Design, synthesis, and biological evaluation of phenyl thiazole-based AR-V7 degraders.

Multiple Splice variants of AR have been reported in the past few years. These splice variants are upregulated in most cases of CRPC resulting in poor prognosis. Most of these variants lack the ligand binding domain (LBD) but still bind to DNA resulting in constitutive activation of downstream targets. The AR-V7 splice variant has been characterized extensively and current clinical trials in CRPC are exploring the use of AR-V7 as a biomarker. New therapeutic molecules that selectively target AR-V7 are also being explored. However, there is a dearth of information available on the selectivity, phenotypic responses in AR-V7 dependent cell lines and pharmacokinetic properties of such molecules. Using our proprietary computational algorithms and rational SAR optimization, we have developed a potent and selective AR-V7 degrader from a known AR DNA binding domain (DBD) binder. This molecule effectively degraded AR-V7 in a CRPC cell line and demonstrated good oral bioavailability in mouse PK studies. This tool compound can be used to evaluate the pharmacological effects of AR-V7 degraders. Further exploration of SAR can be pursued to develop more optimized lead compounds.

Benzimidazole derivatives as potential dual inhibitors for PARP-1 and DHODH.

Poly (ADP-ribose) polymerases (PARPs) play diverse roles in various cellular processes that involve DNA repair and programmed cell death. Amongst these polymerases is PARP-1 which is the key DNA damage-sensing enzyme that acts as an initiator for the DNA repair mechanism. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the pyrimidine biosynthetic pathway which is an important target for anti-hyperproliferative and anti-inflammatory drug design. Since these enzymes share a common role in the DNA replication and repair mechanisms, it may be beneficial to target both PARP-1 and DHODH in attempts to design new anti-cancer agents. Benzimidazole derivatives have shown a wide variety of pharmacological activities including PARP and DHODH inhibition. We hereby report the design, synthesis and bioactivities of a series of benzimidazole derivatives as inhibitors of both the PARP-1 and DHODH enzymes.

Anticancer activity of Neptunia oleracea methanolic extracts.

Neptunia oleracea Lour (water mimosa) is an edible medicinal plant used in treating various diseases. According to Phytochemical and Ethnobotanical Databases, Neptunia oleracea Lour is used in curing earaches, dysentery, syphilis, and tumour. The present study was aimed at demonstrating the anticancer activity of the Neptunia oleracea Lour methanolic extract. The methanolic extract was isolated and its anti-proliferative activity was studied on haematological cancer cell lines. The activity of the extract was further evaluated using cell cycle analysis and apoptosis assays. In addition to this, effect of the extract on c-Myc and PErk1/2 modulation was also evaluated. Neptunia oleracea Lour extract induced cell death in cancer cells while sparing normal cells. An increase in cleaved PARP and reduction in BCL-2 levels observed upon treatment. Neptunia oleracea causes reduction in c-Myc levels and pERK1/2 protein levels. Thus, our work highlights the methanolic extract of Neptunia oleracea Lour as a promising anti-cancer agent.

Dihydroorotate Dehydrogenase Inhibitors Promote Cell Cycle Arrest and Disrupt Mitochondria Bioenergetics in Ramos Cells.

