Novel Small Molecule ROCK2 Inhibitor GNS-3595 Attenuates Pulmonary Fibrosis in Preclinical Studies.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that leads to respiratory decline due to scarring and thickening of lung tissues. Multiple pathways contribute to the fibrotic process in this disease, such as inflammation, epithelial to mesenchymal transition and oxidative stress. The RhoA/ROCK signaling pathway is a key regulator of profibrotic signaling, as it affects the organization of actin-myosin and the remodeling of the extracellular matrix. ROCK1/2, a downstream effector of RhoA, is overexpressed in IPF patients and is a promising target for IPF therapy. However, due to hypotensive side effects of ROCK1/2 inhibitors, selective ROCK2 compounds are being explored. In this study, we report the discovery of GNS-3595, a potent and selective ROCK2 inhibitor that has ~80-fold selectivity over ROCK1 at physiological concentrations of ATP. GNS-3595 effectively inhibited ROCK2-mediated phosphorylation of myosin light chain (p-MLC) and reduced the expression of fibrosis-related proteins, such as collagen, fibronectin, and alpha-smooth muscle actin (α-SMA) in various in vitro cellular models. GNS-3595 also prevented transforming growth factor beta (TGF-ß)-induced fibroblast-to-myofibroblast transition (FMT). Additionally, in a bleomycin-induced mouse model of pulmonary fibrosis, therapeutic exposure to GNS-3595, suppressed lung fibrosis, stabilized body weight loss, and prevented fibrosis-induced lung weight gain. Transcriptome and protein expression analysis from lung tissues showed that GNS-3595 can revert the fibrosis-related gene expression induced by bleomycin. These results indicate that GNS-3595 is a highly potent, selective, and orally active ROCK2 inhibitor with promising therapeutic efficacy against pulmonary fibrosis.

AZD4320, A Dual Inhibitor of Bcl-2 and Bcl-xL, Induces Tumor Regression in Hematologic Cancer Models without Dose-limiting Thrombocytopenia.

PURPOSE: Targeting Bcl-2 family members upregulated in multiple cancers has emerged as an important area of cancer therapeutics. While venetoclax, a Bcl-2-selective inhibitor, has had success in the clinic, another family member, Bcl-xL, has also emerged as an important target and as a mechanism of resistance. Therefore, we developed a dual Bcl-2/Bcl-xL inhibitor that broadens the therapeutic activity while minimizing Bcl-xL-mediated thrombocytopenia. EXPERIMENTAL DESIGN: We used structure-based chemistry to design a small-molecule inhibitor of Bcl-2 and Bcl-xL and assessed the activity against in vitro cell lines, patient samples, and in vivo models. We applied pharmacokinetic/pharmacodynamic (PK/PD) modeling to integrate our understanding of on-target activity of the dual inhibitor in tumors and platelets across dose levels and over time. RESULTS: We discovered AZD4320, which has nanomolar affinity for Bcl-2 and Bcl-xL, and mechanistically drives cell death through the mitochondrial apoptotic pathway. AZD4320 demonstrates activity in both Bcl-2- and Bcl-xL-dependent hematologic cancer cell lines and enhanced activity in acute myeloid leukemia (AML) patient samples compared with the Bcl-2-selective agent venetoclax. A single intravenous bolus dose of AZD4320 induces tumor regression with transient thrombocytopenia, which recovers in less than a week, suggesting a clinical weekly schedule would enable targeting of Bcl-2/Bcl-xL-dependent tumors without incurring dose-limiting thrombocytopenia. AZD4320 demonstrates monotherapy activity in patient-derived AML and venetoclax-resistant xenograft models. CONCLUSIONS: AZD4320 is a potent molecule with manageable thrombocytopenia risk to explore the utility of a dual Bcl-2/Bcl-xL inhibitor across a broad range of tumor types with dysregulation of Bcl-2 prosurvival proteins.

Structure-Based Design and Identification of FT-2102 (Olutasidenib), a Potent Mutant-Selective IDH1 Inhibitor.

