Design, synthesis and evaluation of inhibitors of the SARS-CoV-2 nsp3 macrodomain.

A series of amino acid based 7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low µM Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization.

Design, Synthesis and Evaluation of Inhibitors of the SARS-CoV2 nsp3 Macrodomain.

A series of amino acid based 7H -pyrrolo[2,3- d ]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp 22 and the amide backbone NH of Ile 23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe 157 and Asp 156 , part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low µM Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization.

Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself.

Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

Outcome of glansectomy and skin grafting in the management of penile cancer.

Purpose. To report outcome data for patients with penile cancer treated surgically with glansectomy and skin grafting. Materials and Methods. We retrospectively reviewed data on all patients undergoing surgical management of penile cancer by a single surgeon between 1998 and 2008. Outcomes in patients who underwent glansectomy and skin grafting were analysed. Results. Between 1998 and 2008 a total of 25 patients with a mean age 60 (39-83) underwent glansectomy and skin grafting. Six patients had carcinoma in situ (CIS); the stage in the remaining patients ranged from T1G1 to T3G3. Mean followup for patients was 28 months (range 6-66). Disease specific survival was 92% with 2 patients who had positive nodes at lymph node dissection developing groin recurrence. One patient developed a local recurrence requiring a partial penectomy. Conclusions. Penile preserving surgery with glansectomy and skin grafting is a successful technique with minimal complications for local control of penile carcinoma arising on the glans. Careful followup to exclude local recurrence is required.

Morphological expression of KIT positive interstitial cells of Cajal in human bladder.

PURPOSE: We investigated the 3-dimensional morphological arrangement of KIT positive interstitial cells of Cajal in the human bladder and explored their structural interactions with neighboring cells. MATERIALS AND METHODS: Human bladder biopsy samples were prepared for immunohistochemistry/confocal or transmission electron microscopy. RESULTS: Whole mount, flat sheet preparations labeled with anti-KIT (Merck, Darmstadt, Germany) contained several immunopositive interstitial cell of Cajal populations. A network of stellate interstitial cells of Cajal in the lamina propria made structural connections with a cholinergic nerve plexus. Vimentin positive cells of several morphologies were present in the lamina propria, presumably including fibroblasts, interstitial cells of Cajal and other cells of mesenchymal origin. Microvessels were abundant in this region and branched, elongated KIT positive interstitial cells of Cajal were found discretely along the vessel axis with each perivascular interstitial cell of Cajal associated with at least 6 vascular smooth muscle cells. Detrusor interstitial cells of Cajal were spindle-shaped, branched cells tracking the smooth muscle bundles, closely associated with smooth muscle cells and vesicular acetylcholine transferase nerves. Rounded, nonbranched KIT positive cells were more numerous in the lamina propria than in the detrusor and were immunopositive for anti-mast cell tryptase. Transmission electron microscopy revealed cells with the ultrastructural characteristics of interstitial cells of Cajal throughout the human bladder wall. CONCLUSIONS: The human bladder contains a network of KIT positive interstitial cells of Cajal in the lamina propria, which make frequent connections with a cholinergic nerve plexus. Novel perivascular interstitial cells of Cajal were discovered close to vascular smooth muscle cells, suggesting interstitial cells of Cajal-vascular coupling in the bladder. KIT positive detrusor interstitial cells of Cajal tracked smooth muscle bundles and were associated with nerves, perhaps showing a functional tri-unit controlling bladder contractility.

Nicorandil induced penile ulceration.

We report the unusual complication of penile ulceration caused by Nicorandil, a nicotinamide ester used in the treatment of symptomatic angina pectoris.

The utility of prostate-specific antigen velocity thresholds in clinical practice: a population-based analysis.

OBJECTIVE: To investigate the ability of prostate-specific antigen velocity (PSAV) to predict prostate cancer, and assess the test characteristics of several PSAV thresholds for identifying prostate cancer and high-grade cancers. PATIENTS AND METHODS: From a population-based database of PSA results, men with an initial PSA level of <10.0 ng/mL, taken between I January 1994 and 31 December 2003, were identified. Those with three or more PSA tests before diagnosis, taken over > or =18 months, were included. Men were followed for a diagnosis of prostate cancer or histologically confirmed benign disease until 31 December 2003. RESULTS: In all, 24 709 men were included, with 716 (2.9%) diagnosed with prostate cancer and 1488 (6.0%) with benign histology. The mean (10.38 vs 0.43 ng/mL/year) and median (1.47 vs 0.03 ng/mL/year) PSAV were considerably higher in men with prostate cancer than in those with no cancer (P < 0.001). There was no PSAV threshold that could reliably identify prostate cancer or high-grade cancers without requiring many men to proceed to prostate biopsy. CONCLUSION: In this population, PSAV had additional value over one PSA value in identifying men with prostate cancer. Many men with prostate cancer might have a 'normal' (<0.75 ng/mL/year) PSAV. As with total PSA level, there was no PSAV threshold that could reliably predict prostate cancer, but rather a continuum of risk of cancer associated with PSAV level.