BACKGROUND: The re-emerging of targeting Dihydroorotate Dehydrogenase (DHODH) in cancer treatment particularly Acute Myelogenous Leukemia (AML) has corroborated the substantial role of DHODH in cancer and received the attention of many pharmaceutical industries. OBJECTIVE: The effects of Brequinar Sodium (BQR) and 4SC-101 on lymphoblastoid cell lines were investigated. METHODS: DHODH expression and cell proliferation inhibition of lymphoblastoid and lymphoma cell lines were analyzed using Western blot analysis and XTT assay, respectively. JC-1 probe and ATP biochemiluminescence kit were used to evaluate the mitochondrial membrane potential and ATP generation in these cell lines. Furthermore, we explored the cell cycle progression using Muse™ Cell Cycle Kit. RESULTS: Ramos, SUDHL-1 and RPMI-1788 cells are fast-growing cells with equal expression of DHODH enzyme and sensitivity to DHODH inhibitors that showed that the inhibition of DHODH was not cancer-specific. In ATP depletion assay, the non-cancerous RPMI-1788 cells showed only a minor ATP reduction compared to Ramos and SUDHL-1 (cancer) cells. In the mechanistic impact of DHODH inhibitors on non-cancerous vs cancerous cells, the mitochondrial membrane potential assay revealed that significant depolarization and cytochrome c release occurred with DHODH inhibitors treatment in Ramos but not in the RPMI-1788 cells, indicating a different mechanism of proliferation inhibition in normal cells. CONCLUSION: The findings of this study provide evidence that DHODH inhibitors perturb the proliferation of non-cancerous cells via a distinct mechanism compared to cancerous cells. These results may lead to strategies for overcoming the impact on non-cancerous cells during treatment with DHODH inhibitors, leading to a better therapeutic window in patients.

Selective Vulnerability to Pyrimidine Starvation in Hematologic Malignancies Revealed by AG-636, a Novel Clinical-Stage Inhibitor of Dihydroorotate Dehydrogenase.

Agents targeting metabolic pathways form the backbone of standard oncology treatments, though a better understanding of differential metabolic dependencies could instruct more rationale-based therapeutic approaches. We performed a chemical biology screen that revealed a strong enrichment in sensitivity to a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic versus solid tumor origin. Differential AG-636 activity translated to the in vivo setting, with complete tumor regression observed in a lymphoma model. Dissection of the relationship between uridine availability and response to AG-636 revealed a divergent ability of lymphoma and solid tumor cell lines to survive and grow in the setting of depleted extracellular uridine and DHODH inhibition. Metabolic characterization paired with unbiased functional genomic and proteomic screens pointed to adaptive mechanisms to cope with nucleotide stress as contributing to response to AG-636. These findings support targeting of DHODH in lymphoma and other hematologic malignancies and suggest combination strategies aimed at interfering with DNA-damage response pathways.

Discovery of CA-4948, an Orally Bioavailable IRAK4 Inhibitor for Treatment of Hematologic Malignancies.

Small molecule potent IRAK4 inhibitors from a novel bicyclic heterocycle class were designed and synthesized based on hits identified from Aurigene's compound library. The advanced lead compound, CA-4948, demonstrated good cellular activity in ABC DLBCL and AML cell lines. Inhibition of TLR signaling leading to decreased IL-6 levels was also observed in whole blood assays. CA-4948 demonstrated moderate to high selectivity in a panel of 329 kinases as well as exhibited desirable ADME and PK profiles including good oral bioavailability in mice, rat, and dog and showed >90% tumor growth inhibition in relevant tumor models with excellent correlation with in vivo PD modulation. CA-4948 was well tolerated in toxicity studies in both mouse and dog at efficacious exposure. The overall profile of CA-4948 prompted us to select it as a clinical candidate for evaluation in patients with relapsed or refractory hematologic malignancies including non-Hodgkin lymphoma and acute myeloid leukemia.

Synergistic inhibition of melanoma xenografts by Brequinar sodium and Doxorubicin.

Malignant melanoma continues to be a fatal disease for which novel and long-term curative breakthroughs are desired. One such innovative idea would be to assess combination therapeutic treatments - by way of combining two potentially effective and very different therapy. Previously, we have shown that DHODH inhibitors, A771726 and Brequinar sodium (BQR) induced cell growth impairment in melanoma cells. Similar results were seen with DHODH RNA interference (shRNA). In the present study, we showed that combination of BQR with doxorubicin resulted in synergistic and additive cell growth inhibition in these cells. In addition, in vivo studies with this combination of drugs demonstrated an almost 90% tumor regression in nude mice bearing melanoma tumors. Cell cycle regulatory proteins, cyclin B1 and its binding partner pcdc-2 and p21 were significantly downregulated and upregulated respectively following the combined treatment. Given that we have observed synergistic effects with BQR and doxorubicin, both in vitro and in vivo, these drugs potentially represent a new combination in the targeted therapy of melanoma.