Inhibition of mutant IDH1 is being evaluated clinically as a treatment option for oncology. Here we describe the structure-based design and optimization of quinoline lead compounds to identify FT-2102, a potent, orally bioavailable, brain penetrant, and selective mIDH1 inhibitor. FT-2102 has excellent ADME/PK properties and reduces 2-hydroxyglutarate levels in an mIDH1 xenograft tumor model. This compound has been selected as a candidate for clinical development in hematologic malignancies, solid tumors, and gliomas with mIDH1.

Discovery and Optimization of Quinolinone Derivatives as Potent, Selective, and Orally Bioavailable Mutant Isocitrate Dehydrogenase 1 (mIDH1) Inhibitors.

Mutations at the arginine residue (R132) in isocitrate dehydrogenase 1 (IDH1) are frequently identified in various human cancers. Inhibition of mutant IDH1 (mIDH1) with small molecules has been clinically validated as a promising therapeutic treatment for acute myeloid leukemia and multiple solid tumors. Herein, we report the discovery and optimization of a series of quinolinones to provide potent and orally bioavailable mIDH1 inhibitors with selectivity over wild-type IDH1. The X-ray structure of an early lead 24 in complex with mIDH1-R132H shows that the inhibitor unexpectedly binds to an allosteric site. Efforts to improve the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) properties of 24 yielded a preclinical candidate 63. The detailed preclinical ADME and pharmacology studies of 63 support further development of quinolinone-based mIDH1 inhibitors as therapeutic agents in human trials.

Towards the next generation of dual Bcl-2/Bcl-xL inhibitors.

Structural modifications of the left-hand side of compound 1 were identified which retained or improved potent binding to Bcl-2 and Bcl-xL in in vitro biochemical assays and had strong activity in an RS4;11 apoptotic cellular assay. For example, sulfoxide diastereomer 13 maintained good binding affinity and comparable cellular potency to 1 while improving aqueous solubility. The corresponding diastereomer (14) was significantly less potent in the cell, and docking studies suggest that this is due to a stereochemical preference for the RS versus SS sulfoxide. Appending a dimethylaminoethoxy side chain (27) adjacent to the benzylic position of the biphenyl moiety of 1 improved cellular activity by approximately three-fold, and this activity was corroborated in cell lines overexpressing Bcl-2 and Bcl-xL.

Mitigation of cardiovascular toxicity in a series of CSF-1R inhibitors, and the identification of AZD7507.

The potent and selective 3-amido-4-anilinoquinoline CSF-1R inhibitor AZ683 suffered from cardiovascular liabilities, which were linked to the off-target activities of the compound and ion channel activity in particular. Less basic and less lipophilic examples from both the quinoline and cinnoline series demonstrated cleaner secondary pharmacology profiles. Cinnoline 31 retained the required potency and oral PK profile, and was progressed through the safety screening cascade to be nominated into development as AZD7507.

3-amido-4-anilinocinnolines as a novel class of CSF-1R inhibitor.

3-Amido-4-anilinocinnolines have been identified as potent and highly selective inhibitors of CSF-1R. The synthesis and SAR of these compounds is reported, along with some physical property, pharmacokinetic and kinase selectivity data.

Syntheses of potent, selective, and orally bioavailable indazole-pyridine series of protein kinase B/Akt inhibitors with reduced hypotension.

Compound 7 was identified as a potent (IC50 = 14 nM), selective, and orally bioavailable (F = 70% in mouse) inhibitor of protein kinase B/Akt. While promising efficacy was observed in vivo, this compound showed effects on depolarization of Purkinje fibers in an in vitro assay and CV hypotension in vivo. Guided by an X-ray structure of 7 bound to protein kinase A, which has 80% homology with Akt in the kinase domain, our efforts have focused on structure-activity relationship (SAR) studies of the phenyl moiety, in an attempt to address the cardiovascular liability and further improve the Akt potency. A novel and efficient synthetic route toward diversely substituted phenyl derivatives of 7 was developed utilizing a copper-mediated aziridine ring-opening reaction as the key step. To improve the selectivity of these Akt inhibitors over other protein kinases, a nitrogen atom was incorporated into selected phenyl analogues of 7 at the C-6 position of the methyl indazole scaffold. These modifications resulted in the discovery of inhibitor 37c with greater potency (IC50 = 0.6 nM vs Akt), selectivity, and improved cardiovascular safety profile. The SARs, pharmacokinetic profile, and CV safety of selected Akt inhibitors will be discussed.

