Paul J. Lewi

High-resolution structures of HIV-1 reverse transcriptase/TMC278 complexes: Strategic flexibility explains potency against resistance mutations.

TMC278 is a diarylpyrimidine (DAPY) nonnucleoside reverse transcriptase inhibitor (NNRTI) that is highly effective in treating wild-type and drug-resistant HIV-1 infections in clinical trials at relatively low doses (∼25–75 mg/day). We have determined the structure of wild-type HIV-1 RT complexed with TMC278 at 1.8 Å resolution, using an RT crystal form engineered by systematic RT mutagenesis. This high-resolution structure reveals that the cyanovinyl group of TMC278 is positioned in a hydrophobic tunnel connecting the NNRTI-binding pocket to the nucleic acid-binding cleft. The crystal structures of TMC278 in complexes with the double mutant K103N/Y181C (2.1 Å) and L100I/K103N HIV-1 RTs (2.9 Å) demonstrated that TMC278 adapts to bind mutant RTs. In the K103N/Y181C RT/TMC278 structure, loss of the aromatic ring interaction caused by the Y181C mutation is counterbalanced by interactions between the cyanovinyl group of TMC278 and the aromatic side chain of Y183, which is facilitated by an ∼1.5 Å shift of the conserved Y183MDD motif. In the L100I/K103N RT/TMC278 structure, the binding mode of TMC278 is significantly altered so that the drug conforms to changes in the binding pocket primarily caused by the L100I mutation. The flexible binding pocket acts as a molecular “shrink wrap” that makes a shape complementary to the optimized TMC278 in wild-type and drug-resistant forms of HIV-1 RT. The crystal structures provide a better understanding of how the flexibility of an inhibitor can compensate for drug-resistance mutations.

Evolution of anti-HIV drug candidates. Part 3: diarylpyrimidine (DAPY) analogues

The synthesis and anti-HIV-1 activity of a series of diarylpyrimidines (DAPYs) are described. Several members of this novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) are extremely potent against both wild-type and a panel of clinically significant single- and double-mutant strains of HIV-1.

DNA Synthesis and Mitoses in Coronary Collateral Vessels of the Dog

DNA synthesis and mitotic activity in coronary collateral arterioles was assessed in dogs at different time intervals after gradual ameroid constriction of the left circumflex coronary artery. Labeling of nuclei and the mitotic index were highest at 3 weeks after implantation of the constrictor and gradually declined thereafter. Labeling persisted for at least 8 weeks but radioactive DNA was not found 12 months after coronary artery constriction nor in controls or animals with sham operations. Proliferative activity was, at any time interval, highest at the level of the smallest diameters of the collateral vessels. Labeled nuclei and mitoses were found in endothelial, medial, and adventitial cells. Myocardial mesenchymal cells also incorporated tritiated thymidine. The data provide evidence that after constriction of a major coronary artery, the coronary collateral vessels enlarge by an active growth process that follows the basic laws of cell kinetics.

Myocardial steal produced by coronary vasocilation in chronic coronary artery occlusion.

