Stereoisomers of cyclic urea HIV-1 protease inhibitors: synthesis and binding affinities

We have synthesized stereoisomers of cyclic urea HIV-1 protease inhibitors to study the effect of varying configurations on binding affinities. Four different synthetic approaches were used to prepare the desired cyclic urea stereoisomers. The original cyclic urea synthesis using amino acid starting materials was used to prepare three isomers. Three additional isomers were prepared by synthetic routes utilizing L-tartaric acid and D-sorbitol as chiral starting materials. A stereoselective hydroxyl inversion of the cyclic urea trans-diol was used to prepare three additional isomers. In all 9 of the 10 possible cyclic urea stereoisomers were prepared, and their binding affinities are described.     

Comparative molecular field analysis (CoMFA) of a series of symmetrical bis-benzamide cyclic urea derivatives as HIV-1 protease inhibitors

Production of the sex pheromone bombykol in the silkworm, Bombyx mori, is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN). It has been suggested that the external signal of PBAN in this species is transmitted to the intracellular cascade reactions consisting of Ca2+/calmodulin (CaM) complex and phosphoprotein phosphatase. To demonstrate the molecular mechanisms regulated by PBAN, we attempted to characterize CaM in the pheromone gland of B. mori. By using ion-exchange and RP-HPLC, B. mori CaM was purified from the cytosolic fraction of the pheromone gland. The primary structure was deduced by composition/sequence analysis and mass spectrometric analysis of the fragment peptides obtained from enzymatic and chemical fragmentations. The amino acid sequence of B. mori CaM was identical with Drosophila CaM deduced from the CaM gene of D. melanogaster, suggesting that insects have well conserved the molecule of CaM.     

Crystal structures of complexes of a peptidic inhibitor with wild-type and two mutant HIV-1 proteases

Crystal structures of the protease of human immunodeficiency virus type 1 (HIV-1) and two mutant proteases, V82D and V82N, have been determined. In all three cases the enzyme forms a complex with the peptidic inhibitor U-89360E. All structures have been determined to 2.3 A resolution and have satisfactory agreement factors: 0.173 for wild type, 0.175 for V82D, and 0.182 for V82N. Comparison of the three crystal structures provides explanations which are consistent with the known kinetic properties of these mutant enzymes with the U-89360E inhibitor [Lin, Y., Lin, X., Hong, L., Foundling, S., Heinrikson, R. L., Thaisrivongs, S., Leelamanit, W., Raterman, D., Shah, M., Dunn, B.M., & Tang, J. (1995) Biochemistry 34, 1143-1152]. Unfavorable van der Waals interactions between the inhibitor and the mutated side chains at position 82 are consistent with diminished affinity for the inhibitor by the mutant enzymes. If a mutation is potentially resistant to an inhibitor, the mutant enzyme should not only have an increased Ki for the inhibitor but should also preserve considerable catalytic capability. The V82D mutant possesses these qualities. In the V82D crystal structure, a water molecule, which connects the protease flap to the inhibitor, is missing or of low occupancy. Absence of this bridge may be important in determining catalytic capability. Moreover, mutation at position 82 induces change in two polypeptide backbone regions, 35-41 and 67-68, which may be related to protease flap mobility.     

Nonpeptide cyclic cyanoguanidines as HIV-1 protease inhibitors: synthesis, structure-activity relationships, and X-ray crystal structure studies

Comparison of the high-resolution X-ray structures of the native HIV-1 protease and its complexes with the inhibitors suggested that the enzyme flaps are flexible. The movement at the tip of the flaps could be as large as 7 A. On the basis of this observation, cyclic cyanoguanidines have been designed, synthesized, and evaluated as HIV-1 protease (PR) inhibitors. Cyclic cyanoguanidines were found to be very potent inhibitors of HIV-1 protease. The choice of cyclic cyanoguanidines over cyclic guanidines was based on the reduced basicity of the former. X-ray structure studies of the HIV PR complex with cyclic cyanoguanidine demonstrated that in analogy to cyclic urea, cyclic cyanoguanidines also displace the unique structural water molecule. The structure-activity relationship of the cyclic cyanoguanidines is compared with that of the corresponding cyclic urea analogues. The differences in binding constants of the two series of compounds have been rationalized using high-resolution X-ray structure information.     

