Nanodiscs provide a useful approach for experimental studies focused on elucidating the structure and function of membrane proteins. The self-assembly process offers an efficient way for optimizing the composition and stoichiometry of the lipid mixture, scaffold protein, and the target membrane protein, resulting in soluble lipoprotein particles with a highly homogeneous size distribution. As a result, this system is now commonly used for many biophysical and biochemical applications, resulting in hundreds of new publications every year. In this review, we give an overview of some of the recent results that describe various aspects of lipid-protein interactions being investigated using the Nanodisc system, focusing on the novel methods and presenting some of the advantages of this methodology.

CYP3A4 is the main xenobiotic metabolizing enzyme in the human body, as it is in volved in metabolism of ~30% of drugs on the market as well as of many other compounds, from small molecules such as ethanol to large drugs such as erythromycin and cyclosporine [...].

The human cytochrome P450 CYP17A1 plays a critical role in the production of steroid hormones, converting pregnenolone to dehydroepiandrosterone and progesterone to androstenedione. Sequential reactions catalyzed by CYP17A1 are hydroxylation at C17 position, followed by C17 - C20 carbon‑carbon bond scission. The mechanism of the lyase reaction is still debated, with two proposed reaction pathways favoring either a peroxo- (Compond 0) or iron-oxo (Compound 1) driven catalysis. In this review we summarize the results obtained through collaboration between the Sligar laboratory at University of Illinois and the Kincaid laboratory at Marquette University over the last 15 years. We used a combination of spectroscopic and functional studies of human CYP17A1 incorporated in lipid Nanodiscs, mimicking the native membrane environment, to dissect the elementary steps of P450 reaction cycle and characterize the iron‑oxygen intermediates in the presence of substrates for both reactions catalyzed by CYP17A1. In addition, we used the mutations E305G and T306A to probe the effect of perturbing the proton delivery required for the formation of Compound 1, but not for Compound 0, and the mutation N202S involved in substrate positioning at the active site. Resonance Raman spectra, in combination with cryo-radiolytic reduction of the oxy-complex of CYP17A1, provided a detailed picture of hydrogen bonding and protonation of peroxo- and hydroperoxo- intermediates and identified a new transient hemiketal complex on the peroxo-driven pathway of lyase reaction. These results consistently demonstrated the predominant role of the peroxo-driven catalysis for the lyase reaction in CYP17A1 incorporated in lipid Nanodiscs.

