Myelodysplastic syndromes disable human CD271+VCAM1+CD146+ niches supporting normal hematopoietic stem/progenitor cells.

Mesenchymal stem/stromal cells (MSCs) within the bone marrow microenvironment (BMME) support normal hematopoietic stem and progenitor cells (HSPCs). However, the heterogeneity of human MSCs has limited the understanding of their contribution to clonal dynamics and evolution to myelodysplastic syndromes (MDS). We combined three MSC cell surface markers, CD271, VCAM-1 (Vascular Cell Adhesion Molecule-1) and CD146, to isolate distinct subsets of human MSCs from bone marrow aspirates of healthy controls (Control BM). Based on transcriptional and functional analysis, CD271+CD106+CD146+ (NGFR+/VCAM1+/MCAM+/Lin-; NVML) cells display stem cell characteristics, are compatible with murine BM-derived Leptin receptor positive MSCs and provide superior support for normal HSPCs. MSC subsets from 17 patients with MDS demonstrated shared transcriptional changes in spite of mutational heterogeneity in the MDS clones, with loss of preferential support of normal HSPCs by MDS-derived NVML cells. Our data provide a new approach to dissect microenvironment-dependent mechanisms regulating clonal dynamics and progression of MDS.

Prodrugs of a 1'-CN-4-Aza-7,9-dideazaadenosine C-Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV.

A discovery program targeting respiratory syncytial virus (RSV) identified C-nucleoside 4 (RSV A2 EC50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (1, GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1-NTP, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4. A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.

Reduction of leukemic burden via bone-targeted nanoparticle delivery of an inhibitor of C-chemokine (C-C motif) ligand 3 (CCL3) signaling.

Leukemias are challenging diseases to treat due, in part, to interactions between leukemia cells and the bone marrow microenvironment (BMME) that contribute significantly to disease progression. Studies have shown that leukemic cells secrete C-chemokine (C-C motif) ligand 3 (CCL3), to disrupt the BMME resulting in loss of hematopoiesis and support of leukemic cell survival and proliferation. In this study, a murine model of blast crisis chronic myelogenous leukemia (bcCML) that expresses the translocation products BCR/ABL and Nup98/HoxA9 was used to determine the role of CCL3 in BMME regulation. Leukemic cells derived from CCL3-/- mice were shown to minimally engraft in a normal BMME, thereby demonstrating that CCL3 signaling was necessary to recapitulate bcCML disease. Further analysis showed disruption in hematopoiesis within the BMME in the bcCML model. To rescue the altered BMME, therapeutic inhibition of CCL3 signaling was investigated using bone-targeted nanoparticles (NP) to deliver Maraviroc, an inhibitor of C-C chemokine receptor type 5 (CCR5), a CCL3 receptor. NP-mediated Maraviroc delivery partially restored the BMME, significantly reduced leukemic burden, and improved survival. Overall, our results demonstrate that inhibiting CCL3 via CCR5 antagonism is a potential therapeutic approach to restore normal hematopoiesis as well as reduce leukemic burden within the BMME.

Chronic Stress Exposure Suppresses Mammary Tumor Growth and Reduces Circulating Exosome TGF-ß Content via ß-Adrenergic Receptor Signaling in MMTV-PyMT Mice.

Preclinical models of breast cancer have established mechanistic links between psychological stress and cancer progression. However, epidemiological evidence linking stress and cancer is equivocal. We tested the impact of stress exposure in female mice expressing the mouse mammary tumor virus polyoma middle-T antigen (MMTV-PyMT), a spontaneous model of mammary adenocarcinoma that mimics metastatic hormone receptor-positive human breast cancer development. MMTV-PyMT mice were socially isolated at 6 to 7 weeks of age during premalignant hyperplasia. To increase the potency of the stressor, singly housed mice were exposed to acute restraint stress (2 hours per day for 3 consecutive days) at 8 to 9 weeks of age during early carcinoma. Exposure to this dual stressor activated both major stress pathways, the sympathetic nervous system and hypothalamic-pituitary-adrenal axis throughout malignant transformation. Stressor exposure reduced mammary tumor burden in association with increased tumor cleaved caspase-3 expression, indicative of increased cell apoptosis. Stress exposure transiently increased tumor vascular endothelial growth factor and reduced tumor interleukin-6, but no other significant alterations in immune/inflammation-associated chemokines and cytokines or changes in myeloid cell populations were detected in tumors. No stress-induced change in second-harmonic generation-emitting collagen, indicative of a switch to a metastasis-promoting tumor extracellular matrix, was detected. Systemic indicators of slowed tumor progression included reduced myeloid-derived suppressor cell (MDSC) frequency in lung and spleen, and decreased transforming growth factor ß (TGF-ß) content in circulating exosomes, nanometer-sized particles associated with tumor progression. Chronic ß-adrenergic receptor (ß-AR) blockade with nadolol abrogated stress-induced alterations in tumor burden and cleaved caspase-3 expression, lung MDSC frequency, and exosomal TGF-ß content. Despite the evidence for reduced tumor growth, metastatic lesions in the lung were not altered by stress exposure. Unexpectedly, ß-blockade in nonstressed mice increased lung metastatic lesions and splenic MDSC frequency, suggesting that in MMTV-PyMT mice, ß-AR activation also inhibits tumor progression in the absence of stress exposure. Together, these results suggest stress exposure can act through ß-AR signaling to slow primary tumor growth in MMTV-PyMT mice.

