Rethinking the Philosophy of Antimicrobal Drug Design
By Glen D. Armstrong, PhD
To cause infections, microbial pathogens must first tightly adhere to host cell surfaces. This allows pathogenic microbes to oppose cleansing action -- such as the flushing of urine or tears, peristaltic movement of the gut or mucociliary function of respiratory epithelial cells -- that would otherwise quickly eliminate them from their host's body. Many pathogenic bacteria express specialized surface proteins, called adhesins, which mediate their binding to host cells. Most, but not all, microbial adhesins are long filamentous structures microbiologists call pili or fimbriae. On the host cell, the complex carbohydrate sequences of glycoproteins or glycolipids represent receptors to which many microbial pili are known to bind. It is this binding of pili to complex carbohydrate receptors that allows many microbes to successfully colonize host cell surfaces. With few exceptions, microbial exotoxins bind to host cell glycolipids, thus mediating exotoxin access to their host cell targets.
In theory, therefore, soluble carbohydrate receptor analogs might competitively antagonize the pathophysiological effects of exotoxins or inhibit bacterial colonization of host cell surfaces.
The continuing rise in microbial resistance to antibiotic drugs is a major concern to all healthcare practitioners. It has stimulated infection control professionals and medical microbiologists to seek new ways of preventing or treating classical and emerging infectious diseases.
The merits of education and the enforcement of infection control measures are undisputed. In many cases, the simplest and most economical of measures, frequent handwashing for example, have been shown to have profound positive effects.1 Recent advances in vaccine development have contributed significantly to infection control. The application of immunization to a particular problem is only practical in cases where a subject's immune system is functioning normally. In many cases, it is simply not practical to develop vaccines for infectious diseases where the annual incidence would be less than the anticipated rate of adverse effects in a mass immunization program. Effective drugs will remain a last resort in cases where immunization does not represent a viable option or when other control measures fail.
The main problem associated with current antimicrobial drugs is the huge capacity of microorganisms to become resistant to these drugs, thereby eventually rendering them ineffective. In the presence of such drugs, microbes which are unaffected by them soon become the predominant members of the population. The acquisition of microbial resistance mechanisms requires the pharmaceutical industry to search for new derivatives to which microbial resistance has not developed. In most cases, this enterprise involves chemically altering the structures of known drugs or creating completely synthetic agents with suitable antimicrobial properties. Nonetheless, the principles of drug discovery have not changed and continue to involve the perpetual quest for agents designed to prevent the in-vivo replication of pathogenic microbes. Perhaps it is time to rethink the philosophy of antimicrobial drug design and investigate the feasibility of producing agents that will not create environments favoring the proliferation of genotypically resistant microbes.
Recent advances in the fields of bacterial genomics, proteomics and molecular biology have allowed us the benefit of a much more thorough understanding of pathogenic mechanisms and the intricate interplay between microbes and their hosts.2 These studies reveal potential methods of interfering with the mechanisms of the infection process in ways that may not promote the proliferation of microbes insensitive to the desired inhibitory effects of these agents. Such novel agents should probably be referred to as anti-infective rather than antimicrobial since they will be designed to inhibit the infection process rather than the microbes themselves.
In virtually every example, the attachment of pathogenic microorganisms or their toxic exoproducts to host cells represents the first step in the process leading to a clinically apparent condition. My research is based on the observation that many pathogenic microbes or their exotoxins attach to complex carbohydrate sequences, also referred to as glycans, displayed on host cell surfaces.3 These attachment processes are highly specific, usually involving a microbial protein which recognizes a specific host cell complex carbohydrate sequence or sequences.4 It is conceivable that soluble carbohydrate receptor-based drugs designed to competitively inhibit these microbial attachment processes may be therapeutically beneficial in cases where conventional antibiotics are, for one reason or another, ineffective.5 The rest of this article will elaborate on how we and others have been testing this concept.
