Discovery of an Aldo-Keto reductase 1C3 (AKR1C3) degrader.

Aldo-keto reductase 1C3 (AKR1C3) is a protein upregulated in prostate cancer, hematological malignancies, and other cancers where it contributes to proliferation and chemotherapeutic resistance. Androgen receptor splice variant 7 (ARv7) is the most common mutation of the AR receptor that confers resistance to clinical androgen receptor signalling inhibitors in castration-resistant prostate cancer. AKR1C3 interacts with ARv7 promoting stabilization. Herein we report the discovery of the first-in-class AKR1C3 Proteolysis-Targeting Chimera (PROTAC) degrader. This first-generation degrader potently reduced AKR1C3 expression in 22Rv1 prostate cancer cells with a half-maximal degradation concentration (DC50) of 52 nM. Gratifyingly, concomitant degradation of ARv7 was observed with a DC50 = 70 nM, along with degradation of the AKR1C3 isoforms AKR1C1 and AKR1C2 to a lesser extent. This compound represents a highly useful chemical tool and a promising strategy for prostate cancer intervention.

Discovery of the Next Generation of Non-peptidomimetic Neurolysin Activators with High Blood-Brain Barrier Permeability: a Pharmacokinetics Study in Healthy and Stroke Animals.

PURPOSE: There is growing interest in seeking pharmacological activation of neurolysin (Nln) for stroke treatment. Discovery of central nervous system drugs remains challenging due to the protection of the blood-brain barrier (BBB). The previously reported peptidomimetic Nln activators display unsatisfactory BBB penetration. Herein, we investigate the next generation of non-peptidomimetic Nln activators with high BBB permeability. METHODS: A BBB-mimicking model was used to evaluate their in vitro BBB permeability. Protein binding, metabolic stability, and efflux assays were performed to determine their unbound fraction, half-lives in plasma and brains, and dependence of BBB transporter P-glycoprotein (P-gp). The in vivo pharmacokinetic profiles were elucidated in healthy and stroke mice. RESULTS: Compounds KS52 and KS73 out of this generation exhibit improved peptidase activity and BBB permeability compared to the endogenous activator and previous peptidomimetic activators. They show reasonable plasma and brain protein binding, improved metabolic stability, and independence of P-gp-mediated efflux. In healthy animals, they rapidly distribute into brains and reach peak levels of 18.69% and 12.10% injected dose (ID)/ml at 10 min. After 4 h, their total brain concentrations remain 7.78 and 12.34 times higher than their A50(minimal concentration required for enhancing 50% peptidase activity). Moreover, the ipsilateral hemispheres of stroke animals show comparable uptake to the corresponding contralateral hemispheres and healthy brains. CONCLUSIONS: This study provides essential details about the pharmacokinetic properties of a new generation of potent non-peptidomimetic Nln activators with high BBB permeability and warrants the future development of these agents as potential neuroprotective pharmaceutics for stroke treatment.

Selective carbonic anhydrase IX and XII inhibitors based around a functionalized coumarin scaffold.

Inhibition of specific carbonic anhydrase (CA) enzymes is a validated strategy for the development of agents to target cancer. The CA isoforms IX and XII are overexpressed in various human solid tumors wherein they play a critical role in regulating extracellular tumor acidification, proliferation, and progression. A series of novel sulfonamides based on the coumarin scaffold were designed, synthesized and characterized as potent and selective CA inhibitors. Selected compounds show significant activity and selectivity over CA I and CA II to target the tumor-associated CA IX and CA XII with high inhibition activity at the single digit nanomolar level. Twelve compounds were identified to be more potent compared with acetazolamide (AAZ) control to inhibit CA IX while one was also more potent than AAZ to inhibit CA XII. Compound 18f (Ki's = 955 nM, 515 nM, 21 nM and 5 nM for CA's I, II, IX, and XII, respectively) is highlighted as a novel CA IX and XII inhibitor for further development.

Chlorinated benzothiadiazines inhibit angiogenesis through suppression of VEGFR2 phosphorylation.

