Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

From Lab Formulation Development to CTM Manufacturing of KO-947 Injectable Drug Products: a Case Study and Lessons Learned.

Formulation development of KO-947-K mesylate injectable drug products was described. Solution formulations were initially attempted, and key parameters such as drug concentration, buffer, pH, complexing agent, and tonicity modifying agent were carefully evaluated in the lab setting, mainly focusing on solubility and chemical stability. A lead solution formulation was advanced to a scaleup campaign. An unexpected stability issue was encountered, and the root cause was attributed to the heterogeneous liquid freezing process of the formulated solution at -20°C, which had not been captured in the lab setting. A lyophilized product was then designed to overcome the issue and supplied to the phase I clinical trial.

Eosinophilic fasciitis associated with generalized morphea and IgA nephropathy.

Eosinophilic fasciitis (EF) is a rare connective tissue disease characterized by increased peripheral blood eosinophils and diffuse fasciitis, generalized morphea (GM) is a subtype of localized scleroderma, and IgA nephropathy is a chronic glomerulonephritis caused by abnormal deposition of IgA in the mesangial area of the glomeruli. We describe a 49-year-old male patient with hard skin, cutaneous hyperpigmentation, and proteinuria. The patient had suffered from a long disease course of hard skin, while urine protein was newly detected. Finally, the clinical presentation and physical examination, limb MRI, skin biopsy, and renal biopsy confirmed the diagnosis of eosinophilic fasciitis associated with generalized morphea and IgA nephropathy. This case is the first report of EF associated with GM and IgA nephropathy.

Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation.

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7ß, 27-dihydroxycholesterol (7ß, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7ß, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7ß, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

Fluoro analogs of bioactive oxy-sterols: Synthesis of an EBI2 agonist with enhanced metabolic stability.

Synthesis of several 7-hydroxy oxysterols and their potential roles as signaling molecules in the innate and adaptive immune responses is discussed. Discovery of a new, fluorinated, synthetic analog of the 7α,25-dihydroxycholesterol-the endogenous ligand of GPR 183 (EBI2), a G-protein coupled receptor highly expressed upon Epstein-Barr virus infection is described. Fluoro oxysterol 12 showed good metabolic stability while maintaining excellent EBI2 agonist activity.

Allyl-Assisted, Cu(I)-Catalyzed Azide-Alkyne Cycloaddition/Allylation Reaction: Assembly of the [1,2,3]Triazolo-4,5,6,7-tetrahydropyridine Core Structure.

We report a Cu(I)-catalyzed azide-alkyne-allyl halide three-component reaction for a one-pot synthesis of 1,4-disubstituted 5-allyl-1,2,3-triazoles. The allyl moiety provides not only the electrophile but also a coordinating ligand to Cu, which is essential for the reaction to occur under mild conditions. A concise synthesis of a potential drug candidate 1 is realized based on this key reaction.

[An analysis of abnormal magnetic resonance imaging of sacroiliac joints in patients misdiagnosed as spondyloarthritis].

OBJECTIVE: To study the imaging features of sacroiliac joints (SIJ) in patients who were misdiagnosed as spondyloarthritis (SpA). METHODS: A total of 34 patients with chief complaint of back pain and misdiagnosed as SpA from January 2007 to April 2013 in Department of Rheumatology Chinese PLA General Hospital were enrolled. The imaging, clinical manifestations, laboratory examinations data were analyzed. RESULT: The main reason for misdiagnosis as SpA was because of sacroiliitis presenting on imaging. The final diagnoses included 24 patients as SIJ infection, 4 patients as neoplastic diseases, 2 patients as metabolic bone diseases, 2 patients as sacroiliac joint degeneration, 1 patient as gout of sacroiliac joint, 1 patient as diffuse idiopathic bone hypertrophy. For patients with infection, there were 10 patients receiving X-ray and 22 patients receiving CT of SIJ. However, 5 and 7 patients had negative results respectively. These patients with infection had abnormalities in MRI including all with bone marrow edema, 21 patients with erosion of bone and joint, 22 patients with muscle involved. As to the patients with malignancies, SIJ CT scan appeared normal. Bone marrow edema and erosion in MRI were found in all neoplasm patients expect one as ependymoma. Adjacent muscles were involved in the patient with Ewing's sarcoma. Either X-ray or CT in other patients demonstrated obvious abnormalities, but only mild erosion of bone was found in MRI. CONCLUSION: Bone marrow edema of SIJ in MRI represented not only in patients with SpA. Rheumatologists should analyze the clinical manifestations and laboratory examinations comprehensively in order to avoid the misdiagnoses.