Enterohemorrhagic E. coli (EHEC) cause a condition called hemorrhagic colitis (HC) in humans.6 Although several EHEC serotypes have been identified, E. coli O157:H7 serotype is isolated from the majority of HC cases in North America, Europe and Japan.7 Patients who develop EHEC-mediated HC are also at risk of developing a potentially life-threatening post-infection complication known as the hemolytic-uremic syndrome (HUS). This serious complication of HC is directly linked to the production of potent EHEC cytotoxins.8 These represent a family of cytotoxins which are related to the Shiga toxin (Stx) produced by Shigella dysenteriae Type 1. One of the EHEC cytotoxins, Stx1, is virtually identical to the Stx expressed by Shigella dysenteriae9-10 and was formerly called Shiga-like toxin I (SLT-I). This cytotoxin was also called Verotoxin 1 (VT 1) because its cytotoxic activity was initially described in African green monkey (Vero) kidney cells.11 The other EHEC cytotoxin, formerly called SLT-II or VT 2 but now referred to as Stx2, is more distantly related to Stx1. In addition, several variant forms of Stx2 have been described in EHEC isolates. Regardless of their relationships, though, all the EHEC Shiga toxins share structural and functional similarities.8
The EHEC Shiga toxins are classical hexameric "A/B5" exotoxins consisting of one enzymatically active A subunit and 5 identical B subunits.12-13 Although the cytotoxic action of EHEC Shiga toxins is expressed by their A subunits in the cytoplasm of host cells, absent the B pentamer that binds to glycan receptors on the surface of the host cell,3 the A subunit is atoxic. The Stx B pentamer is critical to the intoxication process because it acts as a mediator for A subunit binding to and translocation into the host cell cytoplasm. Given its critical binding function, it seemed feasible that soluble B pentamer glycan receptor analogs might competitively inhibit Stx binding to and intoxication of host cells. The therapeutic benefits of such Stx receptor analogs might be to prevent HUS developing in subjects suffering from HC.
Typically, protein-carbohydrate interactions are of the low affinity variety.14 Unlike enzymes, which contain a deep cleft or tunnel into which the substrate tightly binds, the carbohydrate ligand binding domains of proteins such as the EHEC Shiga toxins usually occur as shallow depressions on the surface of the protein. Much of the binding energy in carbohydrate-protein interactions is derived from the displacement of disordered water molecules from the depression in the surface of the protein by the ordered hydroxyl groups displayed by the glycan sequence.15 Nature compensates for the weak binding by simply multiplying the number of equivalent ligand binding domains in carbohydrate-binding proteins. This allows these proteins to achieve tight binding by simultaneously engaging multiple receptor sequences.
We have used the X-ray crystal structure of the Stx1 B pentamer, complexed with soluble glycan receptor analogs,16 as a template for creating a symmetrical soluble inhibitor, we call Starfish, capable of simultaneously embracing the multiple domains found on the receptor binding face of this protein.17 The resulting Starfish analog displayed a solid phase binding inhibition constant that was superior, by 10 million times, to that of a monovalent inhibitor capable of engaging only one of the multiple Stx1 B pentamer receptor binding domains. In further co-incubation studies,17 the polyvalent carbohydrate-based soluble inhibitor protected Vero cells, at concentrations in the ?M range, from a lethal challenge dose of both Stx1 and Stx2.
After we disclosed our multivalent inhibitor in the literature, a group of Japanese investigators presented data demonstrating that ?M doses of a polyvalent inhibitor they called "Super Twig" protected mice from lethal challenge doses of both Stx1 and Stx2 [(Nishikawa, K., et al. Development of a new type of Shiga toxin neutralizer, carbosilane dendrimer, which completely rescues the lethality of Shiga toxin 2 in mice. 2000. Abstract 415, 4th International Symposium and Workshop on Shiga toxin [Verocytotoxin], producing Escherichia coli infections, Kyoto, Japan).] In this report, the inhibitor, like the toxins, was administered intravenously and appeared to be well tolerated by the mice. Since the Japanese report, we have obtained compelling evidence demonstrating the efficacy of symmetrical polyvalent inhibitors in protecting mice from the lethal effects of Stx118 and now Stx2 (in preparation). The dosages used in our in vivo experiments were equivalent to a few milligrams of inhibitor per Kg of body weight and so far, we, like the Japanese, have not observed any toxic effects of these carbohydrate-based inhibitors in mice.
Others19 have used an approach similar to ours to develop effective inhibitors for two additional bacterial exotoxins in the A/B5 classification group; Cholera toxin and the Heat-labile (LT) toxins expressed by enterotoxigenic E. coli (ETEC), for example. It would seem that, at least for bacterial exotoxins that display symmetrical arrays of multiple ligand-binding sites, it is generally feasible to produce carbohydrate-based soluble inhibitors that have exceedingly beneficial effects at pharmacological dosages. These recent findings bode well for potentially commercializing polyvalent carbohydrate-based receptor analogs for treating diseases caused by EHEC, ETEC, and, possibly, Vibrio cholerae.