Cyclic HIV-1 protease inhibitors derived from mannitol: synthesis, inhibitory potencies, and computational predictions of binding affinities

The molecular characteristics of midbrain dopamine (DA) neurons have been extensively studied in Parkinson's disease (PD). No such studies of the characteristics of midbrain DA neurons in Alzheimer's disease (AD) or Alzheimer's disease with parkinsonism (AD/Park) have been published. We examined the levels of tyrosine hydroxylase (TH) protein, and the expression of TH and dopamine transporter (DAT) mRNAs, in midbrain neurons of PD, AD, and AD/Park cases. In PD, the loss of TH protein in the ventral tier of the substantia nigra pars compacta (SNpc) of the PD group in accompanied by severe losses in the number of neurons that express TH mRNA and DAT mRNA (74% loss). Remaining neurons show a shift to higher concentrations of TH mRNA but a shift to lower concentrations of DAT mRNA per cell. Hence, there is evidence that compensation in the remaining neurons can elevate concentrations of TH mRNA and lower DAT mRNA. Alternatively, there may be a predilection for a loss of neurons with high levels of DAT mRNA and low TH mRNA levels within the SNpc of PD cases. There was no change in TH protein but an elevation of TH mRNA concentrations per neuron without any change in concentrations of DAT mRNA in the AD group. The AD/Park group did not exhibit changes in the level of TH protein, but showed a small loss (26%) of neurons in the SNpc and a greater loss in other regions of the midbrain (43-53%). Remaining DA neurons showed a marked shift to lower concentrations of DAT mRNA per neuron and a nonsignificant shift in cellular concentration of TH mRNA to higher levels. This is consistent with our previous work showing that with AD/Park there is a significant reduction in the number of DAT sites located on DA terminals in the striatum, but the midbrain neurons have not died. Our results indicate that the differential regulation of mRNAs encoding TH and DAT is similar in the parkinsonian disorders (PD and AD/Park) even though the degree of cell death is very different. This might suggest that compensatory events occur in these DA neurons in AD/Park that are similar to those in PD and that result in differential effects on mRNAs encoding TH and DAT proteins.     

3-Alkanoyl-5-hydroxymethyl tetronic acid homologues and resistomycin: new inhibitors of HIV-1 protease. I. Fermentation, isolation and biological activity

In the course of a screening program for HIV-1 protease inhibiting activity, six new homologues of 3-alkanoyl-5-hydroxymethyl tetronic acids (1 approximately 6) and the known natural product resistomycin (7) were isolated from cultures of the Actinomycete strain DSM 7357. The substituted tetronic acids belong to a recently described structural class of secondary metabolites. The HIV-1 activity of resistomycin (7) has not been reported before.     

Unintegrated circular HIV-1 DNA in the peripheral mononuclear cells of HIV-1-infected subjects: association with high levels of plasma HIV-1 RNA, rapid decline in CD4 count, and clinical progression to AIDS

We observed 36 HIV-infected patients to evaluate whether the presence of tandem 2-long terminal repeat circular unintegrated HIV-1 DNA (2-LTR) in peripheral blood mononuclear cells (PBMC) at baseline was associated with acceleration of HIV disease. Detection of 2-LTR at baseline correlated with high plasma HIV-1 RNA levels (p < .01), recovery of culturable HIV-1 from plasma (p = .02), and progression to AIDS during follow-up (p = .01). More patients with 2-LTR (68%) than without 2-LTR (31%) had a decline in CD4 levels of >50 cells/mm3 over the first 18 months of follow-up (p = .04), and the average annual CD4 decline was 35% in patients with 2-LTR compared with 16% in those without 2-LTR (p = 0.06). Detection of 2-LTR in PBMC at baseline was an independent predictor of high plasma HIV-1 RNA levels and subsequent CD4 cell decline in this cohort of patients with predominantly nonsyncytium-inducing (NSI) isolates at baseline. The presence of 2-LTR in PBMC appears to be reflective of ongoing HIV-1 replication, as measured by plasma HIV-1 RNA levels, and identifies persons at risk for immunologic and clinical decline.     

Cyclic HIV protease inhibitors: design and synthesis of orally bioavailable, pyrazole P2/P2' cyclic ureas with improved potency

Highly potent HIV-1 protease (HIVPR) inhibitors have been designed and synthesized by introducing bidentate hydrogen-bonding oxime and pyrazole groups at the meta-position of the phenyl ring on the P2/P2' substituents of cyclic ureas. Nonsymmetrical cyclic ureas incorporating 3(1H)-pyrazolylbenzyl as P2 and hydrophilic functionalities as P2' show potent protease inhibition and antiviral activities against HIV and have good oral bioavailabilities. The X-ray structure of HIVPR.10A complex confirms that the two pyrazole rings of 10A form bidentate hydrogen bonds with the side-chain oxygen (C=O) and backbone nitrogen (N-H) of Asp30/30' of HIVPR.     

Application of structural modeling with latent variables to adolescent drug use: A reply to Huba, Wingard, and Bentler.

