD2, BRD3, BRD4 and the testis-specific BRDT protein, are epigenetic readers and master transcription coactivators. Extremely potent and efficacious small-molecule PROTAC degraders of the BET proteins, based on available, potent and selective BET inhibitors, have been reported. BET degraders differ from BET inhibitors in their cellular potency, phenotypic effects, pharmacokinetic properties and toxicity profiles. Herein, we provide a review of BET degraders and the differential outcome observed in the cellular and animal models for BET degraders in comparison to BET inhibitors.

Development of an Imidazopyridazine-Based MNK1/2 Inhibitor for the Treatment of Lymphoma.

The mitogen-activated protein kinase-interacting protein kinases (MNKs) are the only kinases known to phosphorylate eukaryotic translation initiation factor 4E (eIF4E) at Ser209, which plays a significant role in cap-dependent translation. Dysregulation of the MNK/eIF4E axis has been found in various solid tumors and hematological malignancies, including diffuse large B-cell lymphoma (DLBCL). Herein, structure-activity relationship studies and docking models determined that 20j exhibits excellent MNK1/2 inhibitory activity, stability, and hERG safety. 20j exhibits strong and broad antiproliferative activity against different cancer cell lines, especially GCB-DLBCL DOHH2. 20j suppresses the phosphorylation of eIF4E in Hela cells (IC50 = 90.5 nM) and downregulates the phosphorylation of eIF4E and 4E-BP1 in A549 cells. In vivo studies first revealed that ibrutinib enhances the antitumor effect of 20j without side effects in a DOHH2 xenograft model. This study provided a solid foundation for the future development of a MNK inhibitor for GCB-DLBCL treatment.

Comprehensive Analyses of the Effects of the Small-Molecule Inhibitor of the UHM Domain in the Splicing Factor U2AF1 in Leukemia Cells.

Mutations in RNA splicing factor genes including SF3B1, U2AF1, SRSF2, and ZRSR2 have been reported to contribute to development of myeloid neoplasms including myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML). Chemical tools targeting cells carrying these mutant genes remain limited and underdeveloped. Among the four proteins, mutant U2AF1 (U2AF1mut) acquires an altered 3' splice site selection preference and co-operates with the wild-type U2AF1 (U2AF1wt) to change various gene isoform patterns to support MDS cells survival and proliferation. U2AF1 mutations in MDS cells are always heterozygous and the cell viability is reduced when exposed to additional insult affecting U2AF1wt function. To investigate if the pharmacological inhibition of U2AF1wt function can provoke drug-induced vulnerability of cells harboring U2AF1 mut , we conducted a fragment-based library screening campaign to discover compounds targeting the U2AF homology domain (UHM) in U2AF1 that is required for the formation of the U2AF1/U2AF2 complex to define the 3' splice site. The most promising hit (SF1-8) selectively inhibited growth of leukemia cell lines overexpressingU2AF1 mut and human primary MDS cells carrying U2AF1 mut . RNA-seq analysis of K562-U2AF1mut following treatment with SF1-8 further revealed alteration of isoform patterns for a set of proteins that impair or rescue pathways associated with endocytosis, intracellular vesicle transport, and secretion. Our data suggested that further optimization of SF1-8 is warranted to obtain chemical probes that can be used to evaluate the therapeutic concept of inducing lethality to U2AF1 mut cells by inhibiting the U2AF1wt protein.

Targeting inhibitors of apoptosis proteins (IAPs) for new breast cancer therapeutics.

Apoptosis resistance is a hallmark of human cancer. Research in the last two decades has identified key regulators of apoptosis, including inhibitor of apoptosis proteins (IAPs). These critical apoptosis regulators have been targeted for the development of new cancer therapeutics. In this article, we will discuss three members of IAP proteins, namely XIAP, cIAP1 and cIAP2, as cancer therapeutic targets and the progress made in developing new cancer therapeutic agents to target these IAP proteins.

Correction to "Profiling the Binding Activities of Peptides and Inhibitors to the U2 Auxiliary Factor Homology Motif (UHM) Domains".

[This corrects the article DOI: 10.1021/acsmedchemlett.2c00537.].

Profiling the Binding Activities of Peptides and Inhibitors to the U2 Auxiliary Factor Homology Motif (UHM) Domains.

RNA splicing is a biological process to generate mature mRNA (mRNA) by removing introns and annexing exons in the nascent RNA transcript and is executed by a multiprotein complex called spliceosome. To aid RNA splicing, a class of splicing factors use an atypical RNA recognition domain (UHM) to bind with U2AF ligand motifs (ULMs) in proteins to form modules that recognize splice sites and splicing regulatory elements on mRNA. Mutations of UHM containing splicing factors have been found frequently in myeloid neoplasms. To profile the selectivity of UHMs for inhibitor development, we established binding assays to measure the binding activities between UHM domains and ULM peptides and a set of small-molecule inhibitors. Additionally, we computationally analyzed the targeting potential of the UHM domains by small-molecule inhibitors. Our study provided the binding assessment of UHM domains to diverse ligands that may guide development of selective UHM domain inhibitors in the future.