Heme-binding biguanides inhibit cytochrome P450 monooxygenase-mediated biosynthesis of the epoxy fatty acid (EpFA) (±)14,15-EET, which promotes progression of ER+HER2- breast cancer as well as regulatory T cell (Treg) proliferation. However, there are no known strategies to exploit this vulnerability. Metformin has shown promise but lacks sufficient potency and clinical utility. Through structure-based design, heme binding hexyl cubane derivatives of metformin were identified, which potently inhibited (±)14,15-EET biosynthesis. Fluorination of the hexyl moiety improved pharmacokinetics (PK) and reduced toxicity. Among the fluorinated derivatives, C5F2-HCB potently inhibited (±)14,15-EET biosynthesis (IC50=3.1 uM for C5F2-HCB vs. 5 mM for metformin). C5F2-HCB and metformin are candidate agents for TME activation from “cold” to “hot” due to their inhibition of EETs biosynthesis and EET-driven oxidative phosphorylation (OXPHOS) which promotes tumor hypoxia. We then tested whether C5F2-HCB or metformin could inhibit immune exclusion in the immunologically “cold” ovarian dependent STAT1 KO SSM2ucd mammary carcinoma model, in part, by reducing Tregs and/or increasing CD8 or CD4 tumor infiltrating T cells (TIL). We hypothesized that biguanides, by reducing EETs, would suppress Tregs and promote effector T cells in the TME. We also hypothesized that C5F2-HCB would be more effective than metformin in modulating the TME, based on greater potency for inhibition of (±)14,15-EET biosynthesis. Both biguanides were tested at their maximum tolerated dose (C5F2-HCB; 18 mg/kg daily and metformin; 50 mg/kg every other day). In the SSM2ucd allograft, all combination treatment groups with biguanides reduced tumor growth relative to the IgG control at the endpoint (day 63) : metformin + IgG (51.5±9.8% of control), C5F2-HCB + IgG (53.3±12.8%), metformin + anti-PD-1 (50.4±11.5%), and C5F2-HCB + anti-PD-1 (49.6±12.8%) [(P < 0.05) for all comparisons]. In contrast, anti-PD-1 antibody alone failed to reduce tumor growth at the endpoint. Flow cytometry analysis of the TME revealed that only C5F2-HCB + IgG treatment increased CD8+ tumor-infiltrating lymphocytes (TILs) compared to all other treatment groups (>2-fold; P < 0.05). Additionally, only C5F2-HCB + anti-PD-1 reduced intratumoral Tregs (>3-fold; P < 0.05). C5F2-HCB in combination with IgG or anti-PD-1 reduced intratumoral macrophages by >50% (P < 0.001) and reduced the M2/M1 macrophage ratio by 32-61% (P < 0.05) relative to other groups. No effects on CD4+ T cells were observed. Furthermore, C5F2-HCB and IgG or anti-PD-1 increased intratumoral neutrophils by 75- 120% compared to the anti-PD-1 or IgG (P < 0.05). (Multiple comparisons tested by ANOVA). Relative to IgG alone, C5F2-HCB and metformin inhibited growth of the SSM2ucd allograft in combination with either anti-PD1 or IgG. In contrast to metformin, only C5F2-HCB modulated the TME, including increased CD8+ TILs with IgG, reduced Tregs with anti-PD-1, and decreased M2/M1 macrophage ratio with IgG and anti-PD-1. C5F2-HCB, in contrast tometformin, was effective in modulating an immune excluded TME and may contribute to a strategy for clinical translation. Zhijun Guo, Jianxun Lei, Joshua McCarra, Qing Cao, Michael J. Pryzbilla, Brenda Koniar, Beverly Norris, Robert J. Schumacher, Swaathi Jayaraman, John R. Hawse, Antonino B. D'Assoro, Ilia G. Denisov, Stephen G. Sligar, Kathryn L. Schwertfeger, Michael A. Farrar, Dipak Panigrahy, Hammock D. Bruce, Elizabeth A. Ambrose, Gunda I. Georg, Goetz P. Matthew, David A. Potter. C5F2-hexyl-(cuban-1-yl-methyl)-biguanide (C5F2-HCB) overcomes an immune excluded tumor microenvironment (TME) and suppresses tumor growth in the ovarian dependent ER+HER2- SSM2ucd mammary carcinoma allograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB010.

Nanodiscs represent a versatile tool for studies of membrane proteins and protein-membrane interactions under native-like conditions. Multiple variations of the Nanodisc platform, as well as new experimental methods, have been recently developed to understand various aspects of structure, dynamics and functional properties of systems involved in signaling, transport, blood coagulation and many other critically important processes. In this mini-review, we focus on some of these exciting recent developments that utilize the Nanodisc platform.

This article continues the series of works by the authors on the approximation of the electronic terms of a diatomic molecules and their vibrational structure by the Morse formula, which is the simplest anharmonic approximation of the real term U(r). Depending on the choice of parameters, the approximation has two alternative solutions M1(r) and M2(r), with different patterns of deviations from the real term and its vibrational structure; some typical deviations for M1(r) and M2(r) are described. The difference delta(r)=U(r)-M(r) quantitatively shows the changes in the shape of the terms during approximation and can serve as a reference point when choosing its type for solving a specific problem. We introduced an empirical anharmonicity function -2 omega ex(v), which characterizes the positions of vibrational levels in the potential well; it demonstrates the distortion of the vibrational structure of the term U(r) during the approximation. Based on the data from literature, the functions delta(r) and -2 omega ex(v) were constructed for more than 20 molecules and a preliminary classification of electronic terms according to their characteristic features is described. Here we present a group of simple terms with minimal deviations from the Morse shape.