SummaryChronic occlusion of the circumflex branch of the left coronary artery was produced in dogs. All animals developed a collateral circulation fast and efficient enough to prevent myocardial infarction. Tracer microsphere experiments together with 133-Xenon studies showed a homogeneous distribution of flow over the entire left ventricle. Drug-induced coronary vasodilation caused a grossly non-homogenous distribution of coronary blood flow. The collateral dependent areas of the heart received much less blood than the areas which are supplied by normal arterial channels. Within the collateral-dependent area the endocardial myocardium received less blood than the epicardial layers. Previously well perfused areas can become underperfused during overperfusion of normal myocardium. Similar changes were not observed in hearts with normal coronary arteries.Nitroclycerine did not produce changes in blood flow in hearts with coronary artery occlusion. Consequently changes in the distribution of myocardial flow were not observed with this drug. A computer model is presented for the explanation of the experimental findings which facilitated the analysis of contributing factors.ZusammenfassungBei Hunden wurde ein chronischer Verschluß des ramus circumflexes der linken Koronararterie erzeugt. Bei allen Tieren entwickelte sich ein Kollateralkreislauf, der das Auftreten von Infarkten verhinderte. Die Durchblutung des linken Ventrikels bei chronischem Koronarverschluß war normal und die Durchblutungsverteilung war homogen, wie aus der 133-Xenon clearance zusammen mit der Gewebekonzentration von Tracer Microspheren hervorging. Pharmakologisch induzierte Koronardilation verursachte eine ungleichmäßige Durchblutungsverteilung. Das von Kollateralen abhängige Perfusionsgebiet erhielt bedeutend weniger Blut als der Teil des Myokards, der über normale Koronararterien versorgt wird. Innerhalb des kollateralabhängigen Myokards erhielt das subendokardiale Gebiet bedeutend weniger Blut pro Zeiteinheit als das subepikardiale Gebiet. Zonen, die vor der Dilation normal und homogen durchblutet waren, erhielten während der Dilatation weniger Blut. Derartige Verteilungsänderungen wurden nicht beobachtet bei Herzen mit normalen Koronararterien.Nitroglycerin verursachte keine Umverteilung der Durchblutung bei Herzen mit chronischem Koronarverschluß. Nitroglycerin verursachte allerdings auch keine nennenswerte Koronardilatation. Ein Komputermodell wird beschrieben zum besseren Verständnis der haemodynamischen Zusammenhänge bei Koronardilatation.

In Vitro Evaluation of Nonnucleoside Reverse Transcriptase Inhibitors UC-781 and TMC120-R147681 as Human Immunodeficiency Virus Microbicides

ABSTRACT The nonnucleoside reverse transcriptase inhibitors UC-781 and TMC120-R147681 (Dapivirine) effectively prevented human immunodeficiency virus (HIV) infection in cocultures of monocyte-derived dendritic cells and T cells, representing primary targets in sexual transmission. Both drugs had a favorable therapeutic index. A 24-h treatment with 1,000 nM UC-781 or 100 nM TMC120-R147681 prevented cell-free HIV infection, whereas 10-fold-higher concentrations blocked cell-associated HIV.

PREDICTIVE VALUE OF GLASGOW COMA SCORE FOR AWAKENING AFTER OUT-OF-HOSPITAL CARDIAC ARREST

The Glasgow coma score (GCS) during days 1-6 after cardiac arrest was used to predict neurological outcome in 360 resuscitated victims of out-of-hospital cardiac arrest. A predictive rule based on the best GCS of 216 patients resuscitated in 1983-84 (prediction group) was constructed, and its predictive power was tested on 133 patients treated in 1985 (test group). Neurological outcome was correctly predicted 2 days after cardiac arrest in 80% of the prediction group, with a best GCS of 10 or above and 4 or below as cutoff points. For patients with a best GCS of 5-9, prediction of outcome was possible 6 days after cardiac arrest, with a best GCS of 8 during the first 6 days as the single cutoff point. The rule was then validated in the test group: the sensitivity was 96%; the specificity 86%; the negative predictive value 97%; and the positive predictive value 77%. These data suggest that this simple GCS-based rule can be helpful in predicting outcome in patients resuscitated after out-of-hospital cardiac arrest, but confirmation of these data is required in a prospective study in a larger number of patients.

Evolution of anti-HIV drug candidates. Part 2: Diaryltriazine (DATA) analogues.

A synthesis program directed toward improving the stability of imidoyl thiourea based non-nucleoside reverse transcriptase inhibitors (NNRTIs) led to the discovery of diaryltriazines (DATAs), a new class of potent NNRTIs. The synthesis and anti-HIV structure-activity relationship (SAR) studies of a series of DATA derivatives are described.

