Guanylyl cyclases

A majority of today's medicines act via receptors, either membrane bound or soluble receptors. These receptors are the communication portals for cells with mostly signals going into the cell but sometimes also signaling from the inside-out. Our lab is mainly focusing on the soluble guanylyl cyclase (sGC) receptor as well as the atrial natriuretic peptide receptor. Both receptors are guanylyl cyclases (GC) and wonderful complex signal transduction systems to probe scientifically with the strong potential of generating mechanistic insights that can be pharmaceutically exploited. These receptors are guanylyl cyclase receptors involved numerous physiological processes including blood pressure regulation and as such have received strong pharmaceutical attention as they are both drug targets. Our lab is one of the world leaders in GC structural biology having crystallographically characterized 4 different domains of guanylyl cyclases or homologs thereof: 1) heme-nitric-oxide-oxygen binding domain (H-NOX) homologous to sGC in complex with NO and CO (Ma et al., EMBO J 2007); 2) H-NOX-associated domain (H-NOXA) dimer homologous to sGC revealing a PAS-fold (Ma et al., JBC 2008); 3) sGC's coiled-coil (CC) domain (Ma et al., BMC Struct. Biol. 2010) ; 4) membrane GC's atrial natriuretic peptide (ANP) receptor hormone binding domain (van den Akker et al., Nature 2000). Our results gained new mechanistic insights into the structure and activation of these receptors and can be used to develop novel therapeutic modulators for cardiovascular diseases or other repercussions of impaired GC signaling. All of these steps are structurally not well understood making these receptors ideal candidates for a concerted multi-disciplinary approach to gain mechanistic insights. Our current projects range from relatively straight forward experiments such as site-directed mutagenesis and activity assays to probe specific questions, to biochemically characterizing and crystallizing the remaining individual domains, to our most ambitious long term goals of crystallizing the entire receptor and discovering new pharmaceutically interesting effectors based on our structural investigations.

Our lab has determined the structure of a sGC activator bound to the heme domain revealing insights into the activation of sGC. This sGC activator, BAY58-2667, was in clinical trials for decompensated heart failure and the insights gained from our structure could lead to improvements into the design of this class of activators. We extended these studies by determining the complex with BAY 60-2770 and also gained insights into regulation by S-nitrosylation. This work was in collaboration with the labs of Annie Beuve (UMDNJ), Johannes-Peter Stasch (Bayer Pharmaceuticals), and Jonathan Stamler (Case Western Reserve University); this work was published in JBC (Sayed et al., 2007; Martin et al. 2010; Kumar et al. 2013).

beta-Lactamases

Bacterial infections are arguably some of the most serious threats to humankind to date. The options for treating infections have dwindled substantially and the reports of antibiotic resistant pathogens are increasing at an alarming rate. Once potent antibiotics such as penicillin nowadays are hardly effective by themselves including against certain strains of Bacillus anthracis, the causative agent of anthrax. This antibiotic resistance is due in large part to bacterial beta-lactamases capable of degrading penicillin-like drugs. A powerful avenue for treating infections was the administration of a beta-lactamase inhibiting co-drug in addition to prescribing penicillin-like antibiotics. There are currently three beta-lactamase inhibitors on the market, tazobactam, sulbactam, and clavulanic acid each with an annual sales of close to or over a billion dollar. The bacterial response to this co-drug combination was not too surprising and beta-lactamase variants were soon found to confer Detailed structural knowledge on how these inhibitors function and how mutations in beta-lactamases confer resistance to these inhibitors is desperately needed. Protein crystallography has been a tremendous tool to study beta-lactamases with and without substrates or inhibitors yet the complexity of the enzymatic degradation pathway of such compounds has often precluded obtaining clear crystallographic snapshots of reaction intermediates. A novel solution to this underlying problem of not knowing what intermediates are formed at what rate in the crystal once soaking is commenced has been developed by our collaborative team including Drs. Paul Carey, Marion Helfand, and Robert Bonomo. This innovative technique, termed Raman crystallography, has led to the identification of the trans-enamine intermediate peaking at 20-30 minutes inside the deacylation deficient E166A mutant of SHV-1 beta-lactamase. Our lab subsequently determined the 1.63 Ang crystal structure of this intermediate complex for tazobactam yielding a wealth of detailed information of how this drug inhibits this enzyme resulting in ideas on how to rationally improve this drug (tazobactam has an annual sales of close to a billion dollars in the US). Click here to view structure or download coordinates. This work was published in Biochemistry