Baseline prostate-specific antigen level and risk of prostate cancer and prostate-specific mortality: diagnosis is dependent on the intensity of investigation.

BACKGROUND: When considering prostate biopsy, men and their physicians must balance the potential benefits of early diagnosis of localized cancer with the implications of overdiagnosis of clinically insignificant cancers. We investigated the risk of prostate cancer and prostate cancer-specific and all-cause mortality by baseline prostate-specific antigen (PSA) level in a population-based cohort study in Northern Ireland, where PSA screening is not recommended and where low to moderately raised (<10.0 ng/mL) PSA levels were not routinely investigated. METHODS: From a regional electronic database of PSA results, men who had their initial PSA between January 1, 1994 and December 31, 1998 were identified and followed for diagnosis of prostate cancer and prostate cancer-specific and all-cause mortality until December 31, 2003. RESULTS: 68,354 men (mean age, 65.2 years) were included, with 50,676 (74.1%) having a baseline PSA of <4.0 ng/mL; 402 (0.8%) of these were subsequently diagnosed with prostate cancer. PSA level was positively associated with risk of prostate cancer and prostate-specific mortality. In men with baseline PSA <4.0 ng/mL, the rate of prostate cancer and high-grade cancer diagnosis was <2 and <1 cases per 1,000 person-years, respectively, whereas prostate-specific mortality was very low (0.18 cases per 1,000 person-years) compared with overall mortality (28.71 cases per 1,000 person-years). CONCLUSION: Following a PSA result, men need to be aware not only of the risk of prostate cancer but also of having cancer that may cause them harm during their lifetime or, more importantly, kill them. These data should inform and reassure men of their risk of clinically significant prostate cancer.

Methods of calculating prostate-specific antigen velocity.

OBJECTIVES: Numerous methods of calculating PSA velocity (PSAV) are used and have the potential to produce differing PSAV results from the same PSA data. We calculated PSAV using three common methods and compared differences between the methods and their predictive value for prostate cancer diagnosis. METHODS: From a population-based database of PSA results, men with initial PSA<10.0 ng/ml and a subsequent diagnosis of prostate cancer or benign histology were identified. Those with > or =3 PSA tests before diagnosis carried out over a minimum of 18 mo were included. PSAV was calculated by using three methods: The differences between the methods and test characteristics of each method were compared. RESULTS: Of the 2204 men included, 716 (32.5%) were diagnosed with prostate cancer and 1488 (67.5%) benign histology. PSAV differed markedly in each method of calculation. The LR and FL methods had similar predictive values, which were higher than the AE method. There was strong agreement for cancer diagnosis between LR and FL (kappa=0.85), with weaker agreement between LR/AE and FL/AE (kappa=0.69 and 0.66, respectively). CONCLUSIONS: Methods used to calculate PSAV using the same PSA data can produce markedly different results. Linear regression should be the method of choice for calculating PSAV. Using first and last PSA values only may be adequate for everyday clinical use, as long as measurements are separated by a sufficiently long time period.

Synergism between radiotherapy and vascular risk factors in the accelerated development of atherosclerosis: a report of three cases.

Radiotherapy is commonly used in the management of testicular tumors. However, to date the risk of radiation-induced vascular occlusive disease in men following radiotherapy for testicular cancer has not been regarded as a major factor in their long-term care. Several animal studies have shown the importance of established vascular risk factors such as hypercholesterolemia and hypertension in the pathogenesis of radiation-induced atherosclerosis. This report presents three cases of premature chronic iliofemoral arterial disease presenting 5,13, and 16 years following exposure to therapeutic irradiation for the treatment of testicular cancer. The patients were in the age group of 40-45 years and all demonstrated associated known atherosclerotic risk factors. The patients had received radiotherapy in the dose of 3,500-4,000 rads in a standard "dog-leg" fashion to the ipsilateral aortoiliac lymphatic chain. Our results showed that young men treated with radiotherapy for testicular cancer may be targeted from the outset for atherosclerotic risk factor reduction to minimize the risk of development of late chronic occlusive arterial disease. It may be that a cohort of men so treated with historical regimes of radiotherapy and now entering middle age should be screened for arterial disease and risk factor reduction.