The Chemokine CCL3 Regulates Myeloid Differentiation and Hematopoietic Stem Cell Numbers.

The chemokine CCL3 is frequently overexpressed in malignancies and overexpression leads to microenvironmental dysfunction. In murine models of chronic myelogenous leukemia (CML), CCL3 is critical for the maintenance of a leukemia stem cell population, and leukemia progression. With CCL3 implicated as a potentially viable therapeutic target, it is important to carefully characterize its role in normal hematopoietic homeostasis. CCL3-/- mice were used to evaluate the role of CCL3 in regulating hematopoietic stem and progenitor cell (HSPC) populations. CCL3-/- mice had loss of mature myeloid populations, while myeloid progenitors and HSPCs were increased, and microenvironmental populations were unchanged. These data show that CCL3 promotes myeloid lineage differentiation and the size of the HSPC pool independent of the supportive bone marrow microenvironment. Our results demonstrate a previously unrecognized role of CCL3 in the maintenance of homeostatic hematopoiesis that should be evaluated when targeting CCL3 signaling for the treatment of hematologic malignancy.

Discovery of a 2'-fluoro-2'-C-methyl C-nucleotide HCV polymerase inhibitor and a phosphoramidate prodrug with favorable properties.

A series of 2'-fluorinated C-nucleosides were prepared and tested for anti-HCV activity. Among them, the triphosphate of 2'-fluoro-2'-C-methyl adenosine C-nucleoside (15) was a potent and selective inhibitor of the NS5B polymerase and maintained activity against the S282T resistance mutant. A number of phosphoramidate prodrugs were then prepared and evaluated leading to the identification of the 1-aminocyclobutane-1-carboxylic acid isopropyl ester variant (53) with favorable pharmacokinetic properties including efficient liver delivery in animals.

Chemotherapeutic drug-specific alteration of microvascular blood flow in murine breast cancer as measured by diffuse correlation spectroscopy.

The non-invasive, in vivo measurement of microvascular blood flow has the potential to enhance breast cancer therapy monitoring. Here, longitudinal blood flow of 4T1 murine breast cancer (N=125) under chemotherapy was quantified with diffuse correlation spectroscopy based on layer models. Six different treatment regimens involving doxorubicin, cyclophosphamide, and paclitaxel at clinically relevant doses were investigated. Treatments with cyclophosphamide increased blood flow as early as 3 days after administration, whereas paclitaxel induced a transient blood flow decrease at 1 day after administration. Early blood flow changes correlated strongly with the treatment outcome and distinguished treated from untreated mice individually for effective treatments.

Discovery of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a potent nucleoside inhibitor of respiratory syncytial virus with excellent selectivity over mitochondrial RNA and DNA polymerases.

Novel 4'-substituted ß-d-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a promising nucleoside lead for RSV.

Discovery of GS-9669, a thumb site II non-nucleoside inhibitor of NS5B for the treatment of genotype 1 chronic hepatitis C infection.

Investigation of thiophene-2-carboxylic acid HCV NS5B site II inhibitors, guided by measurement of cell culture medium binding, revealed the structure-activity relationships for intrinsic cellular potency. The pharmacokinetic profile was enhanced through incorporation of heterocyclic ethers on the N-alkyl substituent. Hydroxyl groups were incorporated to modulate protein binding. Intrinsic potency was further improved through enantiospecific introduction of an olefin in the N-acyl motif, resulting in the discovery of the phase 2 clinical candidate GS-9669. The unexpected activity of this compound against the clinically relevant NS5B M423T mutant, relative to the wild type, was shown to arise from both the N-alkyl substituent and the N-acyl group.

Discovery of the first C-nucleoside HCV polymerase inhibitor (GS-6620) with demonstrated antiviral response in HCV infected patients.