The inducible prostaglandin E synthase (mPGES-1) in neuroinflammatory disorders.

The cyclooxygenase (COX)/prostaglandin E2 (PGE2) signaling pathway has emerged as a critical target for anti-inflammatory therapeutic development in neurological diseases. However, medical use of COX inhibitors in the treatment of various neurological disorders has been limited due to well-documented cardiovascular and cerebrovascular complications. It has been widely proposed that modulation of downstream microsomal prostaglandin E synthase-1 (mPGES-1) enzyme may provide more specificity for inhibiting PGE2-elicited neuroinflammation. Heightened levels of mPGES-1 have been detected in a variety of brain diseases such as epilepsy, stroke, glioma, and neurodegenerative diseases. Subsequently, elevated levels of PGE2, the enzymatic product of mPGES-1, have been demonstrated to modulate a multitude of deleterious effects. In epilepsy, PGE2 participates in retrograde signaling to augment glutamate release at the synapse leading to neuronal death. The excitotoxic demise of neurons incites the activation of microglia, which can become overactive upon further stimulation by PGE2. A selective mPGES-1 inhibitor was able to reduce gliosis and the expression of proinflammatory cytokines in the hippocampus following status epilepticus. A similar mechanism has also been observed in stroke, where the overactivation of microglia by PGE2 upregulated the expression and secretion of proinflammatory cytokines. This intense activation of neuroinflammatory processes triggered the secondary injury commonly observed in stroke, and blockade of mPGES-1 reduced infarction size and edema, suppressed induction of proinflammatory cytokines, and improved post-stroke well-being and cognition. Furthermore, elevated levels of PGE2 have been shown to intensify the proliferation of glioma cells, mediate P-glycoprotein expression at the blood-brain barrier (BBB) and facilitate breakdown of the BBB. For these reasons, targeting mPGES-1, the central and inducible enzyme of the COX cascade, may provide a more specific therapeutic strategy for treating neuroinflammatory diseases.

Discovery of Pyrrolo[2,3-c]pyridines as Potent and Reversible LSD1 Inhibitors.

Lysine specific demethylase 1 (LSD1) acts as an epigenetic eraser by specifically demethylating mono- and histone 3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) residues. LSD1 has been pursued as a promising therapeutic target for the treatment of human cancer, and a number of LSD1 inhibitors have been advanced into clinical development. In the present study, we describe our discovery of pyrrolo[2,3-c]pyridines as a new class of highly potent and reversible LSD1 inhibitors, designed on the basis of a previously reported LSD1 inhibitor GSK-354. Among them, 46 shows an IC50 value of 3.1 nM in inhibition of LSD1 enzymatic activity and inhibits cell growth with IC50 values of 0.6 nM in the MV4;11 acute leukemia cell line and 1.1 nM in the H1417 small-cell lung cancer cell line. Compound 46 (LSD1-UM-109) is a novel, highly potent, and reversible LSD1 inhibitor and serves as a promising lead compound for further optimization.

Prophylactic Mitigation of Acute Graft versus Host Disease by Novel 2-(Pyrrolidin-1-ylmethyl)pyrrole-Based Stimulation-2 (ST2) Inhibitors.

Hematopoietic cell transplantation (HCT) is a proven and potentially curable therapy for hematological malignancies and inherited hematological disease. The main risk of HCT is the development of graft versus host disease (GVHD) acquired in up to 50% of patients. Upregulation of soluble ST2 (sST2) is a key clinical biomarker for GVHD prognosis and was shown to be a potential therapeutic target for GVHD. Agents targeting sST2 to reduce the sST2 level after HCT have the potential to mitigate GVHD progression. Here, we report 32 (or XY52) as the lead ST2 inhibitor from our optimization campaign. XY52 had improved inhibitory activity and metabolic stability in vitro and in vivo. XY52 suppressed proinflammatory T-cell proliferation while increasing regulatory T cells in vitro. In a clinically relevant GVHD model, a 21-day prophylactic regimen of XY52 reduced plasma sST2 and IFN-γ levels and GVHD score and extended survival in mice. XY52 represented a significant improvement over our previous compound, iST2-1, and further optimization of XY52 is warranted. The small-molecule ST2 inhibitors can potentially be used as a biomarker-guided therapy for mitigating GVHD in future clinical applications.

Novel, Brain-Permeable, Cross-Species Benzothiazole Inhibitors of Microsomal Prostaglandin E Synthase-1 (mPGES-1) Dampen Neuroinflammation In Vitro and In Vivo.