Human cytochrome P450 CYP17A1 catalyzes the hydroxylation of pregnenolone and progesterone at the C17 position, with subsequent C17-C20 bond scission, to form dehydroepiandrosterone and androstenedione respectively. The first hydroxylation reaction is faster in H2O than in D2O, while the second carbon‑carbon bond scission event demonstrates an inverse solvent isotope effect, which is more pronounced for 17-hydroxy pregnenolone. In order to better understand the cause of this difference, we compared the optical absorption spectra of oxygenated CYP17A1 with the four substrates (pregnenolone, progesterone, 17-hydroxy pregnenolone and 17-hydroxy progesterone) in both H2O and D2O. We also studied the temperature-dependent decay of the peroxo-ferric and hydroperoxo-ferric intermediates generated by cryoradiolysis of the corresponding oxygenated heme proteins at 77 K. For both pregnenolone and 17-hydroxypregnenolone, annealing of the peroxo-intermediates was observed at lower temperatures in H2O than in D2O. In contrast, no solvent isotope effect was detected when progesterone or 17-hydroxyprogesterone were used as substrates. These differences are attributed to their different positioning in the P450 active site with respect to the heme bound peroxo (Fe-OO-) moiety, which is in agreement with earlier structural and spectroscopic investigations. Analysis of the samples run in both H2O and in D2O, where 17-hydroxyprogesterone is the substrate, demonstrated significant (∼25%) yield of androstenedione product relative to the oxygenated starting material.

The catalytic cycle of the cytochromes P450 (CYP) requires two electrons from a protein redox partner and two protons from water to generate the main catalytic intermediate, a ferryl-oxo complex with π-cation on the heme porphyrin ring, termed Compound 1. The protonation steps are at least partially rate-limiting, therefore the steady-state rates of P450 catalysis are usually slower in deuterated solvent (D2O) by a factor of 1.5–3. However, in several P450 systems a pronounced inverse kinetic solvent isotope effect (KSIE ∼0.4–0.7) is observed, where the reaction is faster in D2O. This raises an important mechanistic question: Is this inverse solvent isotope effect compatible with Compound 1 catalyzed reactions, or is it indicative of another catalytic intermediate being involved? In this communication we use exhaustive numerical modeling of the P450 steady-state kinetics to demonstrate that a significant inverse KSIE cannot be obtained for a pure Compound 1 driven catalytic cycle of P450. Rather, an alternative, protonation independent, catalytic intermediate needs to be introduced. This result is applicable to the broad spectrum of P450s in nature, but as an example we use the extensively documented inverse isotope effect in the human steroid biosynthetic P450 CYP17A1 where the involvement of a heme peroxo anion intermediate has been characterized. Based on this analysis, we show that the observation of an inverse KSIE can be used as a general mechanistic probe for reaction cycle intermediates in the cytochromes P450.

A brief review of the latest results of the application of the approximation of the potential of a diatomic molecule by the Morse model function in applied spectroscopy is presented. The functions of the electronic terms of the diatomic molecules BeH, F 2 , H 2 , HCl, and Be 2 are compared with their two alternative approximations by the Morse function. As a criterion, we used the differences between the original (approximated) term and its Morse models, which, combined with the dependence of the anharmonicity of the original terms on the vibrational quantum number ω e x e (v), allowed to formulate some generalizations about the deformation of the form the original term in the approximations. Simulation always leads to an increase in the bond energy in the range of 7-50% and to an increase in the number of vibrational levels. In favorable cases, the contour shape is reproduced with a deviation of no more than 100-200 cm -1 in the lower part of the potential well. Key words: Morse formula, diatomic molecule, anharmonicity, electronic terms, vibrational structure.

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F' loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation.

The multifunctional cytochrome P450 17A1 (CYP17A1) plays a crucial role in human steroid hormone synthesis (UniProtKB─P05093). It first carries out standard monooxygenase chemistry, converting pregnenolone (PREG) and progesterone (PROG) into 17OH-PREG and 17OH-PROG, utilizing a "Compound I" to initiate hydrogen abstraction and radical recombination in the classic "oxygen rebound" mechanism. Additionally, these hydroxylated products also serve as substrates in a second oxidative cycle which cleaves the 17-20 carbon-carbon bond to form dehydroepiandrosterone and androstenedione, which are key precursors in the generation of powerful androgens and estrogens. Interestingly, in humans, with 17OH-PREG, this so-called lyase reaction is more efficient than with 17OH-PROG, based on Kcat/Km values. In the present work, the asparagine residue at 202 position was replaced by serine, an alteration which can affect substrate orientation and control substrate preference for the lyase reaction. First, we report studies of solvent isotope effects for the N202S CYP17A1 mutant in the presence of 17OH-PREG and 17OH-PROG, which suggest that the ferric peroxo species is the predominant catalytically active intermediate in the lyase step. This conclusion is further supported by employing a combination of cryoradiolysis and resonance Raman techniques to successfully trap and structurally characterize the key reaction intermediates, including the peroxo, the hydroperoxo, and the crucial peroxo-hemiketal intermediate. Collectively, these studies show that the mutation causes active site structural changes that alter the H-bonding interactions with the key Fe-O-O fragment and the degree of protonation of the reactive ferric peroxo intermediate, thereby impacting lyase efficiency.

