The use of interstitial radiation therapy in the treatment of persistent, localized, and unresectable cancer in children

Two children with cancer that persisted after multiple exploratory laparotomies, external beam radiation therapy, and multidrug chemotherapy had gold 198 (198Au) seeds implanted into their localized but unresectable tumor. Both children are alive, are receiving no therapy, and are disease-free more than 2 years later. These two cases indicate the value of interstitial implant therapy in the treatment of some children with cancer.     

Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase

Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery.Small molecules have long proven their utility as an alternative to genetic mutation in perturbing and understanding biological systems (1). Interactions between small molecules are comparable to those observed among genetic mutations. In combination, one molecule may enhance (synergism) or suppress (antagonism) the effect of another molecule. In recent years, drug combinations have been extensively used to explore connections between cellular processes. Specifically, synergies have provided unique means to expose pathway interactions (2, 3) and, in turn, understand the mode of action of small molecules (4, 5). Underpinning these interactions are complex genetic networks that define the outcome of drug combinations (6, 7). Indeed, synergistic pairs often exhibit their effects by targeting related processes and impacting genetic interactions that bridge those pathways. Further, synergistic drug combinations are now essential therapeutic strategies in the clinic in areas such as cancer, HIV, and infectious diseases (8). Where antagonistic interactions are generally undesirable from a therapeutic perspective, the utility of this class of interactions to reveal biological function and underlying network connectivity has been largely overlooked. Like synergism, antagonism typically has a genetic basis that reflects relationships between cellular targets and mechanisms of drug action (7). Recent reports of antagonistic drug interactions have shed light on novel connections that govern important bacterial physiology. For example, the suppressive effects of antibiotics targeting protein and DNA synthesis in Escherichia coli revealed antagonistic connections between the regulation of ribosomal genes and the DNA stress response (9). A more recent study surveyed suppressive interactions among antifungals and described the mechanism of the suppressive activities of bromopyruvate and staurosporine (10). Interestingly, but perhaps counterintuitively, other studies have suggested that antagonistic drug pairs can even slow the evolution of drug resistance (11, 12). Nevertheless, the utility of antagonism among small molecules has yet to be fully explored as a tool to study biological function. Certainly, there have been no systematic searches for antagonistic interactions to exploit suppressive network connections and, in turn, uncover novel inhibitors of the targeted pathways.Bacterial cell wall synthesis is an antibacterial target that is celebrated for its druggability and, increasingly, for its genetic complexity. Indeed, the dispensability of wall teichoic acid (WTA) genes in gram-positive bacteria has emerged in recent years as a prototypical example of genetic antagonism. WTAs are phosphate-rich polymers that make up a large proportion of the cell wall of gram-positive bacteria and, in the pathogen Staphylococcus aureus, have a key role in cell division and virulence (13, 14). The synthesis of WTA in S. aureus is initiated by the action of two nonessential gene products: TarO and TarA. TarO (undecaprenyl-phosphate N-acetylglucosaminyl 1-phosphate transferase) transfers an N-acetyl-glucosamine-1-phosphate moiety to an undecaprenyl phosphate carrier lipid, followed by the transfer of N-acetylmannosamine by TarA (N-acetylglucosaminyldiphospho-undecaprenol N-acetyl-β-d-mannosaminyltransferase). The resulting glycolipid is a substrate for so-called late-step gene products that append a polymer with ribitol-phosphate repeats before export to the external surface and attachment to peptidoglycan (PG). Paradoxically, the genes encoding the late-steps in WTA synthesis have an essential phenotype, but become dispensable in strains with a deletion in either of the early-step genes, tarO or tarA (encoding for an N-acetylglucosamine-1-phosphate transferase and an N-acetylmannosamine transferase, respectively) (15). Interestingly, blocking the early steps in WTA biosynthesis leads to β-lactam sensitivity in methicillin-resistant S. aureus (MRSA) (2, 16). These observations highlight the complexity of cell wall synthesis in gram-positive bacteria and provide a rationale for combination