Hepatitis C virus (HCV) infection presents an unmet medical need requiring more effective treatment options. Nucleoside inhibitors (NI) of HCV polymerase (NS5B) have demonstrated pan-genotypic activity and durable antiviral response in the clinic, and they are likely to become a key component of future treatment regimens. NI candidates that have entered clinical development thus far have all been N-nucleoside derivatives. Herein, we report the discovery of a C-nucleoside class of NS5B inhibitors. Exploration of adenosine analogs in this class identified 1'-cyano-2'-C-methyl 4-aza-7,9-dideaza adenosine as a potent and selective inhibitor of NS5B. A monophosphate prodrug approach afforded a series of compounds showing submicromolar activity in HCV replicon assays. Further pharmacokinetic optimization for sufficient oral absorption and liver triphosphate loading led to identification of a clinical development candidate GS-6620. In a phase I clinical study, the potential for potent activity was demonstrated but with high intra- and interpatient pharmacokinetic and pharmacodynamic variability.

Preclinical characterization of GS-9669, a thumb site II inhibitor of the hepatitis C virus NS5B polymerase.

GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC(50)) of ≤ 11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4. The M423T mutation is readily generated clinically upon monotherapy with the thumb site II inhibitors filibuvir and lomibuvir, and it is notable that GS-9669 exhibited only a 3-fold loss in potency against this variant in the genotype 1b replicon. Rather than M423T, resistance predominantly tracks to residues R422K and L419M and residue I482L in GT 1b and 1a replicons, respectively. GS-9669 exhibited at least additive activity in combination with agents encompassing four other direct modes of action (NS3 protease, NS5A, NS5B via an alternative allosteric binding site, and NS5B nucleotide) as well as with alpha interferon or ribavirin in replicon assays. It exhibited high metabolic stability in in vitro human liver microsomal assays, which, in combination with its pharmacokinetic profiles in rat, dog, and two monkey species, is predictive of good human pharmacokinetics. GS-9669 is well suited for combination with other orally active, direct-acting antiviral agents in the treatment of genotype 1 chronic HCV infection. (This study has been registered at ClinicalTrials.gov under registration number NCT01431898.).

Novel, potent and orally bioavailable indolizidinone-derived inhibitors of the hepatitis C virus NS3 protease.

A novel, potent, and orally bioavailable class of product-like inhibitors of the HCV NS3 protease was discovered by constraining the P2-P3 amide bond and the P3 hydrocarbon substituent to the protease-bound conformation. This preorganization was accomplished by incorporation of the P2-P3 amide into a six-membered ring attached to the P2-proline 5-position. Isothermal calorimetric characterization of the role of hydrocarbon substitution of this six-membered ring, upon binding the HCV NS3 protease, was found to be exclusively entropic in nature. The synthesis, preliminary SAR and pharmacokinetic profiling of this compact, indolizidinone-derived scaffold are described.

The total synthesis of (-)-crispatene.

The total synthesis of the molluscan polypropionate (-)-crispatene is described. The synthesis features a palladium-catalyzed cross-coupling to establish a sensitive conjugated tetraene and its Lewis acid-catalyzed cycloisomerization to yield the bicyclo[3.1.0]hexene core of the natural product. The absolute configuration of (-)-crispatene and related molecules is established.

Wavelength-dependent photofragmentation and photoionization of gaseous (eta4-cycloocta-1,5-diene)(eta5-cyclopentadienyl)cobalt.

Gas-phase photoreactions and photoproducts of the mixed-ligand compound (eta(4)-cycloocta-1,5-diene)(eta(5)-cyclopentadienyl)cobalt are reported. Significant amounts of the monoligated complexes CoCOD and CoCp are produced, and the relative amounts are wavelength dependent. The COD ligand (with the weakest metal-ligand bonds) is always preferentially labilized as expected, but the relative amounts of the CoCOD and CoCp fragments change by 1 order of magnitude as the excitation wavelength is changed. The gas-phase photoreactions exhibit other surprising features that are uncommon in the photoreactions of organometallic compounds in the gas phase. Significant amounts of the intact cation are formed, in contrast to most reported reactions where fragmentation of the weak metal-ligand bonds precedes ionization. Most surprisingly, fragmentation of the ligands occurs while the ligands are still coordinated. The resulting metal complexes contain ligand fragments that remain coordinated to the metal. Breaking several carbon-carbon bonds with retention of weaker metal-ligand bonds is unexpected. For example, C(5)H(5) undergoes fragmentation while still coordinated to the cobalt, yielding smaller compounds such as Co(CH)(+), Co(C(2)H(2))(+), Co(C(3)H(3))(+), and Co(C(4)H(6))(+). Correspondingly, coordinated COD yields Co(C(6)H(6))(+), Co(C(5)H(5))(+), Co(C(4)H(6))(+), Co(C(3)H(3))(+), Co(C(2)H(2))(+), and Co(CH)(+). The wavelength dependence of the ligand labilization is examined by utilizing mass-selected resonance enhanced multiphoton ionization spectroscopy. Both broad bands and sharp lines are observed in the mass-selected excitation spectra. The action spectra obtained in the gas phase while monitoring the cobalt ion follow the absorption onset found in solution. The changes in fragmentation pathways are interpreted in terms of the initially accessed excited state.