Zetomipzomib (KZR-616) attenuates lupus in mice via modulation of innate and adaptive immune responses.

Zetomipzomib (KZR-616) is a selective inhibitor of the immunoproteasome currently undergoing clinical investigation in autoimmune disorders. Here, we characterized KZR-616 in vitro and in vivo using multiplexed cytokine analysis, lymphocyte activation and differentiation, and differential gene expression analysis. KZR-616 blocked production of >30 pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs), polarization of T helper (Th) cells, and formation of plasmablasts. In the NZB/W F1 mouse model of lupus nephritis (LN), KZR-616 treatment resulted in complete resolution of proteinuria that was maintained at least 8 weeks after the cessation of dosing and was mediated in part by alterations in T and B cell activation, including reduced numbers of short and long-lived plasma cells. Gene expression analysis of human PBMCs and tissues from diseased mice revealed a consistent and broad response focused on inhibition of T, B, and plasma cell function and the Type I interferon pathway and promotion of hematopoietic cell lineages and tissue remodeling. In healthy volunteers, KZR-616 administration resulted in selective inhibition of the immunoproteasome and blockade of cytokine production following ex vivo stimulation. These data support the ongoing development of KZR-616 in autoimmune disorders such as systemic lupus erythematosus (SLE)/LN.

Co-inhibition of immunoproteasome subunits LMP2 and LMP7 enables prevention of transplant arteriosclerosis.

AIMS: The loss of vascular wall cells in allotransplanted arteries is the initial event leading to transplant arteriosclerosis (TA) and ensuing loss of allograft function. Pharmacological agents able to prevent TA are currently lacking. We previously showed that selective inhibition of the immunoproteasome prevented the chronic rejection of renal allografts. However, the role and mechanisms of selective inhibition of a single immunoproteasome subunit to prevent immune-mediated vascular allograft rejection and TA is not clear. METHODS AND RESULTS: The effect and potential mechanism of combined or individual inhibition of peptidolytically active immunoproteasome LMP7 (ß5i) and LMP2 (ß1i) subunits on immune rejection-mediated TA was investigated using the epoxyketone inhibitor ONX 0914, and the recently developed LMP7-selective inhibitor KZR-329 and LMP2-selective inhibitor KZR-504 in a rat aorta transplantation model. We find that co-inhibition of LMP7 and LMP2 in allogeneic recipients significantly suppressed T-cell activation and function by expressing inhibitory surface markers and then activating inhibitory signals. Moreover, co-inhibition of LMP7 and LMP2 substantially reduced the number of immunoglobulin G-secreting cells and plasma cells and production of alloantibodies through activating the unfolded protein response and incapacitating the survival niche of plasma cells in the bone marrow. Consequentially, the accumulation of inflammatory cytokines, complement, and antibodies is reduced and the apoptosis of vascular wall cells decreased in aortic allografts via LMP7 and LMP2 co-inhibition with ONX 0914 treatment or combined KZR-329 and KZR-504 treatment. However, neither individual inhibition of LMP7 by KZR-329 nor individual inhibition of LMP2 by KZR-504 showed suppression of immune rejection and TA. CONCLUSIONS: We define a critical role of LMP7 and LMP2 in TA and strongly propose co-inhibition of both immunoproteasome subunits as promising therapeutic approach to suppress TA and allograft rejection.

The Impact of Brown Stink Bug (Hemiptera: Pentatomidae) Damage during the Seedling Stage on Field Corn Growth and Yield.

Brown stink bug, Euschistus servus (Say) (Hemiptera: Pentatomidae), is a common insect that can infest corn fields in the Mid-South and Southeastern U.S. Infestations and damage are sporadic, thus little research has been conducted on the impact of brown stink bug infesting corn seedlings. Two experiments were conducted in eleven commercial corn fields in the Mississippi Delta to evaluate the impact of damage from natural stink bug infestations during the seedling stage (

Required Immunoproteasome Subunit Inhibition Profile for Anti-Inflammatory Efficacy and Clinical Candidate KZR-616 ((2 S,3 R)- N-(( S)-3-(Cyclopent-1-en-1-yl)-1-(( R)-2-methyloxiran-2-yl)-1-oxopropan-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-(( S)-2-(2-morpholinoacetamido)propanamido)propenamide).