therapy. Further, the idiosyncratic genetic antagonism of the WTA biosynthetic pathway and interactions with additional components of cell wall synthesis provide a unique opportunity to screen for new chemical matter with utility as probes to better understand this genetic complexity.To this end, we conducted a search for compounds that antagonize the lethal activity of targocil (17) (Scheme 1), a probe of TarG, the essential gene product that makes up the transmembrane transporter that exports WTAs to the cell surface. Screening a library of previously approved drugs we discovered that clomiphene (Scheme 1) a widely used fertility drug, was a potent antagonist of targocil. Mechanistic characterization revealed that its target was the undecaprenyl diphosphate synthase (UppS), responsible for the synthesis of the lipid carrier, undecaprenyl phosphate (Und-P), and we solved a cocrystal structure of clomiphene with UppS from E. coli. We report on the ability of clomiphene to potentiate the activity of β-lactam antibiotics against MRSA, revealing UppS as a key component of the network that supports β-lactam resistance in MRSA. As such, clomiphene is new cell-permeable probe of the synthesis of Und-P and represents a potential lead for antibiotic drug discovery.Open in a separate windowScheme 1.Chemical structures of clomiphene, targocil, and ticlopidine.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Electrochemically addressable trisradical rotaxanes organized within a metal–organic framework

The organization of trisradical rotaxanes within the channels of a Zr₆-based metal–organic framework (NU-1000) has been achieved postsynthetically by solvent-assisted ligand incorporation. Robust Zr^IV–carboxylate bonds are forged between the Zr clusters of NU-1000 and carboxylic acid groups of rotaxane precursors (semirotaxanes) as part of this building block replacement strategy. Ultraviolet–visible–near-infrared (UV-Vis-NIR), electron paramagnetic resonance (EPR), and ¹H nuclear magnetic resonance (NMR) spectroscopies all confirm the capture of redox-active rotaxanes within the mesoscale hexagonal channels of NU-1000. Cyclic voltammetry measurements performed on electroactive thin films of the resulting material indicate that redox-active viologen subunits located on the rotaxane components can be accessed electrochemically in the solid state. In contradistinction to previous methods, this strategy for the incorporation of mechanically interlocked molecules within porous materials circumvents the need for de novo synthesis of a metal–organic framework, making it a particularly convenient approach for the design and creation of solid-state molecular switches and machines. The results presented here provide proof-of-concept for the application of postsynthetic transformations in the integration of dynamic molecular machines with robust porous frameworks.The predictability and reliability with which metal–organic frameworks (MOFs) are assembled (1–11) has accelerated the rate at which porous materials can be developed for applications as diverse as gas storage and separation (7), sensing (8), catalysis (9), and light harvesting (10, 11). Metal oxide joints and organic struts are arranged regularly within MOFs, giving rise to hybrid materials with permanent porosities. It has been proposed (12, 13) that integrating the rigidity and periodicity of MOFs with the addressability and workings of molecular switches and machines, such as bistable mechanically interlocked molecules (MIMs) (14–16), stands a good chance of giving rise to a new class of functional materials that are simultaneously both robust and dynamic. Most switchable MIMs that have been developed operate in solution where they are stochastically oriented and the net movement of a population of switches averages to zero. By integrating such rudimentary molecular switches within highly ordered MOFs, however, they can be organized periodically and precisely in 3D space, allowing their otherwise incoherent motions to be rectified. Although steady progress has been made (17–26) toward this goal in recent years, it still remains a considerable challenge to design such systems that can be addressed by stimuli in the solid state. Electrochemical potential and light would be particularly appealing stimuli on account of their ease of interfacing with current technologies. The avenues of investigation that have been explored up to this point have entailed the de novo assembly of MOFs from MIMs that double as organic struts—ones that are bulkier and considerably more complex than those routinely used in the preparation of MOFs. Consequently, laborious and demanding optimization of solvothermal crystallization conditions has often been necessary to grow MOFs from the MIM