Argues that the application by G. J. Huba et al (see record 1981-32589-001) of latent variable (LV) structural modeling was seriously flawed and that the data employed to test the models was of questionable quality. It is suggested that statements from powerful inferences that can be made from LV models should be moderated. The author argues that the evaluation and testing of LV models should proceed, but with caution and self-criticism; even highly sophisticated methods like LV modeling cannot substitute for the collection of high quality data and other clear operationalizations of constructs and hypotheses. (12 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.     

Plasma levels of soluble CD30, tumour necrosis factor (TNF)-alpha and TNF receptors during primary HIV-1 infection: correlation with HIV-1 RNA and the clinical outcome

OBJECTIVES: The immunological and virological events associated with primary HIV-1 infection have a major impact on the course of HIV-1 disease, and the identification of early predictors during primary HIV infection is critical for the therapeutic strategy. DESIGN AND METHODS: Eighteen consecutive patients with primary HIV infection were followed for a median of 398 days. Clinical status, CD4+ T-cell counts, and plasma samples were obtained weekly from enrollment until week 6, then at weeks 12, 24 and 52, and every 6 months thereafter. Seroconversion was assessed by anti-HIV-1/2 antibodies and Western blot analysis. HIV-1 RNA in plasma was quantified by Amplicor HIV Monitor test. Samples were assayed for immune complex-dissociated p24 antigen, tumour necrosis factor (TNF)-alpha, soluble TNF receptor (sTNFR)-1, sTNFR-II, sCD30 and sCD8 by enzyme immunoassays. Outcome was defined as entering clinical category B or C according to the Centers for Disease Control and Prevention criteria. As a control group, we included 23 HIV-1-negative healthy blood donors. RESULTS: Plasma levels of sCD30, TNF-alpha and sTNFR were significantly higher in HIV-1-infected patients than in controls, and were positively correlated with each other and with values of HIV-1 RNA. Patients who developed an outcome (n = 4) had significantly higher levels of sCD30, TNF-alpha and sTNFR compared with those who did not. Multivariate logistic regression analysis showed that sCD30 and TNF-alpha were the best predictors of outcome independently of CD4+ T-cell counts. CONCLUSIONS: During primary HIV infection, a persistent immune activation may be associated with a poor clinical outcome. The identification of sCD30 and TNF-alpha levels in plasma as early predictors of outcome in primary HIV infection, may direct the implementation of early therapeutic strategies in patients with elevated risk of disease progression.     

Odor, drug and toxin analysis with neuronal networks in vitro: extracellular array recording of network responses

Neurons, by virtue of intrinsic electrophysiological mechanisms, represent transducers that report the dynamics of cell death, receptor-ligand interactions, alterations in metabolism, and generic membrane perforation processes. In cell culture, mammalian neurons form fault-tolerant, spontaneously active systems with great sensitivity to their chemical environment and generate response profiles that are often concentration- and substance-specific. Changes in action potential patterns are usually detected before morphological changes and cell damage occur, which provides sensitivity and reversibility. Such biological systems can be used to screen rapidly for novel pharmacological substances, toxic agents, and for the detection of certain odorants. Existing simple culture preparations can already be employed effectively for the detection of chemical compounds. So far, three strategies have been investigated in pilot experiments: (1) Substance-dependent major changes in spontaneous native activity patterns. All synaptically active agents (e.g. glutamate, strychnine, N-methyl D-aspartic acid) as well as metabolic poisons generate such changes. (2) Substance-dependent changes in network oscillations via disinhibition. The regularized, oscillatory activity is altered by synaptically and metabolically active substances, ion channel blockers, and toxins. (3) Detection of paroxysmal responses indicating major, pathological membrane currents in large subpopulation of cells. We have explored these three strategies via 64 channel array recordings using spontaneously active murine spinal cord cultures. The glycine receptor blocker strychnine reliably generated increased multichannel bursting at 5-20 nM and regular, coordinated bursting above 5 microM. During biculline-induced network oscillations many compounds alter oscillation frequencies or terminate activity in a substance-specific manner. Finally, the gp120 protein of the AIDS virus (at 1 microgram/ml) produces massive, unique paroxysmal discharges that may last as long as 2 min. These results indicate that cultured neuronal networks are practical systems that can be used for the detection and identification of a great variety of chemical substances. The concept of dynamic fingerprinting to identify specific compounds is discussed.     

Pulmonary metastases of breast carcinomas: ligandohistochemical, nuclear, and structural analysis of primary and metastatic tumors with emphasis on period of occurrence of metastases and survival

The complexity of modern surgical and analytical methods requires the miniaturisation of many medical devices. The LIGA technique and also mechanical microengineering are well known for the batch fabrication of microsystems. Actuators and sensors are developed based on these techniques. The hydraulic actuation principle is advantageous for medical applications since the energy may be supplied by pressurised balanced salt solution. Some examples are turbines, pumps and valves. In addition, optical sensors and components are useful for analysis and inspection as represented by microspectrometers and spherical lenses. Finally, plastic containers with microporous bottoms allow a 3-dimensional growth of cell culture systems.     

