Unveiling the Crucial Intermediates in Androgen Production

Significance: The human enzyme cytochrome P450 17A1 (CYP17A1) catalyzes the critical step in the biosynthesis of the male sex hormones, and, as such, it is a key target for the inhibition of testosterone production that is necessary for the progression of certain cancers. CYP17A1 catalyzes two distinct types of chemical transformations. The first is the hydroxylation of the steroid precursors pregnenolone and progesterone. The second is a different reaction involving carbon–carbon (C-C) bond cleavage, the mechanism of which has been actively debated in the literature. Using a combination of chemical and biophysical methods, we have been able to trap and characterize the active intermediate in this C-C lyase reaction, an important step in the potential design of mechanism-based inhibitors for the treatment of prostate cancers. Abstract: Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from characterization of key reaction intermediates. The multifunctional cytochrome P450 17A1 (CYP17A1) first catalyzes the typical hydroxylation of its primary substrate, pregnenolone (PREG) and then also orchestrates a remarkable C17–C20 bond cleavage (lyase) reaction, converting the 17-hydroxypregnenolone initial product to dehydroepiandrosterone, a process representing the first committed step in the biosynthesis of androgens. Now, we report the capture and structural characterization of intermediates produced during this lyase step: an initial peroxo-anion intermediate, poised for nucleophilic attack on the C20 position by a substrate-associated H-bond, and the crucial ferric peroxo-hemiacetal intermediate that precedes carbon–carbon (C-C) bond cleavage. These studies provide a rare glimpse at the actual structural determinants of a chemical transformation that carries profound physiological consequences

Evidence That Cytochrome \u3cem\u3eb\u3csub\u3e5\u3c/sub\u3e\u3c/em\u3e Acts as a Redox Donor in CYP17A1 Mediated Androgen Synthesis

Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second C--C lyase step, at the expense of glucocorticoid production. Cytochrome b5(cyt b5) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b5 increases androgen biosynthesis. Cyt b5 could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b5 could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b5, we generated a redox-inactive form of cyt b5, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b5), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b5 were enhanced ∼5 fold as compared to reactions in the absence of cyt b5. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b5. Upon addition of Mn-b5 to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b5-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal Fe--S vibrational frequency. Thus, although Mn-b5 binds to CYP17A1, it is unable to enhance the lyase reaction, strongly suggesting that cyt b5 has a redox effector role in enhancement of the CYP17A1 mediated lyase reaction necessary for androgen synthesis

Experimental study of microwave pulse compression using a five-fold helically corrugated waveguide

This paper presents the experimental study of microwave pulse compression using a five-fold helically corrugated waveguide. In the experiment, the maximum power compression ratio of 25.2 was achieved by compressing an input microwave pulse of 80 ns duration and 9.65 GHz to 9.05 GHz frequency swept range into a 1.6 ns Gaussian-envelope pulse. For an average input power of 5.8 kW generated by a conventional traveling wave tube, a peak pulse output power of 144.8 kW was measured corresponding to an energy efficiency of 66.3%

Design and experiments of a five-fold helically corrugated waveguide for microwave pulse compression

Metal waveguide can be used as a dispersive medium to convert long duration, lower power pulses into short, higher peak power pulses. This provides an advanced method to generate radiation with gigawatts power in the millimeter and sub-millimeter wavelength range by compressing a megawatt level long duration pulse. In this paper, a five-fold helically corrugated waveguide operating in X-band was designed and constructed. The experiments conducted show that a 5.75 kW average power microwave pulse with a 6% bandwidth and duration of 80 ns can be compressed into a 144.8 kW, 1.6 ns pulse with a power compression factor of 25.2

Efficiency of Finding Muon Track Trigger Primitives in CMS Cathode Strip Chambers

In the CMS Experiment, muon detection in the forward direction is accomplished by cathode strip chambers~(CSC). These detectors identify muons, provide a fast muon trigger, and give a precise measurement of the muon trajectory. There are 468 six-plane CSCs in the system. The efficiency of finding muon trigger primitives (muon track segments) was studied using~36 CMS CSCs and cosmic ray muons during the Magnet Test and Cosmic Challenge~(MTCC) exercise conducted by the~CMS experiment in~2006. In contrast to earlier studies that used muon beams to illuminate a very small chamber area ($< \! 0.01$~m$^2$), results presented in this paper were obtained by many installed CSCs operating {\em in situ} over an area of $\approx \! 23$~m$^2$ as a part of the~CMS experiment. The efficiency of finding 2-dimensional trigger primitives within 6-layer chambers was found to be~$99.93 \pm 0.03\%$. These segments, found by the CSC electronics within $800$~ns after the passing of a muon through the chambers, are the input information for the Level-1 muon trigger and, also, are a necessary condition for chambers to be read out by the Data Acquisition System