struts.During these last 15 years, we devoted a considerable amount of time and effort in research into manipulating the self-organization of relatively small numbers of bistable MIMs as a means of storing and processing information in defect-tolerant architectures to realize both memory and logic functions in molecular electronic devices (MEDs). We made the decision at the outset of what turned out to be a fruitful collaboration (Fig. 1) with James (Jim) Heath, starting in 2000 with the publication of an article (27) in Science describing the solid-state electronically reconfigurable switch based on a bistable [2]catenane, to work with collections of molecules, rather than with single molecules, for two reasons: one being that we were looking for a relatively simple way of tiling bistable MIMs into device settings where multiple junctions (points) are individually and separately addressable and the other being that isolated single molecules cannot be relied on to reside passively in a particular state: we decided it made much more sense to put our trust in collections of molecules where even if some—obeying the Boltzmann distribution—were out of sync with the rest, then the majority of the molecules would cover for them. The devices, which were chosen right at the beginning, were based on cross-bar architectures (27, 28) for the simple reason that thousands at least, if not hundreds, of molecules could, in principle, be addressed uniquely and separately inside molecular switch tunnel junctions (MSTJs) corresponding to the crossing points of the wires associated with these cross-bar architectures. We established a highly repeatable fabrication method in which the Langmuir–Blodgett (LB) technique is used to self-organize the bistable catenanes and rotaxanes on these cross-bars: the fact that the LB technique is commensurate with the tiling of molecules in two dimensions means that we can deliver monolayers of the bistable MIMs with relative ease and in surprisingly high efficiency to the MSTJs on the cross-bars. We were also to discover, as a result of carrying out an extensive piece of physical organic chemistry (29), that, when these bistable MIMs—which can be switched electrochemically in solution—are introduced into highly viscous polymer matrices, self-assembled as monolayers on gold surfaces courtesy of thiooctic acid appendages, or organized by their hundreds or thousands between two wires (electrodes) at MSTJs, the thermodynamics that characterize their switching remains invariant, whereas their kinetics are influenced as expected: they go from being fast (half-life-times of seconds) in solution to being slow (half-life-times of hours) in cross-bar MSTJs. This program of research hit its high spot in 2007 with the publication of an article (30) in Nature describing a 160-kilobit molecular electronic memory patterned at 100,000,000,000 bits/cm² with bistable [2]rotaxane molecules, which exhibit a footprint somewhere in the region of 2 nm² in an LB film, serving as the data storage elements. The assembled cross-bar memory consisted of 400 Si bottom-nanowire electrodes (16 nm wide, 33 nm pitch) crossed by 400 Ti top-nanowire electrodes (16 nm wide, 33 nm pitch), sandwiching a monolayer of ∼100 bistable [2]rotaxane molecules. There are some major limitations hampering the further development of MEDs based on bistable MIMs: one is the nonscalable LB technique for forming densely packed monolayers of the MIMs free of defects and the other is the fact that the devices tend to peter out after tens, or at the most hundreds, of cycles. A relatively recent attempt (31) to introduce a switchable, multiply bistable polyrotaxane by spin-coating it onto the bottom Si electrode met with only limited success. In retrospect, this observation is hardly all that surprising because structural failure along the polymer backbone is to be expected when it is subjected to repeated dynamics. These lessons have led us to believe (12, 32) that the means of improving device robustness may reside in MOFs that are capable of housing bistable MIMs without compromising their ability to switch, courtesy of the relative movements of their components. A more lengthy discussion of our foray into molecular electronics is presented (32) in Chemical Society Reviews published in 2012. In a perspective entitled “Whence Molecular Electronics?” featured (33) in Science in 2004, it was concluded that

“Molecular electronics will mature into a powerful technology only if its development is based on sound scientific conclusions that have been tried and tested at every step. Reaching these objectives requires a detailed understanding of the molecule / electrode interface, as well as developing methods for manufacturing reliable devices and ensuring their robustness.”