[Clinical significance of high sensitivity C-reactive protein for evaluating the efficacy of etanercept in active ankylosing spondylitis].

OBJECTIVE: To evaluate the therapeutic efficacy of etanercept for active ankylosing spondylitis (AS) at different levels of high sensitivity C-reactive protein (hs-CRP). METHODS: Patients with active AS received a subcutaneous injection of etanercept (50 mg, once per week) for 12 weeks. According to the baseline hs-CRP, they were divided into group A (hs-CRP ≤ 3 mg/L), group B (3 < hs-CRP ≤ 11 mg/L), group C (11< hs-CRP ≤ 26 mg/L) and group D (hs-CRP > 26 mg/L). The following clinical data were recorded: VAS (visual analogue score) of spinal pain, time of morning stiffness, patient global assessment (PGA), BASDAI, BASFI, ASDAS, indices of joint tenderness on baseline and week 12. And the percents of ASAS20, ASAS40, ASAS partial remission, BASDAI20, BASDAI50, ASDAS clinically important improvement and major improvement responders were assessed in all groups. RESULTS: A total of 161 patients finished the study, including group A (n = 18), group B (n = 47), group C (n = 49) and group D (n = 47). Group B was similar with group A. Groups C and D were higher than group A in terms of ASDAS improvement score, percents of ASDAS clinically important improvement and major improvement responders. CONCLUSION: Patients with moderately and significantly elevated hs-CRP may achieve ASDAS improvement more easily than those with normal and mildly elevated levels.

Identification of benzofuran central cores for the inhibition of leukotriene A(4) hydrolase.

Leukotrienes (LT's) are known to play a physiological role in inflammatory immune response. Leukotriene A(4) hydrolase (LTA(4)H) is a cystolic enzyme that stereospecifically catalyzes the transformation of LTA(4) to LTB(4). LTB(4) is a known pro-inflammatory mediator. This paper describes the identification and synthesis of substituted benzofurans as LTH(4)H inhibitors. The benzofuran series demonstrated reduced mouse and human whole blood LTB(4) levels in vitro and led to the identification one analog for advanced profiling. Benzofuran 28 showed dose responsive target engagement and provides a useful tool to explore a LTA(4)H inhibitor for the treatment of inflammatory diseases, such as asthma and inflammatory bowel disease (IBD).

Thalidomide reduces recurrence of ankylosing spondylitis in patients following discontinuation of etanercept.

A previous study showed that most ankylosing spondylitis (AS) patients presented recurrence within 6 months post-discontinuation of etanercept. How to reduce recurrence following discontinuation of etanercept should be further researched. In this study, 111 ankylosing spondylitis patients meeting the Assessment in AS 20 % response (ASAS20) criteria after 12-week administration of etanercept were randomized into three groups: Group I, 150 mg thalidomide once/day; Group II, 1 g sulfasalazine, twice/day; Group III, NSAIDs for the maintenance treatment. The patients were regularly followed up once a month, and AS recurrence was evaluated with the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), the Bath Ankylosing Spondylitis Functional Index (BASFI), the patient global assessment (PGA), and rachialgia. The follow-up lasted for 1 year, and AS recurrence was considered as the end of a visit. Finally, 100 patients completed the follow-up study, of whom 30 were in Group I, 33 in Group II, and 37 in Group III. The average follow-up period was 5.1 ± 3.9 months and the longest lasted for 12 months. At the end of the follow-up study, the recurrence rates in Groups I, II, and III were, respectively, 60.0 % (18/30), 84.8 % (28/33), and 89.2 % (33/37). The recurrence rates of Group I were statistically significantly lower than that of Group II and III (P = 0.0265; P = 0.0053), while there was no significant difference between Group II and Group III. In addition, we found that PGA, C-reactive protein (CRP), and spinal inflammation could be regarded as predictive factors for AS recurrence by analysis with the Cox proportional hazard model. This study points to a new way for maintenance therapy of AS following discontinuation of etanercept and reveals several useful indicators for prediction of AS recurrence.