We analysed the problem of approximation of the potential function of a diatomic molecule by a Morse model function with constant anharmonicity ν x using the Birge-Sponer extrapolation. The analysis of the approximations used in the derivation of the Morse equation shows that the solution of this problem is ambiguous. A scheme for optimizing the selection of initial parameters is proposed, which is illustrated by examples taken from the literature. The advantages of delineation of anharmonicity in the excitation of vibrational levels by deviations of the value ν x from the constant value according to Morse are demonstrated. An attempt is made to use the dimensionless anharmonicity parameter x* as a universal characteristic of the shape features of the electronic term of the molecule. Keywords: Morse potential, diatomic molecule, Birge-Sponer extrapolation, anharmonicity, electronic terms, vibrational structure.

Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C17-C20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.

Abstract Introduction: Small molecule therapeutics of estrogen receptor-positive/HER2-negative breast cancer remains an area of active investigation where novel agents are greatly needed for treatment of hormone therapy resistant metastatic disease. The biguanide hexyl-benzyl-biguanide (HBB) is a potent inhibitor of CYP3A4 arachidonic acid (AA) epoxygenase activity and inhibits breast cancer cell proliferation and MCF-7 breast cancer tumor growth in nude mice. To explore the impact of bioisosteric substitution of the benzyl moiety of HBB with a cubane moiety, we synthesized hexyl-(cuban-1-yl-methyl)-biguanide (HCB) and tested its potency for the inhibition of the cognate CYP3A4 target AA epoxygenase activity as well as breast cancer cell proliferation of hormone therapy sensitive and resistant cell lines. Results: HCB selectively inhibited CYP3A4-mediated biosynthesis of (±)-14,15-EET with an IC50 of 4.7±0.2 uM vs. 64.8±6.5 uM for 8,9-EET and 26.5±1.9 uM for 11,12-EET. At 24 hours, HCB inhibited proliferation of MCF-7 (ER+HER2-), BT474 (ER+HER2+) and MDA-MB-231 (ER-HER2-) cells at IC50 of 8.4±1.2, 11±1.3 and 15±0.9 uM, respectively. At 48 hours, HCB inhibited proliferation of aromatase inhibitor and fulvestrant resistant (LR,FR), and cyclin dependent kinase inhibitor (CDKi) palbociclib resistant (LR,FR,PR) MCF-7 cell lines; LR,FR MCF-7AC1 (IC50 =1.34±0.1 uM) and LR,FR,PR MCF-7AC1 (IC50 =1.64±0.2 uM). Addition of 14,15-EET (1 uM) partially rescues MCF-7 cells from HCB-mediated inhibition of proliferation. OXPHOS is promoted, in part, by EETs. HCB is a potent OXPHOS inhibitor and rapidly inhibits O2 consumption of the MCF-7 and ZR75 (ER+HER2-) cells in a dose-dependent fashion (P<0.05). HCB treatment (10 uM) reduces mitochondrial membrane potential to 57.4±15.3% (P<0.001) of vehicle control in MCF-7 cells. Treatment with HCB at 20 uM for 0.5 hour also causes mitochondrial swelling in MCF-7 cells. HCB (10 uM) activates AMPK within 0.5 hour and increases the level of phosphorylation from 2.4±0.3 to 25.1±6.0 folds in a time dependent fashion in MCF-7 cells from 0.5-24 hours. Conclusion: These results show that HCB inhibits proliferation of ER+HER2- breast cancer cells, in part through inhibition of OXPHOS and suppression of the CYP product 14,15-EET. This inhibition is highly active in hormonal therapy and CDKi resistant ER+HER2- breast cancer cells. These results suggest that HCB is a novel and potent biguanide that has potential to be developed for inhibition of hormone therapy resistant and CDKi resistant breast cancer. Citation Format: Zhijun Guo, Jianxun Lei, Kwon Ho Hong, Beverly Norris, Craig M. Flory, Swaathi Jayaraman, Connor McDermott, Elizabeth Ambrose, Irina Sevrioukova, Tom Poulos, Ilia Denisov, Stephen Sliga, Robert J. Schumacher, Gunda I. Georg, John R. Hawse, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) inhibits hormone therapy resistant breast cancer cells, in part by Inhibiting CYP3A4 arachidonic acid epoxygenase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB078.