Selective immunoproteasome inhibition is a promising approach for treating autoimmune disorders, but optimal proteolytic active site subunit inhibition profiles remain unknown. We reveal here our design of peptide epoxyketone-based selective low molecular mass polypeptide-7 (LMP7) and multicatalytic endopeptidase complex subunit-1 (MECL-1) subunit inhibitors. Utilizing these and our previously disclosed low molecular mass polypeptide-2 (LMP2) inhibitor, we demonstrate a requirement of dual LMP7/LMP2 or LMP7/MECL-1 subunit inhibition profiles for potent cytokine expression inhibition and in vivo efficacy in an inflammatory disease model. These and additional findings toward optimized solubility led the design and selection of KZR-616 disclosed here and presently in clinical trials for treatment of rheumatic disease.

Discovery of Highly Selective Inhibitors of the Immunoproteasome Low Molecular Mass Polypeptide 2 (LMP2) Subunit.

Building upon the success of bortezomib (VELCADE) and carfilzomib (KYPROLIS), the design of a next generation of inhibitors targeting specific subunits within the immunoproteasome is of interest for the treatment of autoimmune disease. There are three catalytic subunits within the immunoproteasome (low molecular mass polypeptide-7, -2, and multicatalytic endopeptidase complex subunit-1; LMP7, LMP2, and MECL-1), and a campaign was undertaken to design a potent and selective LMP2 inhibitor with sufficient properties to allow for sustained inhibition in vivo. Screening a focused library of epoxyketones revealed a series of potent dipeptides that were optimized to provide the highly selective inhibitor KZR-504 (12).

Discovery of a novel class of highly potent, selective, ATP-competitive, and orally bioavailable inhibitors of the mammalian target of rapamycin (mTOR).

A series of novel, highly potent, selective, and ATP-competitive mammalian target of rapamycin (mTOR) inhibitors based on a benzoxazepine scaffold have been identified. Lead optimization resulted in the discovery of inhibitors with low nanomolar activity and greater than 1000-fold selectivity over the closely related PI3K kinases. Compound 28 (XL388) inhibited cellular phosphorylation of mTOR complex 1 (p-p70S6K, pS6, and p-4E-BP1) and mTOR complex 2 (pAKT (S473)) substrates. Furthermore, this compound displayed good pharmacokinetics and oral exposure in multiple species with moderate bioavailability. Oral administration of compound 28 to athymic nude mice implanted with human tumor xenografts afforded significant and dose-dependent antitumor activity.

SAR and in vivo evaluation of 4-aryl-2-aminoalkylpyrimidines as potent and selective Janus kinase 2 (JAK2) inhibitors.

We report the discovery of a series of 4-aryl-2-aminoalkylpyrimidine derivatives as potent and selective JAK2 inhibitors. High throughput screening of our in-house compound library led to the identification of hit 1, from which optimization resulted in the discovery of highly potent and selective JAK2 inhibitors. Advanced lead 10d demonstrated a significant dose-dependent pharmacodynamic and antitumor effect in a mouse xenograft model. Based upon the desirable profile of 10d (XL019) it was advanced into clinical trials.

Discovery of a novel series of potent and orally bioavailable phosphoinositide 3-kinase γ inhibitors.

The phosphoinositide 3-kinases (PI3Ks) have been linked to an extraordinarily diversified group of cellular functions making these enzymes compelling targets for the treatment of disease. A large body of evidence has linked PI3Kγ to the modulation of autoimmune and inflammatory processes making it an intriguing target for drug discovery. Our high-throughput screening (HTS) campaign revealed two hits that were nominated for further optimization studies. The in vitro activity of the first HTS hit, designated as the sulfonylpiperazine scaffold, was optimized utilizing structure-based design. However, nonoptimal pharmacokinetic properties precluded this series from further studies. An overlay of the X-ray structures of the sulfonylpiperazine scaffold and the second HTS hit within their complexes with PI3Kγ revealed a high degree of overlap. This feature was utilized to design a series of hybrid analogues including advanced leads such as 31 with desirable potency, selectivity, and oral bioavailability.

Discovery of a novel class of potent and orally bioavailable sphingosine 1-phosphate receptor 1 antagonists.

A series of subtype selective sphingosine 1-phosphate receptor 1 (S1P(1)) antagonists are disclosed. Our high-throughput screening campaign revealed hit 1 for which an increase in potency and mouse oral exposure was achieved with minor modifications to the chemical scaffold. In vivo efficacy revealed that at high doses compounds 12 and 15 inhibited tumor growth. Further optimization of our lead series led to the discovery of proline derivatives 37 (XL541) and 38 which had similar efficacy as our first generation analogues at significantly lower doses. Analogue 37 displayed excellent pharmacokinetics and oral exposure in multiple species.