[Expression and role of vascular endothelial growth inhibitor in sporadic clear cell renal cell carcinoma].

OBJECTIVE: To evaluate the expression of vascular endothelial growth inhibitor (VEGI) in sporadic clear cell renal cell carcinoma (CCRCC) and explore its relationships between VEGI expression, pathologic grade and tumor staging. METHODS: Western blot and immunohistochemical staining were used to detect the expression of VEGI in CCRCC cell line (786-O cells), CCRCC and paired normal kidney tissues. A total of 50 CCRCC cases were recruited. There were 37 males and 13 females with an average age of 53 ± 12 years. The tumor sizes were < 7 cm (n = 33) and ≥ 7 cm (n = 17). Their pathologic grades were G1 (n = 14), G2 (n = 22) and G3 (n = 14) and pathologic stages pT1 (n = 32), 10 pT2 (n = 10) and pT3 (n = 8). RESULTS: VEGI protein was predominantly located in cytoplasm. Compared with normal kidney tissues(mean optic density (MOD) of VEGI staining: 0.40 ± 0.16), it was lower in CCRCC tissues (MOD: 0.11 ± 0.06, P < 0.01). In addition, the positive rate of VEGI expression, the expression intensity and the MOD of VEGI protein were negatively correlated with the pathologic grade of CCRCC (r = -0.640, P < 0.01; r = -0.831, P < 0.01; r = -0.781, P < 0.01 respectively). The MOD of VEGI expression in ≥ 7 cm tumors (MOD, 0.08 ± 0.04) was significantly lower than that in < 7 cm tumors (MOD: 0.12 ± 0.06, P < 0.05). However, there was no correlations between the VEGI protein level and age, gender and pathologic stage of patients (P > 0.05). CONCLUSION: VEGI protein is predominantly located in cytoplasm. Compared with CCRCC tissues, VEGI protein level is higher in normal ones. In consideration of negative correlations between VEGI expression, pathologic grade and tumor size, it is implied that VEGI may play a negative regulatory role in the occurrence and development of CCRCC.

Identification of structural motifs critical for epstein-barr virus-induced molecule 2 function and homology modeling of the ligand docking site.

Epstein-Barr virus-induced molecule 2 (EBI2) (also known as G-protein-coupled receptor 183) is a G-protein-coupled receptor (GPCR) that is best known for its role in B cell migration and localization. Our recent deorphanization effort led to the discovery of 7α,25-dihydroxycholesterol (7α,25-OHC) as the endogenous ligand for EBI2, which provides a tool for mechanistic studies of EBI2 function. Because EBI2 is the first GPCR known to bind and to be activated by an oxysterol, the goal of this study was to understand the molecular and structural bases for its ligand-dependent activation; this was achieved by identifying structural moieties in EBI2 or in 7α,25-OHC that might affect receptor-ligand interactions. By using a series of chemically related OHC analogs, we demonstrated that all three hydroxyl groups in 7α,25-OHC contributed to ligand-induced activation of the receptor. To determine the location and composition of the ligand binding domain in EBI2, we used a site-directed mutagenesis approach and generated mutant receptors with single amino acid substitutions at selected positions of interest. Biochemical and pharmacological profiling of these mutant receptors allowed for structure-function analyses and revealed critical motifs that likely interact with 7α,25-OHC. By using a hybrid ß(2)-adrenergic receptor-C-X-C chemokine receptor type 4 structure as a template, we created a homology model for EBI2 and optimized the docking of 7α,25-OHC into the putative ligand binding site, so that the hydroxyl groups interact with residues Arg87, Asn114, and Glu183. This model of ligand docking yields important structural insight into the molecular mechanisms mediating EBI2 function and may facilitate future efforts to design novel therapeutic agents that target EBI2.