Angiogenesis inhibitors are a critical pharmacological tool for the treatment of solid tumors. Suppressing vascular permeability leads to inhibition of tumor growth, invasion, and metastatic potential by blocking the supply of oxygen and nutrients. Disruption of the vascular endothelial growth factor (VEGF) signaling pathway is a validated target for the design of antiangiogenic agents. Several VEGFR2 inhibitors have been clinically approved over the past years. Structural analysis of these clinical VEGFR2 inhibitors highlighted key functional group overlap with the benzothiadiazine core contained in a library of in-house compounds. Herein we ascribe anti-angiogenic activity to a series of chlorinated benzothiadiazines. Selected compounds show significant activity to completely ameliorate VEGF-induced endothelial cell proliferation by suppression of VEGFR2 phosphorylation. The scaffold is devoid of activity to inhibit carbonic anhydrases and generally lacks cytotoxicity across a range of cancer and non-malignant cell lines. Assay of activity at 468 kinases shows remarkable selectivity with only four kinases inhibited > 65% at 10 µM concentration, and with significant activity to inhibit TNK2/ACK1 and PKRD2 by > 90%. All four identified kinase targets are known modulators of angiogenesis, thus highlighting compound 17b as a novel angiogenesis inhibitor for further development.

Access to Highly Strained Tricyclic Ketals Derived from Coumarins.

Synthesis of highly strained fused substituted dihydrobenzopyran cyclopropyl lactones derived from coumarin carboxylates are reported. The substrate scope tolerates a variety of 6- and 8-substituents on the coumarin ring. Substitution at the 5- or 7-position is resistant to tricyclic lactone formation except with 7-methyl substitution. Benzamide-containing coumarins afford the tricyclic ketal. A plausible mechanism is proposed for the formation of the fused lactone: intramolecular rearrangement of trans cyclopropyl methyl ketones with phenolic acetate via the formation of a hemiacetal.

Synthesis and biological evaluation of N, N-dialkylcarboxy coumarin-NO donor conjugates as potential anticancer agents.

A series of nitric oxide (NO) donor furoxan conjugates of N, N-dialkylcarboxy coumarins have been synthesized as potential anticancer agents. The synthesized compounds have been tested for their in vitro antiproliferative activities on various cancer and noncancerous cell lines. The candidate derivatives exhibit selectivity towards cancer cells with excellent activities in low nM to µM concentrations. In vitro mechanistic studies indicate that the candidate compounds generate substantial NO, inhibit colony formation, and cause apoptosis in cancer cells. A preliminary in vivo tolerance study of the lead candidate 10 in mice indicates that it is well-tolerated, evidenced by zero mortality and normal body weight gains in treated mice. Further translation of the lead derivative 10 using MDA-MB-231 based tumor xenograft model shows good tumor growth reduction.

Synthesis and biological evaluation of a novel anticancer agent CBISC that induces DNA damage response and diminishes levels of mutant-p53.

A novel nitrogen mustard CBISC has been synthesized and evaluated as an anticancer agent. CBISC has been shown to exhibit enhanced cell proliferation inhibition properties against mutant p53 cell lines colorectal cancer WiDr, pancreatic cancer (MIAPaCa-2 and PANC-1), and triple negative breast cancer (MDA-MB-231 and MDA-MB-468). In vitro mechanism of action studies revealed perturbations in the p53 pathway and increased cell death as evidenced by western blotting, immunofluorescent microscopy and MTT assay. Further, in vivo studies revealed that CBISC is well tolerated in healthy mice and exhibited significant in vivo tumor growth inhibition properties in WiDr and MIAPaCa-2 xenograft models. These studies illustrate the potential utility of CBISC as an anticancer agent.

Case Report: Intra-Tumoral Vaccinations of Quadrivalent HPV-L1 Peptide Vaccine With Topical TLR-7 Agonist Following Recurrence: Complete Resolution of HPV-HR-Associated Gynecologic Squamous Cell Carcinomas in Two Patients.