Open in a separate windowFig. 1.Leaders in the fields of MEDs, the internal dynamics of solids, and robust dynamics in MOFs. From Left to Right: Professors James (Jim) Heath, Omar Yaghi, Miguel Garcia-Garibay, and Stephen (Steve) Loeb.MOFs (1–11) are highly self-assembled crystalline compounds with porous structures comprised of metal-oxide joints and organic struts that extend in all directions throughout the crystal: the molecule is the crystal and the crystal is the molecule! In a perspective on “Robust Dynamics” published (12) in Nature Chemistry in 2010, we proposed, in conjunction (Fig. 1) with Omar Yaghi, coupling the dynamics exhibited by molecular switches and machines in bistable MIMs with the rigid 2D and 3D structures associated with MOFs to yield materials that are intrinsically robust yet exhibit dynamics. The prospect of positioning molecular switches symmetrically within the constitutions of organic struts of MOFs, or attaching them to the metal-oxide joints of MOFs, is an attractive proposition. We argued that, if the mounting of bistable MIMs in MOFs could be achieved, then it would afford us a class of solid-state devices that could open up fresh opportunities in the field of molecular electronics.Two architectural features can be distinguished in the majority of porous MOFs with reference to the pore apertures and the internal sorting and coverage domains. In the sorting domain, molecules are selected at the orifices of the pores, according to their sizes. In the coverage domain, molecules are adsorbed by weak van der Waals forces associated with the struts and metal joints. In 2009, we introduced (34) a third domain, an active one, into a MOF in the form of a crown ether and demonstrated that it is capable of binding methyl viologen (MV²⁺). The docking of MV²⁺ in MOF-1001, where the struts incorporate a bisparaphenylene[34]crown-10 (BPP34C10) established that donor–acceptor molecular recognition between BPP34C10 and MV²⁺, which is well known to occur in solution, can be transferred with its receptor function being preserved into a MOF. The success of this experiment, based on the cubic topology of the archetypal MOF-5, in which paraphenylene struts are joined by Zn₄O(CO₂)₆ cluster joints, places the concept of robust dynamics on a firm footing.Our subsequent attempts to incorporate struts containing a degenerate [2]catenane, in which the BPP34C10 ring, or a 1,5-dioxynaphthalene-containing analog thereof, is interlocked mechanically with the tetracationic cyclophane, cyclobis(paraquat-p-phenylene) (CBPQT⁴⁺), which contains two viologen units, met with only limited success, and that was only after the zinc nitrate used in the synthesis of MOF-1001 was replaced by copper nitrate. (Bold font represents acronyms used to identify chemical compounds.)In 2010, we reported (19) the synthesis and crystal structure of MOF-1011 in which a donor–acceptor [2]catenane, obtained by using a dicarboxylic acid containing a BPP34C10 ring interlocked with a CBPQT⁴⁺ cyclophane, is incorporated into a solid-state, 2D network replete with ordered catenanes—one per copper unit, eight per unit cell, and 81 per 100 nm² of surface—throughout the crystal. An extended strut, wherein 1,5-naphtho-p-phenylene[36]crown-10 (NPP36C10) was grafted into its midriff before being catenated with CBPQT⁴⁺ cyclophanes has been used (18) in the preparation of MOF-1030, which constitutes an example of the ordering of MIMs within a well-defined 3D solid matrix. The catenated MOF-1030, however, brings the prototypically active switching machinery in the struts to a standstill! This phenomenon was not an entirely unfamiliar one. The changes in dynamic behavior that can be expected on transitioning from working in solution, where molecules are free to tumble randomly, to crystalline materials, where they are highly ordered and densely packed, has been investigated systematically (35, 36) by Miguel Garcia-Garibay (Fig. 1) for several years. The additive effect of the many strong short-range interactions between closely packed molecules often arrests large-amplitude molecular motion almost entirely. Rapid dynamics are favored by large free volumes, weak intermolecular interactions, and high symmetry. Not only did the MIM components of MOF-1011 and MOF-1030 fill most of the free volume that would otherwise have been present in the MOF, they also possessed complementary donor and acceptor π-surfaces that could interact favorably with one another.When two—one degenerate and the other nondegenerate—[2]catenanes, containing struts incorporating NPP36C10 and bearing two terphenylene arms were reacted in the presence of copper nitrate, the copper paddlewheel-based