Multivariate analysis of angiographic, histologic, and electrocardiographic data in patients with coronary heart disease

In 61 consecutive patients undergoing aortocoronary bypass grafting, angiographic and electrocardiographic (ECG) changes were studied. Histologic delineation of myocardium was obtained by analysis of transmural biopsy specimens acquired at the time of surgery. The use of principal-component analysis revealed three definite groups of patients. Group I comprised patients with histologic findings associated with severe left anterior descending coronary artery (LAD) stenosis, without abnormal wall motion or ejection fraction. ECG abnormalities were limited to ST changes. Group II comprised patients with severe myocardial cell degeneration with only modest fibrosis associated with severe LAD stenosis and severely impaired wall motion. The incidence of infarction on the ECG was low. Group III patients had important myocardial cell degeneration with severe fibrosis associated with severe LAD stenosis, severely depressed wall motion, and significantly impaired ejection fraction. In this group there was a high incidence of infarction apparent on the ECG. Postoperative follow-up (24 months) showed a total survival of 94.4% in group I, 92.8% in group II, and only 72.7% in group III. This identification of subtypes of coronary artery disease seems to be helpful in estimating patient prognosis after coronary surgery.

Multiple experimental coronary occlusion without infarction: Effects of heart rate and vasodilation

Abstract Chronic occlusion of the left circumflex artery and of the right coronary artery was produced in 21 dogs with the Ameroid technique. Thirteen animals survived and myocardial infarction was avoided in 12 due to the development of a network of collateral vessels. The functional state of the collaterals was tested by physiologic and pharmacologic stimuli using Tracer Microspheres and 133-Xe as indicators of coronary flow and of collateral flow. Increases in heart rate up to 160 beats per minute caused mild coronary vasodilation and homogeneous distribution of left ventricular myocardial flow. Carbochromene caused moderate-to-marked increase in coronary blood flow. Collateral flow rose also but less than coronary flow. Carbochromene plus tachycardia caused a marked increase in coronary flow but collateral flow to the subendocardial layers actually decreased below control values. An explanation of these findings is offered on the basis of relations between the collateral and coronary resistances.

A Series of Diaryltriazines and Diarylpyrimidines Are Highly Potent Nonnucleoside Reverse Transcriptase Inhibitors with Possible Applications as Microbicides

ABSTRACT An in vitro model of monocyte-derived dendritic cells (MO-DC) and CD4+ T cells, representing the primary targets of sexual human immunodeficiency virus (HIV) transmission, was used to evaluate the antiviral and immune suppressive activity of new classes of nonnucleoside reverse transcriptase inhibitors, diaryltriazines (DATAs) and diarylpyrimidines (DAPYs), compared to the reference compounds UC-781 and PMPA. Antiviral activity (as reflected by the 50% effective concentration [EC50]) was determined by treating HIV-infected MO-DC/CD4+-T-cell cocultures with a dose range of a compound during 14 days, followed by analysis of supernatants in HIV p24 antigen enzyme-linked immunosorbent assay. A limited, 24-h treatment evaluated the compounds as microbicides. Viral rescue was evaluated in a PCR by monitoring proviral DNA in secondary cultures with phytohemagglutinin-interleukin-2 blasts. We determined 50% immunosuppressive concentrations in mixed leukocyte cultures of MO-DC and allogeneic T cells, with compound either continuously present or present only during the first 24 h. The EC50 values of DATA and DAPY compounds ranged from 0.05 to 3 nM compared to 50 nM for UC-781 and 89 nM for PMPA. When evaluated in the “microbicide” setting, the most potent compounds completely blocked HIV infection at 10 to 100 nM. The immunosuppressive concentrations were well above the EC50, resulting in favorable therapeutic indices for all compounds tested. The DATA and DAPY compounds described here are more potent than earlier reverse transcriptase inhibitors and show favorable pharmacological profiles in vitro. They could strengthen the antiretroviral armamentarium and might be useful as microbicides.