Integrated database for identifying candidate genes for Aspergillus flavus resistance in maize.

BACKGROUND: Aspergillus flavus Link:Fr, an opportunistic fungus that produces aflatoxin, is pathogenic to maize and other oilseed crops. Aflatoxin is a potent carcinogen, and its presence markedly reduces the value of grain. Understanding and enhancing host resistance to A. flavus infection and/or subsequent aflatoxin accumulation is generally considered an efficient means of reducing grain losses to aflatoxin. Different proteomic, genomic and genetic studies of maize (Zea mays L.) have generated large data sets with the goal of identifying genes responsible for conferring resistance to A. flavus, or aflatoxin. RESULTS: In order to maximize the usage of different data sets in new studies, including association mapping, we have constructed a relational database with web interface integrating the results of gene expression, proteomic (both gel-based and shotgun), Quantitative Trait Loci (QTL) genetic mapping studies, and sequence data from the literature to facilitate selection of candidate genes for continued investigation. The Corn Fungal Resistance Associated Sequences Database (CFRAS-DB) (http://agbase.msstate.edu/) was created with the main goal of identifying genes important to aflatoxin resistance. CFRAS-DB is implemented using MySQL as the relational database management system running on a Linux server, using an Apache web server, and Perl CGI scripts as the web interface. The database and the associated web-based interface allow researchers to examine many lines of evidence (e.g. microarray, proteomics, QTL studies, SNP data) to assess the potential role of a gene or group of genes in the response of different maize lines to A. flavus infection and subsequent production of aflatoxin by the fungus. CONCLUSIONS: CFRAS-DB provides the first opportunity to integrate data pertaining to the problem of A. flavus and aflatoxin resistance in maize in one resource and to support queries across different datasets. The web-based interface gives researchers different query options for mining the database across different types of experiments. The database is publically available at http://agbase.msstate.edu.

A polyether biotoxin binding site on the lipid-exposed face of the pore domain of Kv channels revealed by the marine toxin gambierol.

Gambierol is a marine polycyclic ether toxin belonging to the group of ciguatera toxins. It does not activate voltage-gated sodium channels (VGSCs) but inhibits Kv1 potassium channels by an unknown mechanism. While testing whether Kv2, Kv3, and Kv4 channels also serve as targets, we found that Kv3.1 was inhibited with an IC(50) of 1.2 +/- 0.2 nM, whereas Kv2 and Kv4 channels were insensitive to 1 microM gambierol. Onset of block was similar from either side of the membrane, and gambierol did not compete with internal cavity blockers. The inhibition did not require channel opening and could not be reversed by strong depolarization. Using chimeric Kv3.1-Kv2.1 constructs, the toxin sensitivity was traced to S6, in which T427 was identified as a key determinant. In Kv3.1 homology models, T427 and other molecular determinants (L348, F351) reside in a space between S5 and S6 outside the permeation pathway. In conclusion, we propose that gambierol acts as a gating modifier that binds to the lipid-exposed surface of the pore domain, thereby stabilizing the closed state. This site may be the topological equivalent of the neurotoxin site 5 of VGSCs. Further elucidation of this previously undescribed binding site may explain why most ciguatoxins activate VGSCs, whereas others inhibit voltage-dependent potassium (Kv) channels. This previously undescribed Kv neurotoxin site may have wide implications not only for our understanding of channel function at the molecular level but for future development of drugs to alleviate ciguatera poisoning or to modulate electrical excitability in general.

Total synthesis of gambierol: the generation of the A-C and F-H subunits by using a C-glycoside centered strategy.

Gambierol, a representative of the marine ladder toxin family, consists of eight ether rings, 18 stereocenters, and two challenging pyranyl rings having methyl groups that are in a 1,3-diaxial orientation to one another. Herein we describe the generation of gambierol's A-C and F-H ring systems and demonstrate the versatility of the glycosyl anhydride, enol ether-olefin RCM strategy to fused polycyclic ethers. This work has both enabled us to generate sufficient quantities of the gambierol precursors and has enabled us to better understand the chemical transformations that were key to these efforts. Fundamental work included efforts to C-glycosides and C-ketosides, Claisen rearrangements, and enol ether-olefin RCM reactions.

Total synthesis of gambierol: subunit coupling and completion.