MOF-1050 and MOF-1051 were isolated (24) as crystalline compounds. The solid-state structures of these MOFs reveal that the metal clusters serve to join the heptaphenylene struts into gridlike 2D sheets that are then held together by infinite donor–acceptor stacks involving the [2]catenanes to produce interpenetrated 3D architectures. Our frustration at the fact that π–π stacking interactions always seems to come into play between donor–acceptor catenanes when they are incorporated into MOFs and hence arrest the relative movements of the rings that could lead to switching behavior has found some recompense in MOF-2000, obtained when a mixture of struts containing a crown ether (BPP34C10) and a [2]catenane—with BPP34C10 and CBPQT⁴⁺ mechanically interlocked—are reacted with zinc nitrate in N,N-dimethylformamide at 65 °C. The creation of MOF-2000, whose two components are incorporated always in precisely a 2:1 ratio, even when the ratio of the two struts in the reaction mixture are varied by an order of magnitude, is a profound observation. We recently reported (37) in the Proceedings of the National Academy of Sciences statistical mechanical modeling by Stephen (Steve) Whitelam, which suggests that the robust 2:1 ratio has a nonequilibrium origin that results from kinetic trapping of the two different struts during the growth of the framework of MOF-2000. The timeline presented in Fig. 2 summarizes the progress that has been made in harnessing the switching the switching properties of bistable MIMs in MEDs and the subsequent attempts to house MIMs inside MOFs.Open in a separate windowFig. 2.A timeline from 2000 to 2015 summarizing the progression of bistable MIMs from a single MSTJ fabricated using the LB technique to 160,000 MSTJs in a nanometer-sized device in 2007, followed by a summary of efforts to mount bistable MIMs inside MOFs. The present article describes our most recent contribution to this challenging area of research.While this article was being written up for publication, Stephen Loeb (Fig. 1) has described, in an article (38) published in Nature Chemistry, the design and synthesis of a molecular shuttle that operates inside a Zn-based MOF. A wide range of dynamic ¹³C nuclear magnetic resonance (NMR) experiments was performed on the crystalline MOF that demonstrated that a [24]crown-8 (24C8) ring undergoes a degenerate shuttling motion between two benzimidazole recognition sites in a molecular shuttle positioned in an H-like manner between two triphenyldicarboxylic acid struts used in the synthesis of the MOF. The rate of shuttling of the 24C8 ring in the MOF is associated with an energy barrier of 14.1 kcal/mol, which is considerably higher than the energy barrier of the 7.7 kcal/mol observed for the strut by itself dissolved in a deuterated toluene solution. Although several reasons are advanced for this difference in the free energy of activation between the solution and solid states, an intriguing, but unlikely, possibility is that “one ring cannot move without its motion requiring the next ring to move.”Here, we outline a strategy for the organization of MIMs within the channels of a premade MOF through postsynthetic building block replacement. A semirotaxane (39), which is first formed in solution, binds (Fig. 3A) to coordination sites within the MOF through a functional end group at its nonstoppered end. It is thus immobilized within the pores of the framework while simultaneously undergoing a transformation to a kinetically trapped rotaxane in which the MOF serves as the second stopper. In addition to characterizing the resulting framework by ultraviolet–visible–near-infrared (UV-Vis-NIR), electron paramagnetic resonance (EPR), and ¹H NMR spectroscopies, we also conducted a molecular mechanics investigation to gain further insight into the experimentally observed number of mechanically interlocked (rotaxane) and noninterlocked (dumbbell) components within the pores. We grew electroactive thin films of the MOF crystals and demonstrated that redox-active subunits located on the rotaxane components can be accessed electrochemically in the solid state during cyclic voltammetry.Open in a separate windowFig. 3.(A) Schematic of the SALI approach to organizing rotaxanes inside the channels of a metal–organic framework. A semirotaxane is formed under equilibrium conditions from a ring and a half-dumbbell component with a bulky stoppering group at one end. A functional group (red) at the other end of the linear component acts as an attachment point to anchor the semirotaxane within the NU-1000 MOF. (B) An idealized representation of the structure of NU-1000 based on single-crystal X-ray diffraction (40).     