The human papilloma virus (HPV) high-risk variants (HPV-HR) such as HPV16 and HPV18 are responsible for most HPV related cancers, including anogenital and head and neck cancers. Here, we present two patients with HPV-HR-associated gynecological malignancies who, after failing radiation therapy, were treated with experimental salvage immunotherapy regimen resulting in complete, durable responses in both patients. Each patient was diagnosed with recurrent, radiation-refractory, HPV-HR positive, squamous cell carcinoma of the lower genital tract. Patient A was a 90-year-old, African American, with metastatic vulvar cancer to the right inguinal-femoral triangle and pulmonary metastases. Patient B was a 41-year-old, Caucasian, with a central-recurrence of cervix cancer. Each patient received at least two intratumoral quadrivalent HPV-L1 vaccine (Gardasil™) injections and daily topical TLR-7 agonist (imiquimod) to the tumor surface 2 weeks apart. This combination of intratumoral vaccinations and topical TLR-7 agonist produced unexpected complete resolution of disease in both patients. The importance of radiation therapy, despite being considered a treatment failure by current definitions, cannot be understated. Radiation therapy appears to have offered a therapeutic immune advantage by modifying the tumor microenvironment. This immune protocol has potential to help patients with advanced HPV-HR-related malignancies previously considered incurable.

Synthesis, in vitro, and in vivo evaluation of novel N-phenylindazolyl diarylureas as potential anti-cancer agents.

Novel N-phenylindazole based diarylureas have been designed, synthesized and evaluated as potential anticancer agents. In vitro cell viability studies of these derivatives illustrate good potency with IC50 values in the range of 0.4-50 µM in several cancer cell lines including murine metastatic breast cancer 4T1, murine glioblastoma GL261, human triple negative breast cancer MDA-MB-231, human pancreatic cancer MIAPaCa-2, and human colorectal cancer cell line WiDr. The ester group in the lead compound 8i was modified to incorporate amino-amides to increase solubility and stability while retaining biological activity. Further in vitro studies reveal that lead candidates inhibit tube length in HUVEC cells. In vivo systemic toxicity studies indicate that these candidate compounds are well tolerated in mice without any significant side effects. Anticancer efficacy studies in WiDr tumor xenograft and 4T1 tumor syngraft models demonstrate that the lead candidate 11 exhibits significant antitumor properties as a single agent in these tumor models.

Synthesis and biological evaluation of arylphosphonium-benzoxaborole conjugates as novel anticancer agents.

Arylphosphonium-benzoxaborole conjugates have been synthesized as potential mitochondria targeting anticancer agents. The synthesized compounds have been tested for their effects on cell viability in various solid tumor cell lines including breast cancer 4T1 and MCF-7, pancreatic cancer MIAPaCa-2 and colorectal adenocarcinoma WiDr. Compound 6c is designated as a lead compound for further studies due to its enhanced effects on cell viability in the above-mentioned cell lines. Seahorse Xfe96 based metabolic assays reveal that the lead candidate 6c inhibits mitochondrial respiration in 4T1 and WiDr cell lines as evidenced by the reduction of mitochondrial ATP production and increase in proton leak. Epiflourescent microscopy experiments also illustrate that 6c causes significant mitochondrial fragmentation in 4T1 and WiDr cells, morphologically consistent with programmed cell death. Our current studies illustrate that arylphosphonium-benzoxaborole conjugates have potential to be further developed as anticancer agents.

Development of Novel Analogs of the Monocarboxylate Transporter Ligand FACH and Biological Validation of One Potential Radiotracer for Positron Emission Tomography (PET) Imaging.

Monocarboxylate transporters 1-4 (MCT1-4) are involved in several metabolism-related diseases, especially cancer, providing the chance to be considered as relevant targets for diagnosis and therapy. [18F]FACH was recently developed and showed very promising preclinical results as a potential positron emission tomography (PET) radiotracer for imaging of MCTs. Given that [18F]FACH did not show high blood-brain barrier permeability, the current work is aimed to investigate whether more lipophilic analogs of FACH could improve brain uptake for imaging of gliomas, while retaining binding to MCTs. The 2-fluoropyridinyl-substituted analogs 1 and 2 were synthesized and their MCT1 inhibition was estimated by [14C]lactate uptake assay on rat brain endothelial-4 (RBE4) cells. While compounds 1 and 2 showed lower MCT1 inhibitory potencies than FACH (IC50 = 11 nM) by factors of 11 and 25, respectively, 1 (IC50 = 118 nM) could still be a suitable PET candidate. Therefore, 1 was selected for radiosynthesis of [18F]1 and subsequent biological evaluation for imaging of the MCT expression in mouse brain. Regarding lipophilicity, the experimental log D7.4 result for [18F]1 agrees pretty well with its predicted value. In vivo and in vitro studies revealed high uptake of the new radiotracer in kidney and other peripheral MCT-expressing organs together with significant reduction by using specific MCT1 inhibitor α-cyano-4-hydroxycinnamic acid. Despite a higher lipophilicity of [18F]1 compared to [18F]FACH, the in vivo brain uptake of [18F]1 was in a similar range, which is reflected by calculated BBB permeabilities as well through similar transport rates by MCTs on RBE4 cells. Further investigation is needed to clarify the MCT-mediated transport mechanism of these radiotracers in brain.