Dihydroorotate dehydrogenase Inhibitors Target c-Myc and Arrest Melanoma, Myeloma and Lymphoma cells at S-phase.

Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme in the de novo biosynthesis pathway of pyrimidines. Inhibition of this enzyme impedes cancer cell proliferation but the exact mechanisms of action of these inhibitors in cancer cells are poorly understood. In this study, we showed that cancer cells, namely melanoma, myeloma and lymphoma overexpressed DHODH protein and treatment with A771726 and Brequinar sodium resulted in cell cycle arrest at S-phase. Transfection with DHODH shRNA depleted DHODH protein expression and impeded the proliferation of melanoma cells. shRNA knockdown of DHODH in combination with DHODH inhibitors further reduced the cancer cell proliferation, suggesting that knockdown of DHODH had sensitized the cells to DHODH inhibitors. Cell cycle regulatory proteins, c-Myc and its transcriptional target, p21 were found down- and up-regulated, respectively, following treatment with DHODH inhibitors in melanoma, myeloma and lymphoma cells. Interestingly, knockdown of DHODH by shRNA had also similarly affected the expression of c-Myc and p21 proteins. Our findings suggest that DHODH inhibitors induce cell cycle arrest in cancer cells via additional DHODH-independent pathway that is associated with p21 up-regulation and c-Myc down-regulation. Hence, DHODH inhibitors can be explored as potential therapeutic agents in cancer therapy.

Changes in the carbon dioxide expirogram in response to ozone exposure.

The objectives of this study were to quantify pulmonary responses to ozone (O3) exposure by parameters computed from the carbon dioxide expirogram and to compare these responses to decrements in forced expired spirometry. Anatomical dead space (VD) was determined from the pure dead space and transition regions of the expirogram. Four alternative parameters were computed from the alveolar plateau: slope (S), normalized slope (NS), peripheral cross-sectional area (AP) and well-mixed peripheral volume (VMP). Forty-seven healthy nonsmokers (25 men and 22 women) participated in two research sessions in which they exercised on a cycle ergometer for 1 h while orally inhaling either room air at a minute ventilation of 30.6 +/- 3.6 L or room air mixed with 0.252 +/- 0.029 ppm O3 at a minute ventilation of 29.9 +/- 3.7 L. Carbon dioxide expirograms were measured before exposure, 10 min after exposure and 70 min after exposure. Percent changes (mean +/- SD) in expirogram parameters were significant (P < or = 0.002) at both 10 and 70 min after O3 exposure: VD(-4.2 +/- 5.1, -3.3 +/- 6.9), S(16.4 +/- 17.9, +15.1 +/- 20.2), NS(17.5 +/- 15.4, +15.9 +/- 19.2), AP(-8.1 +/- 7.6, -7.7 +/- 9.8) and VMP(-15.4 +/- 13.0, -13.0 +/- 15.2). Percent decrements of forced expired volume in one second (FEV1) were also significant at both 10 min (-13.3 +/- 13.4) and 70 min (-11.1 +/- 9.2) following O3 exposure. Changes in the expirogram as well as decrements in FEV1 were not significant at either time point after air exposure. Thus, the CO2 expirogram is useful for characterizing the effect of O3 exposure on gas transport, and for supplementing forced expired spirometry that is frequently used to quantify lung mechanics.

Nitric oxide measurements in rat mesentery reveal disrupted venulo-arteriolar communication in diabetes.