Differential gene expression between human schwannoma and control Schwann cells.

The NF2 gene encodes the tumour suppressor protein merlin. The mutation of a single allele of this gene causes the autosomal dominantly inherited disease neurofibromatosis type 2 (NF2), which is characterized mainly by vestibular schwannoma carrying a second hit mutation. Complete lack of merlin is also found in spontaneous schwannomas and meningiomas. As the events leading to schwannoma development are largely unknown we investigated the differences in gene expression between schwannoma cells from NF2 patients and normal human primary Schwann cells by cDNA array analysis. We identified 41 genes whose expression levels differed by more than factor 2. Most of these clones were corroborated by real-time reverse transcription polymerase chain reaction analysis. By this method a total of seven genes with increased and seven genes with decreased mRNA levels in schwannoma compared with normal Schwann cells could be identified. Regulated clones, some of which not been described in Schwann cells earlier, included matrix metalloproteinase's, growth factors, growth factor receptors and tyrosine kinases.

Transforming growth factor beta1 enhances platelet aggregation through a non-transcriptional effect on the fibrinogen receptor.

Upon activation, platelets store and release large amounts of the peptide transforming growth factor beta1 (TGFbeta1). The released TGFbeta1 can then act on nearby vascular cells to mediate subsequent vessel repair. In addition, TGFbeta1 may circulate to bone marrow and regulate megakaryocyte activity. It is not known what effect, if any, TGFbeta1 has on platelets. Adult TGFbeta1-deficient mice exhibit thrombocythemia and a mild bleeding disorder that is shown to result from faulty platelet aggregation. TGFbeta1-deficient platelets are shown to contain functional receptors, and preincubation with recombinant TGFbeta1 improves aggregation, demonstrating that TGFbeta1 plays an active role in platelet aggregation. TGFbeta1-deficient platelets fail to retain bound fibrinogen in response to aggregation agonists, but they possess normal levels of the alpha(IIb)/beta(3) fibrinogen receptor. Signaling from agonist receptors is normal because the platelets change shape, produce thromboxane A(2), and present P-selectin in response to stimulation. Consequently, activation and maintenance of alpha(IIb)/beta(3) into a fibrinogen-binding conformation is impaired in the absence of TGFbeta1. 4-Phorbol 12-myristate 13-acetate treatment and protein kinase C activity measurements suggest a defect downstream of protein kinase C in its activation cascade. Because platelets lack nuclei, these data demonstrate for the first time a non-transcriptionally mediated TGFbeta1 signaling pathway that enhances the activation and maintenance of integrin function.

Molecular basis of cooperative DNA bending and oriented heterodimer binding in the NFAT1-Fos-Jun-ARRE2 complex.

Cooperative DNA binding by transcription factors that bind to separate recognition sites is likely to require bending of intervening sequences and the appropriate orientation of transcription factor binding. We investigated DNA bending in complexes formed by the basic region-leucine zipper domains of Fos and Jun with the DNA binding region of nuclear factor of activated T cells 1 (NFAT1) at composite regulatory elements using gel electrophoretic phasing analysis. The NFAT1-Fos-Jun complex induced a bend at the ARRE2 site that was distinct from the sum of the bends induced by NFAT1 and Fos-Jun separately. We designate this difference DNA bending cooperativity. The bending cooperativity was directed toward the interaction interface between Fos-Jun and NFAT1. We also examined the influence of NFAT1 on the orientation of Fos-Jun heterodimer binding using a novel fluorescence resonance energy transfer assay. The interaction with NFAT1 could reverse the orientation of Fos-Jun heterodimer binding to the ARRE2 site. The principal determinants of both cooperative DNA bending and oriented heterodimer binding were localized to three amino acid residues at the amino-terminal ends of the leucine zippers of Fos and Jun. Consequently, interactions between transcription factors can remodel promoters by altering DNA bending and the orientation of heterodimer binding.