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme of the cyclooxygenase (COX) cascade that generates prostaglandin E2 (PGE2) during inflammatory conditions. PGE2 is known to be a potent immune signaling molecule that mediates both peripheral and central inflammations. Inhibition of mPGES-1, rather than COX, may overcome the cardiovascular side effects associated with long-term COX inhibition by providing a more specific strategy to target inflammation. However, mPGES-1 inhibitor development is hampered by the large differences in cross-species activity due to the structural differences between the human and murine mPGES-1. Here, we report that our thiazole-based mPGES-1 inhibitors, compounds 11 (UT-11) and 19 derived from two novel scaffolds, were able to suppress PGE2 production in human (SK-N-AS) and murine (BV2) cells. The IC50 values of inhibiting PGE2 production in human and murine cells were 0.10 and 2.00 µM for UT-11 and 0.43 and 1.55 µM for compound 19, respectively. Based on in vitro and in vivo pharmacokinetic data, we selected UT-11 for evaluation in a lipopolysaccharide (LPS)-induced inflammation model. We found that our compound significantly suppressed proinflammatory cytokines and chemokines in the hippocampus but not in the kidney. Taken together, we demonstrated the potential of UT-11 in treating neuroinflammatory conditions, including epilepsy and stroke, and warrant further optimization.

Discovery of ARD-2051 as a Potent and Orally Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of Androgen Receptor for the Treatment of Advanced Prostate Cancer.

We report the discovery of ARD-2051 as a potent and orally efficacious androgen receptor (AR) proteolysis-targeting chimera degrader. ARD-2051 achieves DC50 values of 0.6 nM and Dmax >90% in inducing AR protein degradation in both the LNCaP and VCaP prostate cancer cell lines, potently and effectively suppresses AR-regulated genes, and inhibits cancer cell growth. ARD-2051 achieves a good oral bioavailability and pharmacokinetic profile in mouse, rat, and dog. A single oral dose of ARD-2051 strongly reduces AR protein and suppresses AR-regulated gene expression in the VCaP xenograft tumor tissue in mice. Oral administration of ARD-2051 effectively inhibits VCaP tumor growth and causes no signs of toxicity in mice. ARD-2051 is a promising AR degrader for advanced preclinical development for the treatment of AR+ human cancers.

Discovery of ARD-1676 as a Highly Potent and Orally Efficacious AR PROTAC Degrader with a Broad Activity against AR Mutants for the Treatment of AR + Human Prostate Cancer.

We report herein the discovery and extensive characterization of ARD-1676, a highly potent and orally efficacious PROTAC degrader of the androgen receptor (AR). ARD-1676 was designed using a new class of AR ligands and a novel cereblon ligand. It has DC50 values of 0.1 and 1.1 nM in AR+ VCaP and LNCaP cell lines, respectively, and IC50 values of 11.5 and 2.8 nM in VCaP and LNCaP cell lines, respectively. ARD-1676 effectively induces degradation of a broad panel of clinically relevant AR mutants. ARD-1676 has an oral bioavailability of 67, 44, 31, and 99% in mice, rats, dogs, and monkeys, respectively. Oral administration of ARD-1676 effectively reduces the level of AR protein in the VCaP tumor tissue in mice and inhibits tumor growth in the VCaP mouse xenograft tumor model without any sign of toxicity. ARD-1676 is a highly promising development candidate for the treatment of AR+ human prostate cancer.

Computational Cosolvent Mapping Analysis Leads to Identify Salicylic Acid Analogs as Weak Inhibitors of ST2 and IL33 Binding.

Cytokine signaling initiated by the binding of the cytokine receptors to cytokines plays important roles in immune regulation and diseases. Structurally, cytokine receptors interact with cytokines via an extensive, rugged interface that represents a challenge in inhibitor development. Our computational analysis has previously indicated that butyric acid, mimicking acidic residues, preferentially binds to sites in ST2 (Stimulation-2) that interact with acidic residues of IL33, the endogenous cytokine for ST2. To investigate if a charged group in small molecules facilitates ligand binding to ST2, we developed a biochemical homogeneous time resolved fluorescence assay to determine the inhibition of ST2/IL33 binding by five molecules containing an aromatic ring and a charged group. Three molecules, including niacin, salicylic acid, and benzamidine, exhibit inhibition activities at millimolar concentrations. We further employed the computational cosolvent mapping analysis to identify a shared mode of interaction between niacin, salicylic acid, and ST2. The mode of interaction was further confirmed by four analogous compounds that exhibited similar or improved activities. Our study provided the evidence of inhibition of ST2 and IL33 binding by salicylic acid and analogs. The results suggest that biological activity of salicylic acid may be partly mediated through modulating extracellular cytokine receptors and cytokine interaction.