Serial Doppler Gradients Are Predictive of Future Bioprosthetic Valve Degeneration

Doppler echocardiography and color flow imaging are helpful techniques in evaluating the functional status of a bioprosthetic valve. The aim of this study was to determine whether serial Doppler gradients are predictive of future bioprosthetic valve degeneration. We performed serial echo-Doppler studies over a 6-year period (1988–1994) on 228 patients who had undergone mitral (n = 112) or aortic (n = 116) bioprosthetic valve implantation between 1973 and 1994. Thirtynine mitral prostheses and 30 aortic prostheses became dysfunctional and required reoperation. A serial rise in mean gradient of 5 mmHg or more across the mitral valve and 25 mmHg or more across the aortic valve was significantly associated with increased valve degeneration (odds ratio 3.40 and 16.11 and 95% confidence intervals 1.31 and 8.80 and 13.6 and 72.13 for the mitral and aortic valve, respectively). Both aortic and mitral valves began to degenerate after 8 years. Serial echo-Doppler studies showed a rise in transvalvular gradients around the same time. Closer evaluation for prosthetic valve dysfunction should be considered in patients 8 or more years status post surgery, especially those with high transvalvular gradients.     

Potentiation of Aminoglycoside Activity in Pseudomonas aeruginosa by Targeting the AmgRS Envelope Stress-Responsive Two-Component System

A screen for agents that potentiated the activity of paromomycin (PAR), a 4,5-linked aminoglycoside (AG), against wild-type Pseudomonas aeruginosa identified the RNA polymerase inhibitor rifampin (RIF). RIF potentiated additional 4,5-linked AGs, such as neomycin and ribostamycin, but not the clinically important 4,6-linked AGs amikacin and gentamicin. Potentiation was absent in a mutant lacking the AmgRS envelope stress response two-component system (TCS), which protects the organism from AG-generated membrane-damaging aberrant polypeptides and, thus, promotes AG resistance, an indication that RIF was acting via this TCS in potentiating 4,5-linked AG activity. Potentiation was also absent in a RIF-resistant RNA polymerase mutant, consistent with its potentiation of AG activity being dependent on RNA polymerase perturbation. PAR-inducible expression of the AmgRS-dependent genes htpX and yccA was reduced by RIF, suggesting that AG activation of this TCS was compromised by this agent. Still, RIF did not compromise the membrane-protective activity of AmgRS, an indication that it impacted some other function of this TCS. RIF potentiated the activities of 4,5-linked AGs against several AG-resistant clinical isolates, in two cases also potentiating the activity of the 4,6-linked AGs. These cases were, in one instance, explained by an observed AmgRS-dependent expression of the MexXY multidrug efflux system, which accommodates a range of AGs, with RIF targeting of AmgRS undermining mexXY expression and its promotion of resistance to 4,5- and 4,6-linked AGs. Given this link between AmgRS, MexXY expression, and pan-AG resistance in P. aeruginosa, RIF might be a useful adjuvant in the AG treatment of P. aeruginosa infections.