Induction of Immune Responses and Phosphatidylserine Exposure by TLR9 Activation Results in a Cooperative Antitumor Effect with a Phosphatidylserine-targeting Prodrug.

Head and neck cancer is a major cancer type, with high motility rates that reduce the quality of life of patients. Herein, we investigated the effectiveness and mechanism of a combination therapy involving TLR9 activator (CpG-2722) and phosphatidylserine (PS)-targeting prodrug of SN38 (BPRDP056) in a syngeneic orthotopic head and neck cancer animal model. The results showed a cooperative antitumor effect of CpG-2722 and BPRDP056 owing to their distinct and complementary antitumor functions. CpG-2722 induced antitumor immune responses, including dendritic cell maturation, cytokine production, and immune cell accumulation in tumors, whereas BPRDP056 directly exerted cytotoxicity toward cancer cells. We also discovered a novel function and mechanism of TLR9 activation, which increased PS exposure on cancer cells, thereby attracting more BPRDP056 to the tumor site for cancer cell killing. Killed cells expose more PS in tumor for BPRDP056 targeting. Tumor antigens released from the dead cells were taken up by antigen-presenting cells, which enhanced the CpG-272-promoted T cell-mediated tumor-killing effect. These form a positive feed-forward antitumor effect between the actions of CpG-2722 and BPRDP056. Thus, the study findings suggest a novel strategy of utilizing the PS-inducing function of TLR9 agonists to develop combinational cancer treatments using PS-targeting drugs.

Rejuvenating hepatic tumor microenvironment immunity with a phosphatidylserine-targeting small molecule drug conjugate.

We report the design, synthesis and evaluation of a class of phosphatidylserine-targeting zinc (II) dipicolylamine drug conjugates and show that conjugate 16b elicits immune cell infiltration and remodels the "cold" hepatic tumor microenvironment to the inflamed "hot" tumor. Structure-property relationship study via linker modifications and subsequent pharmacokinetics profiling were carried out to improve the solubility and stability of the conjugates in vivo. In a spontaneous hepatocellular carcinoma mouse model, we showed that conjugate 16b exhibited better antitumor efficacy than sorafenib. In particular, significant increase of CD8+ T cell infiltration and granzyme B level was observed, providing insights in sensitizing tumors from intrinsic immune suppressive microenvironment. Evaluation of tumor inflammation-related mRNA expression profile revealed that conjugate 16b, through inductions of key gene expressions including STAT1, CXCL9, CCL5, and PD-L1, rejuvenated tumor microenvironment with enhancement in T cell-, macrophage-, NK cell-, chemokines and cytokines'- functions. Our study establishes that an apoptosis-targeting theranostic enables enrichment of multifaceted immune cells into the tumor mass, which provides potential therapeutic strategies in the combination with immune checkpoint blockade treatment.

Chemoproteomic profiling reveals cellular targets of nitro-fatty acids.

Nitro-fatty acids are a class of endogenous electrophilic lipid mediators with anti-inflammatory and cytoprotective effects in a wide range of inflammatory and fibrotic disease models. While these beneficial biological effects of nitro-fatty acids are mainly attributed to their ability to form covalent adducts with proteins, only a small number of proteins are known to be nitro-alkylated and the scope of protein nitro-alkylation remains undetermined. Here we describe the synthesis and application of a clickable nitro-fatty acid probe for the detection and first global identification of mammalian proteins that are susceptible to nitro-alkylation. 184 high confidence nitro-alkylated proteins were identified in THP1 macrophages, majority of which are novel targets of nitro-fatty acids, including extended synaptotagmin 2 (ESYT2), signal transducer and activator of transcription 3 (STAT3), toll-like receptor 2 (TLR2), retinoid X receptor alpha (RXRα) and glucocorticoid receptor (NR3C1). In particular, we showed that 9-nitro-oleate covalently modified and inhibited dexamethasone binding to NR3C1. Bioinformatic analyses revealed that nitro-alkylated proteins are highly enriched in endoplasmic reticulum and transmembrane proteins, and are overrepresented in lipid metabolism and transport pathways. This study significantly expands the scope of protein substrates targeted by nitro-fatty acids in living cells and provides a useful resource towards understanding the pleiotropic biological roles of nitro-fatty acids as signaling molecules or as multi-target therapeutic agents.