Acute choroidal involvement in lupus nephritis: A case report and review of literature.

BACKGROUND: Systemic lupus erythematosus (SLE), characterized by the production of autoantibodies and widespread deposition of immune complexes, predominantly affects women of childbearing age. More than one-third of SLE patients present ocular manifestations. Choroidal disease is currently not completely understood, and its precise differentiation from central serous chorioretinopathy is rarely achieved. To date, no more than 60 patients with choroidal involvement have been reported. CASE SUMMARY: A 37-year-old Chinese woman experienced decreased visual acuity bilaterally, accompanied by increasing periorbital swelling and severe conjunctival chemosis. Decreased breath sounds in both bases were detected via auscultation, as well as pitting edema in both ankles. SLE and lupus nephritis were diagnosed based on serositis, renal disorder, leukopenia and positive anti-Smith and anti-nuclear antibodies. Lupus choroidopathy was diagnosed based on ocular presentation and imaging. The patient was treated with systemic corticosteroids, spironolactone, hydroxychloroquine (HCQ), mycophenolate mofetil (MMF), and intravenous immunoglobulin. After 4 wk of hospitalization, the patient was discharged. Indocyanine green angiography showed no leakage from choroidal vessels, and ocular coherence tomography detected low amounts of subretinal fluid right before discharge. The patient was prescribed oral methylprednisolone, HCQ, and MMF. Two months after the first visit, ophthalmological examination revealed a visual acuity of 20/20 bilaterally, and SLE disease activity was well controlled; her symptoms disappeared completely. CONCLUSION: Here we presented a case of lupus choroidopathy, successfully treated with systemic corticosteroids, and discussed previously reported cases, focusing on differential diagnosis with a central serous chorioretinopathy.

Similarities and differences between spondyloarthritis in Asia and other parts of the world.

PURPOSE OF REVIEW: Spondyloarthritis (SpA) is a group of diverse interrelated inflammatory arthritides, which share multiple clinical features as well as common genetic predisposing factors. Ankylosing spondylitis (AS) is regarded as the most typical subtype. The purpose of this article is to review relevant studies conducted in Asia and other parts of the world, which may open a window to a better understanding of the epidemiology, clinical feature, diagnosis, and management of this condition. RECENT FINDINGS: The prevalence, clinical feature, diagnosis, and therapy of SpA and its correlation with HLA-B27 in Asia are generally similar to other parts of the world. NSAIDs form the cornerstone of the treatment for AS. The new treatment options with tumor necrosis factor (TNF)-α blocking agents seem a breakthrough for patients with SpA refractory to conventional treatment. Recent results showed that thalidomide was an effective, well tolerated, and economic option for refractory AS patients, especially in maintaining disease remission after etanercept or infliximab treatment was discontinued. SUMMARY: The similarities between spondyloarthritides in Asia and other parts of the world are major and the differences are minor. Because of the major socioeconomic burden and poor access to expensive means of treatment of SpA in Asia, the rheumatologists and physicians in Asia are working hard to look for effective but cheaper alternatively regimens for refractory SpA patients. Thalidomide may be a potentially effective option for patients who cannot afford biologicals in undeveloped areas.

Direct, metal-free amination of heterocyclic amides/ureas with NH-heterocycles and N-substituted anilines in POCl3.