Not all bacterial carbohydrate-binding proteins display the same five-fold rotational symmetry as the A/B5 family of exotoxins. Some bacterial exotoxins, those expressed by Clostridium difficile, the organisms responsible for antibiotic-associated colitis in hospitalized patients or residents of extended-care facilities, for example, contain a linear array of multiple carbohydrate-binding domains.20 Filamentous bacterial adhesions may also display linear arrays of multiple carbohydrate-binding domains.21 Alternately, the pili or fimbriae expressed by some bacterial pathogens may display carbohydrate-binding domains only at their distal tips. In these "tip domain" situations, polyvalency may be achieved not only by the symmetrical arrangement of multiple carbohydrate binding sites at the tips of the individual pili but also by expressing multiple pili per organism.21
The situation presents an even greater challenge to developing effective inhibitor analogs because the flexibility of these filaments contributes to a relative lack of symmetry in these systems. Once an organism has attached itself to a host cell, it may be impossible for a soluble inhibitor to reverse this process. We22, however, have demonstrated that a synthetic glycoconjugate consisting of bovine serum albumin (BSA) containing covalently bound N-acetyllacosaminyl glycoside sequences inhibited bundle-forming pili (BFP) -- mediated attachment of enteropathogenic E. coli (EPEC) to cultured human epithelial cells. This inhibitory activity appeared to depend on the average number, i.e., potential valency, of N-acetyllacosaminyl glycosides attached to each BSA molecule and was specific for this particular carbohydrate sequence. In EPEC, at least, the relative absence of a symmetrical arrangement of the BFP-localized carbohydrate-binding domains did not hinder the inhibitory activity of a BSA glycoconjugate containing multiple glycan ligands.
Further investigation into the EPEC host cell binding inhibition mechanism of BSA glycoconjugates revealed the process was more complex than we originally believed. These studies demonstrated that inhibition likely involved two mechanisms, one involving simple competitive inhibition, and the other involving a time-dependent glycoconjugate-mediated loss of BFP from the surface of the bacteria. At a glance, this would appear to be counterintuitive to the role of BFP in EPEC attachment to host cells. However, EPEC colonize host cells by a complex multi-step process. BFP-mediated attachment represents only the first step in EPEC colonization of host cells. Once this first step has successfully occurred, the continuing presence of BFP on the surface of EPEC may interfere with the subsequent colonization steps. We are speculating, therefore, that EPEC BFP binding to specific carbohydrate receptors may produce an as yet unknown signal informing the bacterium that it has made initial contact with an accommodating host cell and may now discard its BFP in preparation for the next phase of colonization.
Once EPEC have colonized a specific site in the host, it may be necessary for some organisms to be released from this primary colonization site. In the absence of any host response, these released organisms would then be free to colonize additional, perhaps downstream host sites, thereby perpetuating the infection process. Once they have been released from the influence of their host cell glycan receptors, these planktonic organisms may once again produce BFP that would enable them to colonize the new sites in the host. It is during this secondary phase of the infection process, the colonization of additional host sites, that the administration of a multivalent carbohydrate receptor analog may be of benefit in a subject suffering from EPEC-mediated diarrhea.
Others have also noted that EPEC alter the expression of their adhesins in response to contacting host cell surfaces and there is some speculation that glycan receptors may be generally involved in these sensing reactions.23 Many pathogenic microbes, including EPEC, possess the ability to assemble a complex multimeric organelle, called a Type III secretory apparatus, when they contact host cell surfaces.6 This Type III secretory apparatus is necessary for injecting virulence factors into the cytoplasm of host cells. These factors cause host cells to modify their physiological behavior conducive to the pathogenic process. In light of what is known about the possible role of glycan receptors in regulating adhesin expression in EPEC, it is possible that specific host cell surface carbohydrate sequences may also influence the function of microbial Type III secretory systems. Should this assumption prove to be valid, soluble carbohydrate receptor analogs may also eventually be useful for disrupting the function of Type III secretory systems. Such analogs might, for example, trigger Type III-mediated secretion of virulence factors from planktonic bacteria, thereby causing these factors to disperse harmlessly to the exterior of the host cells.
It is unlikely microorganisms will rapidly develop resistance to carbohydrate-based anti-infective drugs because this would involve altering their ability to bind such agents. Organisms that alter their ability to bind to soluble host cell carbohydrate receptor analogs would not gain an advantage over their wild-type siblings because these mutants would also lose their ability to bind or respond properly to host cells. Alternatively, microorganisms may acquire the capacity to express adhesins with a specificity for different host cell receptors. However, although it is quite possible for microbes to acquire the genetic capacity to express complex multi-protein virulence factors such as pili or Type III secretory systems, en block6, this may require a sustained environment which highly favors organisms displaying the new phenotype. It is unlikely the use of carbohydrate-based anti-infective drugs only during the acute phase of an infection would create such a sustained environment. To be of any use to the microbes, the newly acquired adhesins would also have to display a specificity for other unique carbohydrate sequences already present on host cells.
Glen D. Armstrong is a professor in the Department of Medical Microbiology and Immunology at the University of Alberta, Edmonton, AB, Canada.