BPRDP056, a novel small molecule drug conjugate specifically targeting phosphatidylserine for cancer therapy.

Zinc(II)-dipicolylamine (Zn-DPA) has been shown to specifically identify and bind to phosphatidylserine (PS), which exists in bulk in the tumor microenvironment. BPRDP056, a Zn-DPA-SN38 conjugate was designed to provide PS-targeted drug delivery of a cytotoxic SN38 to the tumor microenvironment, thereby allowing a lower dosage of SN38 that induces apoptosis in cancer cells. Micro-Western assay showed that BPRDP056 exhibited apoptotic signal levels similar to those of CPT-11 in the treated tumors growing in mice. Pharmacokinetic study showed that BPRDP056 has excellent systemic stability in circulation in mice and rats. BPRDP056 is accumulated in tumors and thus increases the cytotoxic effects of SN38. The in vivo antitumor activities of BPRDP056 have been shown to be significant in subcutaneous pancreas, prostate, colon, liver, breast, and glioblastoma tumors, included an orthotopic pancreatic tumor, in mice. BPRDP056 shrunk tumors at a lower (~20% only) dosing intensity of SN38 compared to that of SN38 conjugated in CPT-11 in all tumor models tested. A wide spectrum of antitumor activities is expected to treat all cancer types of PS-rich tumor microenvironments. BPRDP056 is a first-in-class small molecule drug conjugate for cancer therapy.

Comparative study between deep learning and QSAR classifications for TNBC inhibitors and novel GPCR agonist discovery.

Machine learning is a well-known approach for virtual screening. Recently, deep learning, a machine learning algorithm in artificial neural networks, has been applied to the advancement of precision medicine and drug discovery. In this study, we performed comparative studies between deep neural networks (DNN) and other ligand-based virtual screening (LBVS) methods to demonstrate that DNN and random forest (RF) were superior in hit prediction efficiency. By using DNN, several triple-negative breast cancer (TNBC) inhibitors were identified as potent hits from a screening of an in-house database of 165,000 compounds. In broadening the application of this method, we harnessed the predictive properties of trained model in the discovery of G protein-coupled receptor (GPCR) agonist, by which computational structure-based design of molecules could be greatly hindered by lack of structural information. Notably, a potent (~ 500 nM) mu-opioid receptor (MOR) agonist was identified as a hit from a small-size training set of 63 compounds. Our results show that DNN could be an efficient module in hit prediction and provide experimental evidence that machine learning could identify potent hits in silico from a limited training set.

Linker Optimization and Therapeutic Evaluation of Phosphatidylserine-Targeting Zinc Dipicolylamine-based Drug Conjugates.

We report that compound 13, a novel phosphatidylserine-targeting zinc(II) dipicolylamine drug conjugate, readily triggers a positive feedback therapeutic loop through the in situ generation of phosphatidylserine in the tumor microenvironment. Linker modifications, pharmacokinetics profiling, in vivo antitumor studies, and micro-Western array of treated-tumor tissues were employed to show that this class of conjugates induced regeneration of apoptotic signals, which facilitated subsequent recruitment of the circulating conjugates through the zinc(II) dipicolylamine-phosphatidylserine association and resulted in compounding antitumor efficacy. Compared to the marketed compound 17, compound 13 not only induced regressions in colorectal and pancreatic tumor models, it also exhibited at least 5-fold enhancement in antitumor efficacy with only 40% of the drug employed during treatment, culminating in a >12.5-fold increase in therapeutic potential. Our study discloses a chemically distinct apoptosis-targeting theranostic, with built-in complementary functional moieties between the targeting module and the drug mechanism to expand the arsenal of antitumor therapy.

