Third generation quinoline-3-carboxamide transcriptional disrupter of HDAC4, HIF-1α, and MEF-2 signaling for metastatic castration-resistant prostate cancer.

BACKGROUND: The quinoline-3-carboxamide, Tasquinimod (TasQ), is orally active as a maintenance therapy with an on-target mechanism-of-action via allosteric binding to HDAC4. This prevents formation of the HDAC4/NCoR1/HDAC3 complex, disrupting HIF-1α transcriptional activation and repressing MEF-2 target genes needed for adaptive survival signaling in the compromised tumor micro environment. In phase 3 clinical testing against metastatic castration-resistant prostate cancer(mCRPC), TasQ (1 mg/day) increased time-to-progression, but not overall survival. METHODS: TasQ analogs were chemically synthesized and tested for activity compared to the parental compound. These included HDAC4 enzymatic assays, qRT-PCR and western blot analyses of gene and protein expression following treatment, in vitro and in vivo efficacy against multiple prostate cancer models including PDXs, pharmacokinetic analyses,AHR binding and agonist assays, SPR analyses of binding to HDAC4 and NCoR1, RNAseq analysis of in vivo tumors, 3D endothelial sprouting assays, and a targeted kinase screen. Genetic knockout or knockdown controls were used when appropriate. RESULTS: Here, we document that, on this regimen (1 mg/day), TasQ blood levels are 10-fold lower than the optimal concentration (≥2 µM) needed for anticancer activity, suggesting higher daily doses are needed. Unfortunately, we also demonstrate that TasQ is an arylhydrocarbon receptor (AHR) agonist, which binds with an EC50 of 1 µM to produce unwanted off-target side effects. Therefore, we screened a library of TasQ analogsto maximize on-target versus off-target activity. Using this approach, we identified ESATA-20, which has ~10-fold lower AHR agonism and 5-fold greater potency against prostate cancer patient-derived xenografts. CONCLUSION: This increased therapeuticindex nominates ESATA-20 as a lead candidate forclinical development as an orally active third generation quinoline-3-carboxamide analog thatretains its on-target ability to disrupt HDAC4/HIF-1α/MEF-2-dependent adaptive survival signaling in the compromisedtumor microenvironment found in mCRPC.

Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia.

Approximately 50% of acute myeloid leukemia (AML) patients do not respond to induction therapy (primary induction failure [PIF]) or relapse after <6 months (early relapse [ER]). We have recently shown an association between an immune-infiltrated tumor microenvironment (TME) and resistance to cytarabine-based chemotherapy but responsiveness to flotetuzumab, a bispecific DART antibody-based molecule to CD3ε and CD123. This paper reports the results of a multicenter, open-label, phase 1/2 study of flotetuzumab in 88 adults with relapsed/refractory AML: 42 in a dose-finding segment and 46 at the recommended phase 2 dose (RP2D) of 500 ng/kg per day. The most frequent adverse events were infusion-related reactions (IRRs)/cytokine release syndrome (CRS), largely grade 1-2. Stepwise dosing during week 1, pretreatment dexamethasone, prompt use of tocilizumab, and temporary dose reductions/interruptions successfully prevented severe IRR/CRS. Clinical benefit accrued to PIF/ER patients showing an immune-infiltrated TME. Among 30 PIF/ER patients treated at the RP2D, the complete remission (CR)/CR with partial hematological recovery (CRh) rate was 26.7%, with an overall response rate (CR/CRh/CR with incomplete hematological recovery) of 30.0%. In PIF/ER patients who achieved CR/CRh, median overall survival was 10.2 months (range, 1.87-27.27), with 6- and 12-month survival rates of 75% (95% confidence interval [CI], 0.450-1.05) and 50% (95% CI, 0.154-0.846). Bone marrow transcriptomic analysis showed that a parsimonious 10-gene signature predicted CRs to flotetuzumab (area under the receiver operating characteristic curve = 0.904 vs 0.672 for the European LeukemiaNet classifier). Flotetuzumab represents an innovative experimental approach associated with acceptable safety and encouraging evidence of activity in PIF/ER patients. This trial was registered at www.clinicaltrials.gov as #NCT02152956.

PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

BACKGROUND: Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. METHODS: Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. RESULTS: Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA <0.1 ng/mL. PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. CONCLUSIONS: In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate cancer to alter total A2M levels, which can potentially alter levels of a variety of growth factors such as IL-6, TGF-beta, basic FGF, and PDGF. Alterations in levels of these cytokines and proteolytic degradation of small peptide hormones may have profound effect on host-cancer interaction.

Proteolysis of complement factors iC3b and C5 by the serine protease prostate-specific antigen in prostatic fluid and seminal plasma.

Prostate-specific Ag (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high- and low-grade prostate cancer throughout the course of disease progression, even in the androgen-ablated state, suggests that PSA has a role in the pathogenesis of disease. Current experimental and clinical evidence suggests that chronic inflammation, regardless of the cause, may predispose men to prostate cancer. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated but not completely understood. In this study, we used a mass spectrometry-based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of Igs and the complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed the high-level expression of C3 in the prostatic fluid and the presence of a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. These results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that could help to create an environment hospitable to malignancy through proteolysis of the complement system.

Trypsin-like proteolytic contamination of commercially available psa purified from human seminal fluid.

BACKGROUND: Prostate-Specific Antigen (PSA) is a serine protease whose expression is maintained in all stages of prostate cancer. A role for PSA in the pathobiology for prostate cancer has not been firmly established. Experimental studies to date support a role for PSA through mechanisms such as release or processing of growth factors and degradation of the extracellular matrix. Exposure of prostate cancer cells to exogenous PSA also results in gene expression changes. These in vitro and biochemical assays rely on the use of commercially available PSA. Contamination of these commercial preparations can significantly impact the results of these in vitro studies. METHODS: We characterized PSA and trypsin-like activity of PSA preparations obtained from three commercial sources: Calbiochem, Fitzgerald, and AbD Serotec. Silver stained gels were used to compare the purity of each preparation and mass spectrometry was performed to characterize contaminating proteases. RESULTS: PSA activity varied between PSA preparations with AbD Serotec PSA having highest degree of activity. Significant trypsin-like activity, which was inhibited by aprotinin, was observed in PSA preparations from Calbiochem and Fitzgerald, but not AbD Serotec. These former two PSA preparations also contained the greatest degree of non-PSA contaminants by silver stain and mass spectrometry. CONCLUSIONS: Commercially available preparations of PSA contain contaminating proteins, including trypsin-like protease activity, that could potentially complicate the interpretation of results obtained from in vitro studies assessing PSA proteolysis of potential protein substrates and effects of PSA on gene expression.

C4-alkylthiols with activity against Moraxella catarrhalis and Mycobacterium tuberculosis.

Antimicrobial resistance represents a global threat to healthcare. The ability to adequately treat infectious diseases is increasingly under siege due to the emergence of drug-resistant microorganisms. New approaches to drug development are especially needed to target organisms that exhibit broad antibiotic resistance due to expression of ß-lactamases which is the most common mechanism by which bacteria become resistant to ß-lactam antibiotics. We designed and synthesized 20 novel monocyclic ß-lactams with alkyl- and aryl-thio moieties at C4, and subsequently tested these for antibacterial activity. These compounds demonstrated intrinsic activity against serine ß-lactamase producing Mycobacterium tuberculosis wild type strain (Mtb) and multiple (n=6) ß-lactamase producing Moraxella catarrhalis clinical isolates.

Prostate-specific antigen: an overlooked candidate for the targeted treatment and selective imaging of prostate cancer.

