The fragment indole-6-carboxylic acid (1F1), previously identified as a flap site binder in a fragment-based screen against HIV protease (PR), has been co-crystallized with pepstatin-inhibited PR and with apo-PR. Another fragment, 3-indolepropionic acid (1F1-N), predicted by AutoDock calculations and confirmed in a novel ‘inhibition of nucleation’ crystallization assay, exploits the same interactions in the flap site in two crystal structures. Both 1F1 and 1F1-N bind to the closed form of apo-PR and to pepstatin:PR. In solution, 1F1 and 1F1-N raise the Tm of apo-PR by 3.5–5 °C as assayed by differential scanning fluorimetry (DSF), and show equivalent low-micromolar binding constants to both apo-PR and pepstatin:PR, assayed by backscattering interferometry (BSI). The observed signal intensities in BSI are greater for each fragment upon binding to apo-PR than to pepstatin-bound PR, consistent with greater conformational change in the former binding event. Together, these data indicate that fragment binding in the flap site favors a closed conformation of HIV PR.
Clinically approved inhibitors of HIV-1 protease function via a competitive mechanism. A particular vulnerability of competitive inhibitors is their sensitivity to increases in substrate concentration, as may occur during virion assembly, budding and processing into a mature, infectious viral particle. Advances in chemical synthesis have led to the development of new chemical libraries with high diversity using rapid in-solution syntheses. These libraries have been previously shown to be effective at disrupting protein-protein and protein-nucleic acid interfaces. We have screened 44,000 compounds from such a library to identify inhibitors of HIV-1 protease. One compound was identified that inhibits wild type protease, as well as a drug-resistant protease with 6 mutations. Moreover, analysis of this compound suggests an allosteric, non-competitive mechanism of inhibition and may represent a starting point for an additional strategy for anti-retroviral therapy.
In intensity modulated radiotherapy (IMRT), targets are treated by multiple beams at different orientations each with spatially-modulated beam intensities. This approach spreads the normal tissue dose to a greater volume and produces a higher dose conformation to the target. In general, inverse planning is used for IMRT treatment planning. The inverse planning requires iterative calculation of dose distribution in order to optimize the intensity profile for each beam and is very computation intensive. In this paper, we propose a single-step method utilizing a figure of merit (FoM) to estimate the beam intensities for IMRT treatment planning. The FoM of a ray is defined as the ratio between the delivered tumour dose and normal tissue dose and is a good index for the dose efficacy of the ray. To maximize the beam utility, it is natural to irradiate the tumour with intensity of each ray proportional to the value of the FoM. The nonuniform beam intensity profiles are then fixed and the weights of the beam are determined iteratively in order to yield a uniform tumour dose. In this study, beams are employed at equispaced angles around the patient. Each beam with its field size that just covers the tumour is divided into a fixed number of beamlets. The FoM is calculated for each beamlet and this value is assigned to be the beam intensity. Various weighting factors are incorporated in the FoM computation to accommodate different clinical considerations. Two clinical datasets are used to test the feasibility of the algorithm. The resultant dose-volume histograms of this method are presented and compared to that of conformal therapy. Preliminary results indicate that this method reduces the critical organ doses at a small expense of uniformity in tumour dose distribution. This method estimates the beam intensity in one single step and the computation time is extremely fast and can be finished in less than one minute using a regular PC.
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