Development of Stem-Cell-Mobilizing Agents Targeting CXCR4 Receptor for Peripheral Blood Stem Cell Transplantation and Beyond.

The function of the CXCR4/CXCL12 axis accounts for many disease indications, including tissue/nerve regeneration, cancer metastasis, and inflammation. Blocking CXCR4 signaling with its antagonists may lead to moving out CXCR4+ cell types from bone marrow to peripheral circulation. We have discovered a novel series of pyrimidine-based CXCR4 antagonists, a representative (i.e., 16) of which was tolerated at a higher dose and showed better HSC-mobilizing ability at the maximal response dose relative to the approved drug 1 (AMD3100), and thus considered a potential drug candidate for PBSCT indication. Docking compound 16 into the X-ray crystal structure of CXCR4 receptor revealed that it adopted a spider-like conformation striding over both major and minor subpockets. This putative binding mode provides a new insight into CXCR4 receptor-ligand interactions for further structural modifications.

Targeting Tumor Associated Phosphatidylserine with New Zinc Dipicolylamine-Based Drug Conjugates.

A series of zinc(II) dipicolylamine (ZnDPA)-based drug conjugates have been synthesized to probe the potential of phosphatidylserine (PS) as a new antigen for small molecule drug conjugate (SMDC) development. Using in vitro cytotoxicity and plasma stability studies, PS-binding assay, in vivo pharmacokinetic studies, and maximum tolerated dose profiles, we provided a roadmap and the key parameters required for the development of the ZnDPA based drug conjugate. In particular, conjugate 24 induced tumor regression in the COLO 205 xenograft model and exhibited a more potent antitumor effect with a 70% reduction of cytotoxic payload compared to that of the marketed irinotecan when dosed at the same regimen. In addition to the validation of PS as an effective pharmacodelivery target for SMDC, our work also provided the foundation that, if applicable, a variety of therapeutic agents could be conjugated in the same manner to treat other PS-associated diseases.

Epigallocatechin-3-gallate inhibits bacterial virulence and invasion of host cells.

Increasing antibiotic resistance and beneficial effects of host microbiota has motivated the search for anti-infective agents that attenuate bacterial virulence rather than growth. For example, we discovered that specific flavonoids such as baicalein and quercetin from traditional medicinal plant extracts could attenuate Salmonella enterica serovar Typhimurium type III protein secretion and invasion of host cells. Here, we show epigallocatechin-3-gallate from green tea extracts also inhibits the activity of S. Typhimurium type III protein effectors and significantly reduces bacterial invasion into host cells. These results reveal additional dietary plant metabolites that can attenuate bacterial virulence and infection of host cells.

Bromomethylthioindole Inspired Carbazole Hybrids as Promising Class of Anti-MRSA Agents.

Series of N-substituted carbazole analogues bearing an indole ring were synthesized as anti-methicillin-resistant Staphylococcus aureus (MRSA) agents from a molecular hybridization approach. The representative compound 19 showed an MIC = 1 µg/mL against a panel of MRSA clinical isolates as it possessed comparable in vitro activities to that of vancomycin. Moreover, compound 19 also exhibited MIC = 1 µg/mL activities against a recent identified Z172 MRSA strain (vancomycin-intermediate and daptomycin-nonsusceptible phenotype) and the vancomycin-resistant Enterococcus faecalis (VRE) strain. In a mouse model with lethal infection of MRSA (4N216), a 75% survival rate was observed after a single dose of compound 19 was intravenously administered at 20 mg/kg. In light of their equipotent activities against different MRSA isolates and VRE strain, the data underscore the importance of designed hybrid series for the development of new N-substituted carbazoles as potential anti-MRSA agents.