We developed an efficient and sensitive probe for drug-drug interactions mediated by human CYP3A4 by using midazolam (MDZ) as a probe substrate. Using global analysis of four parameters over several experimental data sets, we demonstrate that the first MDZ molecule (MDZ1) binds with high affinity at the productive site near the heme iron and gives only hydroxylation at the 1 position (1OH). The second midazolam molecule (MDZ2) binds at an allosteric site at the membrane surface and perturbs the position and mobility of MDZ1 such that the minor hydroxylation product at the 4 position (4OH) is formed in a 1:2 ratio (35%). No increase in catalytic rate is observed after the second MDZ binding. Hence, the site of the 1OH:4OH metabolism ratio is a sensitive probe for drugs, such as progesterone, that bind with high affinity to the allosteric site and serve as effectors. We observe similar changes in the MDZ 1OH:4OH ratio in the presence of progesterone (PGS), suggesting a direct communication between the active and allosteric sites. Mutations introduced into the F-F' loop indicate that residues F213 and D214 are directly involved in allosteric interactions leading to MDZ homotropic cooperativity, and these same residues, together with L211, are involved in heterotropic allosteric interactions in which PGS is the effector and MDZ the substrate. Molecular dynamics simulations provide a mechanistic picture of the origin of this cooperativity. These results show that the midazolam can be used as a sensitive probe for drug-drug interactions in human P450 CYP3A4.

Here, we report the first (pseudo)clathrochelate of iron(II) to undergo a temperature-induced spin crossover (SCO) to an elusive high-spin state "trapped" in one of its solvatomorphs. An SCO-inducing ability of the resulting extended-tripodal (cage) ligand in an otherwise SCO-inactive type of iron(II) complexes arises from its molecular design that adapts to the metal ion in the two spin states by experiencing a massive trigonal twist distortion. Combined with the amenability of this ligand to further functionalization to produce a series of iron(II) (pseudo)clathrochelates with tunable SCO behaviors, it opens up a possibility for this unique class of trigonal-prismatic complexes to soon emerge as effective switchable components for molecular electronics and spintronics.

Using Birge-Sponer extrapolation we have analyzed the approximation of the potential of a real diatomic molecule by the Morse model, which implies a constant value of anharmonicity ωx. The real values of ωx*(v) for each vibrational level are estimated from transition frequencies between neighboring levels. The dependence of ωx* on the vibrational quantum number v up to dissociation is calculated from the literature data for the ground electronic state of H2, O2, Be2, Li2, ArXe, Xe2, Kr2 and the excited state of Li2. Characteristic features of deviations of the anharmonicity parameter x* - x from the Morse model are described.

CYP17A1 is an essential human steroidogenic enzyme, which catalyzes two sequential reactions leading to the formation of androstenedione from progesterone and dehydroepiandrosterone from pregnenolone. The second reaction is the C17-C20 bond scission, which is strongly dependent on the presence of cytochrome b5 and displays a heretofore unexplained more pronounced acceleration when 17OH-progesteone (17OH-PROG) is a substrate. The origin of the stimulating effect of cytochrome b5 on C-C bond scission catalyzed by CYP17A1 is still debated as mostly due to either the acceleration of the electron transfer to the P450 oxy complex or allosteric effects of cytochrome b5 favoring active site conformations that promote lyase activity. Using resonance Raman spectroscopy, we compared the effect of Mn-substituted cytochrome b5 (Mn-Cytb5) on the oxy complex of CYP17A1 with both proteins co-incorporated in lipid nanodiscs. For CYP17A1 with 17OH-PROG, a characteristic shift of the Fe-O mode is observed in the presence of Mn-b5, indicating reorientation of a hydrogen bond between the 17OH group of the substrate from the terminal to the proximal oxygen atom of the Fe-O-O moiety, a configuration favorable for the lyase catalysis. For 17OH-pregnenolone, no such shift is observed, the favorable H-bonding orientation being present even without Mn-Cytb5. These new data provide a precise allosteric interpretation for the more pronounced acceleration seen for the 17OH-PROG substrate.