Human COL6A1: genomic characterization of the globular domains, structural and evolutionary comparison with COL6A2

We studied nephrotic patients hospitalised in internal medicine service at Treichville Teaching hospital from September 1986 to February 1993 for precising the aetiological aspects of black adult patients and their evolutive biological, clinical and epidemiological profile. Secondary Nephrotic syndrome represented 18% of the whole patients with Nephrotic syndrome hospitalised during the same period. In aetiological field it was about: diabetic nephropathy 11 cases (33%); lupus nephritis 7 cases (21%); renal amyloidosis 5 cases (15%); HIV nephropathy 5 cases (15%); schistosomiasis nephrotic syndrome 1 case (3%); pregnancy nephrotic syndrome 1 case (3%); cryoglobulinemia 1 case (3%); malignancy nephrotic syndrome 1 case (3%); nephrosclerosis 1 case (3%).     

A comparative structural study of the steroid epimers: 17 beta-amino-1,3,5(10)-estratrien-3-ol, 17 alpha-amino-1,3,5(10)-estratrien-3-ol, and some derivatives by 1H NMR, and x-ray diffraction analysis

The epimers 17 beta-amino-1,3,5(10)-estratrien-3-ol and 17 alpha-amino-1,3,5(10)-estratrien-3-ol were synthesized. 17 beta-Amino-1,3,5(10)-estratrien-3-ol was prepared by catalytic hydrogenation of the estrone-oxime. The 17 alpha-amino epimer was obtained from estradiol, via tosylate, followed by nucleophilic displacement by sodium azide and subsequent reduction with LiAlH4. They were characterized by spectroscopic methods. Determination of the crystal structures of 3-(toluene-4-sulfonyloxy)-17 alpha-1,3,5(10)-estratriene, 3-(toluene-4-sulfonyloxy)-17-alpha-amino,1,3,5(10)-estratriene hydrochloride, 17 beta-acetylamino-1,3,5(10)-estratriene-3-ol, and 3-acetoxy-17 beta-acetylamino-1,3,5(10)-estratriene enabled us to characterize the structure of the 17 alpha and 17 beta amino epimers for the first time.     

Somatostatin receptor subtypes sst1, sst2, sst3 and sst5 expression in human pituitary, gastroentero-pancreatic and mammary tumors: comparison of mRNA analysis with receptor autoradiography

The aim of this study was to elucidate further the causative mechanism of abnormal coronary vasomotion in patients with syndrome X. In patients with syndrome X, defined as angina pectoris and documented myocardial ischaemia during stress testing with normal findings at coronary angiography, abnormal coronary vasomotion of either the micro- or the macrocirculation has been suggested as the causative mechanism. Accordingly, we evaluated endothelial function, vasodilator reserve, and perfusion heterogeneity in these patients. Twenty-five patients with syndrome X (definitely normal coronary arteriogram, group A), 15 patients with minimal coronary artery disease (group B) and 21 healthy volunteers underwent [13N]ammonia positron emission tomography at rest, during cold pressor stimulation (endothelial function) and during dipyridamole stress testing (vasodilator reserve). Heterogeneity of myocardial perfusion was analysed by parametric polar mapping using a 480-segment model. In both patient groups, resting perfusion was increased compared to the normal subjects: group A, 127+/-31 ml.min-1.100 g-1; group B, 124+/-30 ml.min-1.100 g-1 normal subjects, 105+/-21 ml.min-1.100 g-1 (groups A and B vs normals, P<0.05). These differences were abolished after correction for rate-pressure product. During cold pressor stimulation, the perfusion responses (ratio of cold pressor perfusion to resting perfusion) were similar among the patients and the control subjects (group A, 1.20+/-0.23; group B, 1.24+/-0.22; normal subjects, 1.23+/-0.14). Likewise, during dipyridamole stress testing, perfusion responses were similar among the three groups (group A, 2.71+/-0.67; group B, 2.77+/-1.29; normal subjects, 2. 91+/-1.04). In group A the heterogeneity of resting perfusion, expressed as coefficient of variation, was significantly different from the volunteers (20.1+/-4.5 vs 17.0+/-3.0, P<0.05). In group B (coefficient of variation 19.4+/-3.9) the difference from normal volunteers was not significant. In this study, patients with syndrome X and patients with minimal coronary artery disease showed normal perfusion responses during cold pressor stimulation and dipyridamole stress testing. Our findings therefore suggest that endothelial dysfunction and impaired vasodilator reserve are of no major pathophysiological relevance in patients with syndrome X. Rather, other mechanisms such as increased sympathetic tone and focal release of vasoactive substances may play a role in the pathogenesis of syndrome X.