Antibacterial Flavonoids from Medicinal Plants Covalently Inactivate Type III Protein Secretion Substrates.

Traditional Chinese Medicines (TCMs) have been historically used to treat bacterial infections. However, the molecules responsible for these anti-infective properties and their potential mechanisms of action have remained elusive. Using a high-throughput assay for type III protein secretion in Salmonella enterica serovar Typhimurium, we discovered that several TCMs can attenuate this key virulence pathway without affecting bacterial growth. Among the active TCMs, we discovered that baicalein, a specific flavonoid from Scutellaria baicalensis, targets S. Typhimurium pathogenicity island-1 (SPI-1) type III secretion system (T3SS) effectors and translocases to inhibit bacterial invasion of epithelial cells. Structurally related flavonoids present in other TCMs, such as quercetin, also inactivated the SPI-1 T3SS and attenuated S. Typhimurium invasion. Our results demonstrate that specific plant metabolites from TCMs can directly interfere with key bacterial virulence pathways and reveal a previously unappreciated mechanism of action for anti-infective medicinal plants.

Stem cell mobilizers targeting chemokine receptor CXCR4: renoprotective application in acute kidney injury.

We have discovered a novel series of quinazoline-based CXCR4 antagonists. Of these, compound 19 mobilized CXCR4(+) cell types, including hematopoietic stem cells and endothelial progenitor cells, more efficiently than the marketed 1 (AMD3100) with subcutaneous administration at the same dose (6 mg/kg) in mice. This series of compounds thus provides a set of valuable tools to study diseases mediated by the CXCR4/SDF-1 axis, including myocardial infarction, ischemic stroke, and cancer metastasis. More importantly, treatment with compound 19 significantly lowered levels of blood urea nitrogen and serum creatinine in rats with renal ischemia-reperfusion injury, providing evidence for its therapeutic potential in preventing ischemic acute kidney injury. CXCR4 antagonists such as 19 might also be useful to increase circulating levels of adult stem cells, thereby exerting beneficial effects on damaged and/or inflamed tissues in diseases that currently are not treated by standard approaches.

Function-oriented development of CXCR4 antagonists as selective human immunodeficiency virus (HIV)-1 entry inhibitors.

Motivated by the pivotal role of CXCR4 as an HIV entry co-receptor, we herein report a de novo hit-to-lead effort on the identification of subnanomolar purine-based CXCR4 antagonists against HIV-1 infection. Compound 24, with an EC50 of 0.5 nM against HIV-1 entry into host cells and an IC50 of 16.4 nM for inhibition of radioligand stromal-derived factor-1α (SDF-1α) binding to CXCR4, was also found to be highly selective against closely related chemokine receptors. We rationalized that compound 24 complementarily interacted with the critical CXCR4 residues that are essential for binding to HIV-1 gp120 V3 loop and subsequent viral entry. Compound 24 showed a 130-fold increase in anti-HIV activity compared to that of the marketed CXCR4 antagonist, AMD3100 (Plerixafor), whereas both compounds exhibited similar potency in mobilization of CXCR4(+)/CD34(+) stem cells at a high dose. Our study offers insight into the design of anti-HIV therapeutics devoid of major interference with SDF-1α function.

Therapeutic development in targeting protein-protein interactions with synthetic topological mimetics.

Protein-protein interactions lie at the heart of cellular signaling pathways and the deregulation of which has frequently led to diseases. In contrast to inhibitors that bind to distinctive enzyme active sites, molecules targeting protein surface topologies have been underexploited in drug development. The challenges in developing protein surface antagonists or agonists originate from the relatively large and flat surface areas that lack well-defined cavities required for sufficient binding affinity. In the past decade, our understanding of protein recognition has served as solid basis for the design of synthetic mimetics to modulate these protein-protein interactions. Herein, we summarize recent successes in the development of synthetic α-helix mimetics, proteomimetics, and biologics with the therapeutic potentials of inhibiting tumorgenesis or cancer-related viral infections.