A novel MCT1 and MCT4 dual inhibitor reduces mitochondrial metabolism and inhibits tumour growth of feline oral squamous cell carcinoma.

Monocarboxylate transporters (MCTs) support tumour growth by regulating the transport of metabolites in the tumour microenvironment. High MCT1 or MCT4 expression is correlated with poor outcomes in human patients with head and neck squamous cell carcinoma (HNSCC). Recently, drugs targeting these transporters have been developed and may prove to be an effective treatment strategy for HNSCC. Feline oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy resembling advanced or recurrent HNSCC. The goals of this study were to investigate the effects of a previously characterized dual MCT1 and MCT4 inhibitor, MD-1, in OSCC as a novel treatment approach for feline oral cancer. We also sought to determine the potential of feline OSCC as a large animal model for the further development of MCT inhibitors to treat human HNSCC. In vitro, MD-1 reduced the viability of feline OSCC and human HNSCC cell lines, altered glycolytic and mitochondrial metabolism and synergized with platinum-based chemotherapies. While MD-1 treatment increased lactate concentrations in an HNSCC cell line, the inhibitor failed to alter lactate levels in feline OSCC cells, suggesting an MCT-independent activity. In vivo, MD-1 significantly inhibited tumour growth in a subcutaneous xenograft model and prolonged overall survival in an orthotopic model of feline OSCC. Our results show that MD-1 may be an effective therapy for the treatment of feline oral cancer. Our findings also support the further investigation of feline OSCC as a large animal model to inform the development of MCT inhibitors and future clinical studies in human HNSCC.

Development of Novel Silyl Cyanocinnamic Acid Derivatives as Metabolic Plasticity Inhibitors for Cancer Treatment.

Novel silyl cyanocinnamic acid derivatives have been synthesized and evaluated as potential anticancer agents. In vitro studies reveal that lead derivatives 2a and 2b have enhanced cancer cell proliferation inhibition properties when compared to the parent monocarboxylate transporter (MCT) inhibitor cyano-hydroxycinnamic acid (CHC). Further, candidate compounds exhibit several-fold more potent MCT1 inhibition properties as determined by lactate-uptake studies, and these studies are supported by MCT homology modeling and computational inhibitor-docking studies. In vitro effects on glycolysis and mitochondrial metabolism also illustrate that the lead derivatives 2a and 2b lead to significant effects on both metabolic pathways. In vivo systemic toxicity and efficacy studies in colorectal cancer cell WiDr tumor xenograft demonstrate that candidate compounds are well tolerated and exhibit good single agent anticancer efficacy properties.

Prexasertib treatment induces homologous recombination deficiency and synergizes with olaparib in triple-negative breast cancer cells.

BACKGROUND: Breast cancer remains as one of the most lethal types of cancer in women. Among various subtypes, triple-negative breast cancer (TNBC) is the most aggressive and hard to treat type of breast cancer. Mechanistically, increased DNA repair and cell cycle checkpoint activation remain as the foremost reasons behind TNBC tumor resistance to chemotherapy and disease recurrence. METHODS: We evaluated the mechanism of prexasertib-induced regulation of homologous recombination (HR) proteins using 20S proteasome inhibitors and RT-PCR. HR efficiency and DNA damages were evaluated using Dr-GFP and comet assays. DNA morphology and DNA repair focus studies were analyzed using immunofluorescence. UALCAN portal was used to evaluate the expression of RAD51 and survival probability based on tumor stage, subtype, and race in breast cancer patients. RESULTS: Our results show that prexasertib treatment promotes both post-translational and transcriptional mediated regulation of BRCA1 and RAD51 proteins. Additionally, prexasertib-treated TNBC cells revealed over 55% reduction in HR efficiency compared to control cells. Based on these results, we hypothesized that prexasertib treatment induced homologous recombination deficiency (HRD) and thus should synergize with PARP inhibitors (PARPi) in TNBC cells. As predicted, combined treatment of prexasertib and PARPi olaparib increased DNA strand breaks, γH2AX foci, and nuclear disintegration relative to single-agent treatment. Further, the prexasertib and olaparib combination was synergistic in multiple TNBC cell lines, as indicated by combination index (CI) values. Analysis of TCGA data revealed elevated RAD51 expression in breast tumors compared to normal breast tissues, especially in TNBC subtype. Interestingly, there was a discrepancy in RAD51 expression in racial groups, with African-American and Asian breast cancer patients showing elevated RAD51 expression compared to Caucasian breast cancer patients. Consistent with these observations, African-American and Asian TNBC patients show decreased survival. CONCLUSIONS: Based on these data, RAD51 could be a biomarker for aggressive TNBC and for racial disparity in breast cancer. As positive correlation exists between RAD51 and CHEK1 expression in breast cancer, the in vitro preclinical data presented here provides additional mechanistic insights for further evaluation of the rational combination of prexasertib and olaparib for improved outcomes and reduced racial disparity in TNBC.