OBJECTIVE: Arteriolar tone is partially controlled by diffusing mediators released by closely paired venules and is reported to depend on venular shear and venular leukocyte adherence. In healthy rat mesentery, venule-initiated arteriolar dilation and consequent enhanced capillary flow appear to be tightly regulated by nitric oxide (NO). In contrast, diabetes inhibits NO-dependent vasodilation and is associated with dysfunctional microcirculation. The objective of this study was to investigate venule-dependent NO in diabetes. METHODS: Arteriolar and venular wall concentrations of NO were measured in control and diabetic (streptozotocin-induced) rat mesentery with fluorescent diaminofluorescein-2-diacetate (DAF-2-DA); tissue NO was measured with DAF-2. Venular leukocyte adherence and microvascular shear rates were also measured. RESULTS: Microvascular NO in diabetic rats was found to be significantly lower (<50%) than in controls. In normal rats, arteriolar NO demonstrated a positive correlation with venular NO and venular shear, and a negative correlation with venular leukocyte adherence. Diabetes eliminated all these correlations. No correlation was present between arteriolar NO and arteriolar shear in either normal or diabetic rats. CONCLUSIONS: Arteriolar NO appears to be enhanced by venular shear in normal but not in diabetic rats. This dysfunction could contribute to poor capillary perfusion in diabetes.

Inhibition of leukocyte adherence enables venular control of capillary perfusion in streptozotocin-induced diabetic rats.

OBJECTIVE: Vasoactive molecules can diffuse from venules to dilate closely paired arterioles and enhance capillary perfusion. Venular control of capillary flow has been found to be dependent on nitric oxide (NO), which might be scavenged rapidly in diabetic microvasculature due to the presence of activated leukocytes. This study attempts to improve venular control of capillary flow using fucoidan, which inhibits venular leukocvte adhesion. METHODS: Microvascular red blood cell velocity was measured in the mesentery of streptozotocin-induced diabetic rats, with and without fucoidan treatment, and in normal rats. Arteriolar pathways leading to branching capillaries were videotaped to measure the percent of the surrounding area occupied by a venule (% pairing). Microvascular wall NO was measured using fluorescent diaminofluorescein-2-diacetate in diabetic rats, with and without fucoidan treatment. RESULTS: In normal rats, close pairing of venules to arterioles resulted in faster capillary flow. However, after 4-5 weeks of diabetes, the correlation between capillary velocity and % pairing was no longer significant. Capillary velocity and % pairing decreased approximately 50% in comparison to normal rats. Treatment of diabetic rats with fucoidan restored venular control of capillary flow and increased NO levels. CONCLUSION: Leukocyte-derived mediators that scavenge NO may lead to inadequate venular control of capillary flow in diabetes.

L-arginine and antineutrophil serum enable venular control of capillary perfusion in hypercholesterolemic rats.

OBJECTIVE: The purpose of this study was to investigate and counteract dysfunctional control of capillary flow in hypercholesterolemia. Capillary flow is controlled by arteriolar tone, which in turn is influenced by mediators released from closely paired venules in a mechanism that involves nitric oxide (NO). However, venular control of capillary flow is altered with hypercholesterolemia. METHODS: Rats were given a normal or high-cholesterol diet before measurements of mesenteric capillary red blood cell velocity. The arteriolar pathway leading to the capillary was videotaped to measure the percent of the surrounding area (within 15 |gmm) that was occupied by a venule (% pairing). RESULTS: Venule-paired arterioles were significantly smaller in hypercholesterolemia compared with normocholesterolemia, corresponding to slower capillary flow. A positive correlation between capillary velocity and % pairing observed in normocholesterolemia was not observed during NO synthase inhibition or in hypercholesterolemic rats. However, positive correlations between the two parameters were found in hypercholesterolemia when the rats were given drinking water supplementation of L-arginine or an injection of antineutrophil serum, both of which tended to improve velocity in capillaries branching from venule-paired arteriolar pathways. CONCLUSIONS: Dysfunctional venular control of capillary perfusion in hypercholesterolemia may be a consequence of a neutrophil-mediated deficiency of NO.