Blocking HIV-1 entry by a gp120 surface binding inhibitor.

We report the mode of action of a proteomimetic compound that binds to the exterior surface of gp120 and blocks HIV-1 entry into cells. Using a one cycle time-of-addition study and antibody competition binding studies, we have determined that the compound blocks HIV-1 entry through modulation of key protein-protein interactions mediated by gp120. The compound exhibits anti-HIV-1 replication activities against several pseudotype viruses derived from primary isolates and the resistant strains isolated from existing drug candidates with equal potency. Together, these data provide evidence that the proteomimetic compound represents a novel class of HIV-1 viral entry inhibitor that functions through protein surface recognition in analogy to an antibody.

Alkynyl-farnesol reporters for detection of protein S-prenylation in cells.

Protein S-prenylation is a lipid modification that regulates membrane-protein and protein-protein interactions in cell signaling. Though sites of protein S-prenylation can be predicted based upon conserved C-terminal CaaX or CC/CXC motifs, biochemical detection of protein S-prenylation in cells is still challenging. Herein, we report an alkynyl-isoprenol chemical reporter (alk-FOH) as an efficient substrate for prenyltransferases in mammalian cells that enables sensitive detection of S-farnesylated and S-geranylgeranylated proteins using bioorthogonal ligation methods. Fluorescent detection alleviates the need to deplete cellular isoprenoids for biochemical analysis of S-prenylated proteins and enables robust characterization of S-prenylated proteins, such as effectors that are injected into host cells by bacterial pathogens. This alkynyl-prenylation reporter provides a sensitive tool for biochemical analysis and rapid profiling of prenylated proteins in cells.

Visible fluorescence detection of type III protein secretion from bacterial pathogens.

Type III protein secretion is essential for many gram-negative bacterial infections of host cells and an attractive target for new antibacterial drugs. Here, we describe a bacterial protein effector-carboxypeptidase G2 (CPG2) reporter system for fluorescence and visible detection of type III protein secretion in Salmonella typhimurium. This system provides a general method for measuring protein expression and secretion as well as a high-throughput and quantitative assay for analyzing type III protein secretion inhibitors.

Tandem fluorescence imaging of dynamic S-acylation and protein turnover.

The functional significance and regulation of reversible S-acylation on diverse proteins remain unclear because of limited methods for efficient quantitative analysis of palmitate turnover. Here, we describe a tandem labeling and detection method to simultaneously monitor dynamic S-palmitoylation and protein turnover. By combining S-acylation and cotranslational fatty acid chemical reporters with orthogonal clickable fluorophores, dual pulse-chase analysis of Lck revealed accelerated palmitate cycling upon T-cell activation. Subsequent pharmacological perturbation of Lck palmitate turnover suggests yet uncharacterized serine hydrolases contribute to dynamic S-acylation in cells. In addition to dually fatty-acylated proteins, this tandem fluorescence imaging method can be generalized to other S-acylated proteins using azidohomoalanine as a methonine surrogate. The sensitivity and efficiency of this approach should facilitate the functional characterization of cellular factors and drugs that modulate protein S-acylation. Furthermore, diverse protein modifications could be analyzed with this tandem imaging method using other chemical reporters to investigate dynamic regulation of protein function.

Discovery of a synthetic dual inhibitor of HIV and HCV infection based on a tetrabutoxy-calix[4]arene scaffold.

A potential anti-HIV and HCV drug candidate is highly desirable as coinfection has become a worldwide public health challenge. A potent compound based on a tetrabutoxy-calix[4]arene scaffold that possesses dual inhibition for both HIV and HCV is described. Structural activity relationship studies demonstrate the effects of lower-rim alkylation in maintaining cone conformation and upper-rim interacting head groups on the calix[4]arene play key roles for its potent dual antiviral activities.