The role of prostate-specific antigen (PSA) or kallikrein-related peptidase 3 (KLK3) as a biomarker for prostate cancer is well known; however, the precise physiological role of it's serine protease activity in prostate cancer remains a mystery. PSA is produced at high levels by both androgen-dependent and -independent prostate cancers. Studies have documented high levels of active PSA in the milieu surrounding osseous and soft tissue metastases. This evidence, coupled with growing experimental evidence, suggests that PSA plays an important role in the pathobiology of prostate cancer. These observations support the development of PSA-selective inhibitors as useful tools for the targeted treatment and imaging of prostate cancer. Here, we review the research that has been conducted to date on developing selective inhibitors for PSA. The different approaches used to determine PSA substrate specificity and for creating inhibitors are discussed. In addition, the unique active site characteristics of PSA and how these motifs aided our research in developing PSA targeted agents are highlighted.

Structural optimization, biological evaluation, and application of peptidomimetic prostate specific antigen inhibitors.

Prostate-specific antigen (PSA) is a serine protease produced at high levels by normal and malignant prostate epithelial cells that is used extensively as a biomarker in the clinical management of prostate cancer. To better understand PSA's role in prostate cancer progression, we prepared a library of peptidyl boronic acid-based inhibitors. To enhance selectivity for PSA vs other serine proteases, we modified the P1 site of the inhibitors to incorporate a bromopropylglycine group. This allowed the inhibitors to participate in halogen bond formation with the serine found at the bottom of the specificity pocket. The best of these Ahx-FSQn(boro)Bpg had PSA Ki of 72 nM and chymotrypsin Ki of 580 nM. In vivo studies using PSA-producing xenografts demonstrated that candidate inhibitors had minimal effect on growth but significantly altered serum levels of PSA. Biodistribution of (125)I labeled peptides showed low levels of uptake into tumors compared to other normal tissues.

Biological evaluation of paclitaxel-peptide conjugates as a model for MMP2-targeted drug delivery.

Paclitaxel (PTX) is a highly effective cytotoxic agent widely used for the treatment of several solid tumors. However, PTX shows dose-limiting cytotoxicity and in most cases induces drug resistance followed by failure in treatment. To enhance the therapeutic index of a given drug, various drug delivery methods have been explored to systemically deliver sufficient amount of the drug to the desired site. In the present study, we designed and synthesized two PTX prodrugs by conjugating PTX at different sites with an octapeptide (AcGPLGIAGQ) that can be cleaved by MMP2 at tumor sites. As a result, PTX is expected to be released at the tumor sites, absorbed by the tumor cells, and thereby inhibit the tumor growth. We evaluated the in vitro activities of the two drugs in a panel of drug-sensitive and -resistant cancer cell lines and their in vivo efficacies in a HT1080 fibrosarcoma mouse xenograft model that overexpresses MMP2. Our in vitro results showed that the PTX-AcGPLGIAGQ conjugates inhibited cancer cell proliferation with higher activity compared to that observed for free PTX, both of which were mediated by an arrest of G(2)/M-phase of the cell cycle. Consistent with the in vitro results, treatment with PTX-octapeptide conjugate resulted in extensive areas of necrosis and a lower percentage of proliferating cells in xenograft tumor sections. Together, our results indicate the potential of the tumor-targeted delivery of PTX to exploit the specific recognition of MMP2, reduce toxicity, and selectively kill tumor cells.

Determination of the cation-chelating potential of C-methylthiolated beta-lactams and their sulfones by electrospray ionization mass spectrometry.

The chelation potential of highly lipophilic C-dimethylthiolated monocyclic beta-lactams was examined using electrospray ionization mass spectrometry (ESI-MS). The metal salts NaCl, KCl, CaCl2, ZnCl2, Cu(NO3)2, CdSO4, MnCl2, and Mg(NO3)2 were used for the analysis. The K+ adducts of the compounds studied were more responsive in ESI analysis, compared to their Na+ adducts, regardless of the oxidation state of the sulfur (in the methylthio or the sulfone groups) and the type of the group adjacent to the lactam carbonyl. Opening of the beta-lactam ring, leading to formation of a chargeable N-atom, had little to no effect on the K+ adduct formation. Interactions of the methylthio group with the divalent zinc ion were also observed.