A POCl(3)-mediated, direct amination reaction of heterocyclic amides/ureas with NH-heterocycles or N-substituted anilines is described. Compared to the existing methods, this operationally simple protocol provides unique reactivity and functional group compatibility because of the metal-free, acidic reaction conditions. The yields are generally excellent.

Protecting-group-free synthesis of a dual CCK1/CCK2 receptor antagonist.

In our pursuit of an efficient, protecting-group-free synthesis of the dual CCK1/CCK2 receptor antagonist 1, we have developed chemoselective conditions for sulfonamide formation reaction in pure water and a PhNMe(2) mediated carboxamide formation, both in the presence of a carboxylic acid. Practical synthesis of an unnatural, chiral ß-aryl-α-amino acid is also described.

Practical synthesis of a Cathepsin S inhibitor: route identification, purification strategies, and serendipitous discovery of a crystalline salt form.

A "redox economical" strategy resulted in a concise, modular synthesis of compound 1, a potent Cathepsin S inhibitor. Starting from three building blocks, crude drug substance was prepared in a two-step sequence in high yield. Efficient purification of the crude drug substance was accomplished via the formation of an unusual monoethyl oxalate salt.

Practical and scalable synthesis of a selective CCK1 receptor antagonist.

We describe a practical and scalable route to compound (Z)-1, a selective CCK1 receptor antagonist. Notable features of this concise route are (1) a regioselective construction of the pyrazole core through the reaction of an aryl hydrazine and an elaborated acetylenic ketone, (2) a Tf2O/pyridine mediated Z-selective dehydration of an α-hydroxyester, and (3) a stereoselective hydrolysis. The sequence is high-yielding and amenable for large-scale synthesis.

CuI-catalyzed amination of arylhalides with guanidines or amidines: a facile synthesis of 1-H-2-substituted benzimidazoles.

CuI/L5 (N,N'-dimethylethylenediamine) proves to be an efficient catalyst system for the amination of arylhalides with guanidines. The same catalyst system is then successfully applied to the one-step synthesis of 1-H-2-amino-benzimidazoles through tandem aminations of 1,2-dihaloarenes in modest yields. This methodology is also applicable for the preparation of 1-H or 1-substutituted 2-aryl- or 2-alkyl-benzimidazoles.

Detection of Erosions in Sacroiliac Joints of Patients with Axial Spondyloarthritis Using the Magnetic Resonance Imaging Volumetric Interpolated Breath-hold Examination.

OBJECTIVE: The volumetric interpolated breath-hold examination (VIBE) magnetic resonance imaging (MRI) technique can visualize erosive cartilage defects in peripheral joints. We evaluated the ability of VIBE to detect erosions in sacroiliac joints (SIJ) of patients with axial spondyloarthritis (axSpA) compared to the established T1-weighted MRI sequence and computed tomography (CT). METHODS: MRI (T1-weighted and VIBE) and CT scans of SIJ of 109 patients with axSpA were evaluated by 2 blinded readers based on SIJ quadrants (SQ). Erosions were defined according to Assessment of Spondyloarthritis international Society (ASAS) definitions. Scores were recorded if readers were in agreement. RESULTS: Erosions were less frequently detected by CT (153 SQ) than by T1-weighted MRI (182 SQ; p = 0.008) and VIBE-MRI (199 SQ; p < 0.001 vs CT and p = 0.031 vs T1-weighted MRI). Taking CT as the gold standard, the sensitivity of VIBE-MRI (71.2%) was higher than that for T1-weighted MRI (63.4%), with similar specificity (87.3% vs 88%, respectively). In linear regression analysis, younger age was significantly associated with occurrence of erosions independently in VIBE-MRI (ß = 0.384, p < 0.001) and T1-weighted MRI (ß = 0.369, p < 0.001) compared to CT. CONCLUSION: The VIBE-MRI sequence was more sensitive than T1-weighted MRI in identifying erosive damage in the SIJ, especially in younger patients. This might be due to the ability of VIBE-MRI to identify structural changes in the cartilage that have not yet extended to the underlying bone, where CT seems to be superior.