Novel N,N-dialkyl cyanocinnamic acids as monocarboxylate transporter 1 and 4 inhibitors.

Potent and dual monocarboxylate transporter (MCT) 1 and 4 inhibitors have been developed for the first time as potential anticancer agents based on α-cyanocinnamic acid structural template. Candidate inhibitors 1-9 have been evaluated for in vitro cell proliferation against MCT1 and MCT4 expressing cancer cell lines. Potential MCT1 and MCT4 binding interactions of the lead compound 9 have been studied through homology modeling and molecular docking prediction. In vitro effects on extracellular flux via glycolysis and mitochondrial stress tests suggest that candidate compounds 3 and 9 disrupt glycolysis and OxPhos efficiently in MCT1 expressing colorectal adenocarcinoma WiDr and MCT4 expressing triple negative breast cancer MDA-MB-231 cells. Fluorescence microscopy analyses in these cells also indicate that compound 9 is internalized and concentrated near mitochondria. In vivo tumor growth inhibition studies in WiDr and MDA-MB-231 xenograft tumor models in mice indicate that the candidate compound 9 exhibits a significant single agent activity.

Development and radiosynthesis of the first 18 F-labeled inhibitor of monocarboxylate transporters (MCTs).

Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) are involved in tumor development and progression. Their expression levels are related to clinical disease prognosis. Accordingly, both MCTs are promising drug targets for treatment of a variety of human cancers. The noninvasive imaging of these MCTs in cancers is regarded to be advantageous for assessing MCT-mediated effects on chemotherapy and radiosensitization using specific MCT inhibitors. Herein, we describe a method for the radiosynthesis of [18 F]FACH ((E)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylic acid), as a novel radiolabeled MCT1/4 inhibitor for imaging with PET. A fluorinated analog of α-cyano-4-hydroxycinnamic acid (FACH) was synthesized, and the inhibition of MCT1 and MCT4 was measured via an L-[14 C]lactate uptake assay. Radiolabeling was performed by a two-step protocol comprising the radiosynthesis of the intermediate (E)/(Z)-[18 F]tert-Bu-FACH (tert-butyl (E)/(Z)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylate) followed by deprotection of the tert-butyl group. The radiofluorination was successfully implemented using either K[18 F]F-K2.2.2 -carbonate or [18 F]TBAF. The final deprotected product [18 F]FACH was only obtained when [18 F]tert-Bu-FACH was formed by the latter procedure. After optimization of the deprotection reaction, [18 F]FACH was obtained in high radiochemical yields (39.6 ± 8.3%, end of bombardment (EOB) and radiochemical purity (greater than 98%).

Synthesis and Evaluation of Baylis-Hillman Reaction Derived Imidazole and Triazole Cinnamates as Antifungal Agents.

Allylic acetates derived from Baylis-Hillman reaction undergo efficient nucleophilic isomerization with imidazoles and triazoles to provide imidazolylmethyl and triazolylmethyl cinnamates stereoselectively. Antifungal evaluation of these derivatives against Cryptococcus neoformans exhibits good minimum inhibitory concentration values. These compounds exhibit low toxicity in proliferating MCF-7 breast cancer cell line. Structure activity relationship studies indicate that halogenated aromatic derivatives provide better antifungal activity.