The preceding manuscript detailed our synthesis of the gambierol A-C and F-H ring precursors. Reported herein is a description of the coupling of the two precursors and the conversion of the coupled material into gambierol. Coupling of the subunits involved ester formation, enol ether RCM, and mixed thioketal formation and reduction. By employing this strategy we were able to bring highly advanced subunits into the coupling and, as a result, we were able to minimize the number of post-coupling transformations required to complete gambierol. At the completion of the synthesis, we generated 7.5 mg (1.5 % overall yield) of (-)-gambierol in 44 steps (longest linear sequence).

The total synthesis of gambierol.

This communication describes the total synthesis of the marine polyether toxin, gambierol. This work couples our iterative C-glycoside/enol ether-olefin metathesis strategy to the subunits with a unique olefin metathesis/carbonyl olefination reaction to bring the subunits together.

Total ear canal ablation combining bulla osteotomy and curettage in dogs with chronic otitis externa and media.

Ear canal ablation combining bulla osteotomy and curettage was performed on 44 dogs (n = 72 ears). Indications for the procedure included one or more of the following: chronic nonresponsive otitis externa and/or media (n = 71), tumor in the horizontal portion of the ear canal (n = 1), failed lateral ear resection (n = 11), ossified auricular cartilages secondary to chronic otitis externa (n = 22), failed previous total ear canal ablation (n = 1), and otitis interna (n = 1). In 40 dogs, the surgery was successful in alleviating all clinical signs of otitis externa and media. During the immediate postoperative period, 2 dogs died of causes unrelated to otitis. Complications related to the surgery developed in 9 of the surviving 42 dogs. Ultimately, 95% (40 of the surviving 42) of the dogs were cured by use of this procedure. Surgery successfully resolved the original problems in 97% (66 of 68) of the surgically treated ears of these dogs.

Transarticular pinning for repair of hip dislocation in the dog: a retrospective study of 40 cases.

Transarticular pinning was used for the repair of hip dislocation in 40 dogs. The mean follow-up period was 18.4 months. Satisfactory results were achieved in 80% of the cases. Body weight, ipsilateral femoral head fracture, and hip dysplasia appeared to affect long-term prognosis. The most frequent complication was pin breakage, but this did not affect final outcome and could be avoided by using pins of larger diameter. Osteonecrosis was observed in two cases.

Feline model for the study of frozen osteoarticular hemijoint transplantation: qualitative and quantitative assessment of bone healing.

Twenty-four outbred cats underwent massive osteoarticular allograft and control autograft transplantation, using the right distal femur with its articular cartilage, capsule, and medial collateral ligament intact. The cats were monitored clinically and radiographically for 1 year. Groups of cats (4 allografts and 2 control autografts) were euthanatized at 3-, 6-, 9-, and 12-month intervals. At necropsy, the grafts were photographed and assessed for bone healing and replacement by standard radiography, quantitative 99mTc bone scans, microradiography, and histologic examination of decalcified and nondecalcified specimens. The osteosynthesis site of the allografts usually healed by 5 months, compared with the autografts that healed by 3 months. As illustrated by quantitative bone scans, creeping appositional new bone slowly invaded and replaced the allograft bone. Seemingly, the cat can be used as an acceptable and clinically comparable model for the massive osteoarticular allografts currently being used for the reconstruction of joints damaged or destroyed by neoplasm surgery in limb-sparing procedures in human beings. This model may also be used to assess the rate and method of bone healing.

Radiographic diagnosis and surgical management of fragmented medial coronoid process in dogs.

The diagnosis of fragmented (ununited) medial coronoid process was made in 21 dogs, with 10 dogs having bilateral lesions. Ten of these dogs were presented to surgery. The diagnosis of fragmented medial coronoid process was made in young and middle-aged large- and giant-breed dogs with a history of intermittent or persistent lameness. Diagnosis was based on history as well as results of clinical examination and radiographic study of both elbows. Concomitant elbow lesions seen with fragmented medial coronoid processes were ununited anconeal process and osteochondritis dissecans of the medial humeral condyle. Avulsion of the medial epicondyle and posteromedial supracondylar ridge of the humerus were thought not to have occurred in these cases. Instead, dystrophic ossification of the synovial membrane and periosteal proliferation on the medial epicondyle and posteromedial supracondylar ridge had taken place. It was concluded that, with a questionable radiographic diagnosis of fragmented medial coronoid process, exploratory arthrotomy may be necessary to confirm the diagnosis. Without surgery, the eventual outcome of fragmented coronoid process is degenerative joint disease. Even with degenerative joint disease, however, clinical improvement was seen following removal of the fragment of the medial coronoid process.