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

Massive pulmonary thromboembolism after manipulation of an unstable pelvic fracture: a case report and review of the literature.

Acute pulmonary thromboembolism has not been reported in association with manipulation of an unstable pelvic fracture. We report a case of pulmonary thromboembolism following manipulation of an unstable pelvic fracture one week after injury.

Onset and progression of pathological lesions in transforming growth factor-beta 1-deficient mice.

Null-mutant (knockout) mice were obtained through disruption of the sixth exon of the endogenous transforming growth factor-beta 1 allele in murine embryonic stem cells via homologous recombination. Mice lacking transforming growth factor-beta 1 (mutants) were born grossly indistinguishable from wild-type littermates. With time, mutant mice exhibited a wasting phenotype that manifested itself in severe weight loss and dishevelled appearance (between 15 and 36 days of age). Examination of these moribund mice histologically revealed that transforming growth factor-beta 1-deficient mice exhibit a moderate to severe, multifocal, organ-dependent, mixed inflammatory cell response adversely affecting the heart, stomach, diaphragm, liver, lung, salivary gland, and pancreas. Because of the known multifunctional nature of transforming growth factor-beta 1 on the control of growth and differentiation of many different cell types, it is important to determine the degree to which the inflammatory response interacts with or masks other deficiencies that are present. To this end, we examined the extent and nature of the inflammatory lesions in different ages of neonatal knockout mice (5, 7, 10, and 14 days of age) and older moribund mice (> 15 days of age) and compared them with the histology seen in wild-type normal animals. Mild inflammatory infiltrates were first observed in 5-day mutant mice in the heart, by day 7 in the lung, salivary gland, and pancreas, and by day 14 inflammatory lesions were found in almost all organs examined. Moderate to severe inflammation was not present until the mice were 10 to 14 days old. In the older animals, there was a slight increase in the severity of the inflammatory lesions as the mice aged.

Inhibition of Rhizobium etli Polysaccharide Mutants by Phaseolus vulgaris Root Compounds.

Crude bean root extracts of Phaseolus vulgaris were tested for inhibition of the growth of several polysaccharide mutants of Rhizobium etli biovar phaseoli CE3. Mutants deficient only in exopolysaccharide and some mutants deficient only in the O-antigen of the lipopolysaccharide were no more sensitive than the wild-type strain to the extracts, whereas mutants defective in both lipopolysaccharide and exopolysaccharide were much more sensitive. The inhibitory activity was found at much higher levels in roots and nodules than in stems or leaves. Inoculation with either wild-type or polysaccharide-deficient R. etli did not appear to affect the level of activity. Sequential extractions of the crude root material with petroleum ether, ethyl acetate, methanol, and water partitioned inhibitory activity into each solvent except methanol. The major inhibitors in the petroleum ether and ethyl acetate extracts were purified by C(18) high-performance liquid chromatography. These compounds all migrated very similarly in both liquid and thin-layer chromatography but were distinguished by their mass spectra. Absorbance spectra and fluorescence properties suggested that they were coumestans, one of which had the mass spectrum and nuclear magnetic resonances of coumestrol. These results are discussed with regard to the hypothesis that one role of rhizobial polysaccharides is to protect against plant toxins encountered during nodule development.

Infection of soybean and pea nodules by Rhizobium spp. purine auxotrophs in the presence of 5-aminoimidazole-4-carboxamide riboside.

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth.

Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease.