Based on this tazobactam complex, we have designed a novel inhibitor SA2-13 with an intend to stabilize the trans-enamine intermediate. Our designed SA2-13 compound yielded a 10-fold improvement of the longevity of the trans-enamine intermediate and, unlike the starting tazobactam compound, SA2-13 could now readily be trapped in wt SHV-1 crystals. This work was published in JACS and in collaboration with Dr. John Buynak. Click here to view structure or download coordinates.

We also determined the crystal structure of the beta-lactamase KPC-2. KPC-2 has special hydrolytic properties in that it can also hydrolyze carbapenems and cephamycins which have a bulky alpha-substituent on the beta-lactam ring. The ability to hydrolyze carbapenems is particularly worrisome since carbapenem antibiotics constitute our 'last resort' antibiotics. KPC-2 belongs therefore to the class of carbapenemases and its presence has been linked to numerous difficult-to-treat Klebsiella outbreaks in New York, Israel, and elsewhere. This rapid spread is further facilitates since KPC-2 is the first plasmid-encoded class A carbapenemase. Our structure elucidates the structural basis of the carbapenemase activity and could aid in the development of more potent novel beta-lactam antibiotics and inhibitors.
Our structure was published in Biochemistry and is a collaboration with Dr. Bonomo (see Publications link). Click here to view structure or download coordinates.

We currently have a four-pronged approach of developing novel potent beta-lactamases inhibitors as we explore: 1) C6-substituted penicillin sulfones (Nottingham et al. Bioorg & Med Chem. Lett 2011), 2) 6-alkylidene-2'-substituted penicillanic acid sulfones (Bou et al., JACS 2010; Pattanaik et al. JBC 2009), 3) penam sulfones (Padayatti et al., JACS 2006; Sampson et al., Antimicrob. Agents and Chemotherap. 2011), and 4) boronic acid transition state analogs (Ke et al., Antimicrob Agents and Chemotherap. 2011). These classes of compounds are developed to target the most clinically urgent class A, class C, and class D beta-lactamases and carbapenemases.

SOFTWARE: DDQ is a crystal structure validation tool developed by van den Akker

Example of a well-refined crystal structure revealing strong, round, positive electron density peaks for water molecules at stereochemically appropriate positions (as well as absence of shift peaks; 1MYR PDBid).


DDQ (Difference Density Quality) automatically assesses:

  • the local correctness of macro-molecular crystal structures (via absence of nearby shift peaks and presence of stereochemically correctly position water peaks in an unbiased 'hydrated' difference map where water molecules have been deliberately removed from the structure factor calulcations)
  • the global correctness of macro-molecular crystal structures and will give you a ranking on how your structure compares to other PDB structure of similar resolution (ranking as follows: worst < bottom 25% < below average < above average < top 25% < best).
  • whether crystallographic refinement has been completed satisfactorily (i.e. a. no shift peaks, b. many strong correctly positioned water peaks, c. absence of additional positive density due to unmodeled residues/ligands)

  • Click here to download article describing the DDQ method (van den Akker & Hol Acta Cryst. D55, 206-218 ,1999).
    Click here to download program.

    COLLABORATORS

    Please click here to visit Dr.Robert Bonomo's website http://www.case.edu/med/id/faculty/bonomo_robert.html

    Please click here to visit Dr. Paul Carey's website http://www.cwru.edu/med/biochemistry/faculty/carey.html

    Please click here to visit Dr. John Buynak's website http://faculty.smu.edu/jbuynak/

    Please click here to visit Dr. Fabio Prati's website http://personale.unimore.it/Rubrica/dettaglio/fprati

    Additional collaborations. We have additional crystallographic and/or in silico drug screening collaborations with the labs of Sam Mesiano, Arne Rietsch, Jonathan Stamler, Dennis Stuehr, David Wald, and two pharmaceutical companies.