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional growth factor that has profound regulatory effects on many developmental and physiological processes. Disruption of the TGF-beta 1 gene by homologous recombination in murine embryonic stem cells enables mice to be generated that carry the disrupted allele. Animals homozygous for the mutated TGF-beta 1 allele show no gross developmental abnormalities, but about 20 days after birth they succumb to a wasting syndrome accompanied by a multifocal, mixed inflammatory cell response and tissue necrosis, leading to organ failure and death. TGF-beta 1-deficient mice may be valuable models for human immune and inflammatory disorders, including autoimmune diseases, transplant rejection and graft versus host reactions.

Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart.

Several clones were isolated from a rat genomic library in order to further characterize a region of variability within the third membrane-spanning region of the fourth motif (IVS3) of the L-type voltage-dependent calcium channel. We report here that this diversity arises from alternative splicing of a primary transcript containing a single pair of adjacent exons each encoding a unique sequence for the IVS3 region. Definitive proof of a mutually exclusive splicing mechanism was obtained by genomic mapping of flanking upstream and downstream exons and by extensive sequence analysis of the relevant exon/intron boundaries. S1 nuclease protection experiments revealed that both variant forms of the IVS3 were equally expressed in newborn and fetal rat heart, whereas only a single isoform predominated in adult rat heart. The results demonstrate the existence of an important developmentally regulated switch mediated by alternatively spliced exons in cardiac tissue at a time when major changes in excitation occur.

Molecular cloning of multiple subtypes of a novel rat brain isoform of the alpha 1 subunit of the voltage-dependent calcium channel.

Several cDNAs encoding an isoform of the alpha 1 subunit of the voltage-dependent calcium channel were isolated from rat brain cDNA libraries. The complete nucleotide sequence of 6975 bp encodes a protein of 1634 amino acids, which corresponds to an Mr of 186,968. The protein exhibits 71% and 76% homology to skeletal and cardiac alpha 1 subunits, respectively. When compared with skeletal and cardiac alpha 1 isoforms, the rat brain protein is intermediate in size at the amino terminus and shorter at the carboxyl terminus. Multiple subtypes of this alpha 1 isoform cDNA were characterized. These are indicative of alternative splicing of a primary transcript and encode three variants between motif I and motif II and two within the S3 region of motif IV. Thus, multiple isoforms of this rat brain alpha 1 subunit are possible.

Rhizobium leguminosarum exopolysaccharide mutants: biochemical and genetic analyses and symbiotic behavior on three hosts.

Ten independently generated mutants of Rhizobium leguminosarum biovar phaseoli CFN42 isolated after Tn5 mutagenesis formed nonmucoid colonies on all agar media tested and lacked detectable production of the normal acidic exopolysaccharide in liquid culture. The mutants were classified into three groups. Three mutants harbored Tn5 insertions on a 3.6-kilobase-pair EcoRI fragment and were complemented to have normal exopolysaccharide production by cosmids that shared an EcoRI fragment of this size from the CFN42 genome. The Tn5 inserts of five other mutants appeared to be located on a second, slightly smaller EcoRI fragment. Attempts to complement mutants of this second group with cloned DNA were unsuccessful. The mutations of the other two mutants were located in apparently adjacent EcoRI fragments carried on two cosmids that complemented those two mutants. The latter two mutants also lacked O-antigen-containing lipopolysaccharides and induced underdeveloped nodules that lacked nitrogenase activity on bean plants. The other eight mutants had normal lipopolysaccharides and wild-type symbiotic proficiencies on bean plants. Mutants in each of these groups were mated with R. leguminosarum strains that nodulated peas (R. leguminosarum biovar viciae) or clovers (R. leguminosarum biovar trifolii). Transfer of the Tn5 mutations resulted in exopolysaccharide-deficient R. leguminosarum biovar viciae or R. leguminosarum biovar trifolii transconjugants that were symbiotically deficient in all cases. These results support earlier suggestions that successful symbiosis with peas or clovers requires that rhizobia be capable of acidic exopolysaccharide production, whereas symbiosis with beans does not have this requirement.