Current Pharmaceutical Biotechnology - Volume 3, Issue 2, 2002

β-lactams and glycopeptides antibiotics directed against enzymes involved in bacterial cell wall synthesis have generated bacterial resistance. Search for new antibiotic molecules is widely focused on bifunctional Penicillin-Binding Proteins (PBPs), with particular emphasis on their glycosyltransferase activity. This function catalyzes glycan chain polymerization of the cell wall peptidoglycan. This review summarizes recent results about biochemical characterization of bifunctional PBPs and enzymatic properties of the glycosyltransferase domain. Moenomycin, a well studied glycosyltransferase activity inhibitor has provided useful informations about lipid binding properties and about cellular role of bifunctional PBPs. These enzymes were shown to be a part of the multienzymatic complex involved in peptidoglycan biosynthesis. Furthermore, bifunctional PBPs are also present in the protein complex located at the site of septation during cell division. The glycosyltransferase domain of bifunctional PBPs remains unsufficently characterized: the structural analysis may lead to the development of novel antibacterials and to the understanding of the enzymatic properties, while genetic and cellular studies focused on bifunctional PBPs will provide a wealth of knowledge regarding cell growth and division.

Intrinsic and acquired multidrug resistance in Gram-negative bacteria owes much to the synergy between limited outer membrane permeability and energy-dependent multidrug efflux. The importance of the outer membrane vis-a-vis resistance is aptly demonstrated by the impact of mutational changes in outer membrane constituents on drug susceptibility. Changes in lipopolysaccharide (LPS) that correlate with increased drug susceptibility confirm, for example, the significance of this macromolecule in the intrinsic antimicrobial resistance of Gram-negative bacteria. Alterations in LPS and porins correlating with increased resistance to a variety of antimicrobials are also known and highlight the significance of the outer membrane vis-a-vis acquired antimicrobial resistance. Efflux systems accommodating a range of structurally distinct antimicrobials, including antibiotics, detergents, dyes, biocides and aromatic hydrocarbons have been identified in a number of Gram-negative organisms. Mutational studies have confirmed the importance of these systems to intrinsic and acquired antimicrobial resistance in important disease-causing organisms. As such, strategies aimed at thwarting efflux and or the outer membrane barrier are effective at reversing antimicrobial resistance in these organisms.

Proteins and peptides that form membrane-spanning pores and channels comprise a diverse class of molecules ranging from short peptides that are unregulated and create nonselective pathways to large ion channel proteins that are highly regulated and exhibit exquisite selectivity for particular ions. The diversity of regulation and selectivity, together with recent advances in protein “re-engineering” technology, provide a strong framework on which to build custom molecules with wide-ranging biotechnological application. Here we review a selection of pore-forming peptides and proteins from a number of different species to highlight their structural and functional diversity. The current and potential uses of native and re-engineered molecules are discussed together with a novel strategy to re-engineer α- hemolysin to create targeted and regulable cell-killing agents termed proimmunolysins. Numerous pore-forming peptides are currently in development as antimicrobial agents with potential application as anti-tumorigenic agents. In addition to their roles as biotherapeutic agents, pore-forming proteins are also being developed as biosensors for a range of different analytes. Recent examples of this technology include the use of α-hemolysin with an adapter molecule to create sensors for organic molecules and gramicidin as a general-purpose sensor for a range of analytes. These approaches promise to deliver a configurable binding site for analytes encoded in a readily measured electrical signal. The number of applications for pore-forming molecules is sure to grow in both quantity and diversity with increased knowledge of the fundamental structure and function of pores.

Carbapenem-hydrolyzing ß-lactamases of several Ambler molecular classes have been reported as the source of acquired ß-lactam antibiotic resistance in Gram negative bacteria. The metallo-enzymes of Ambler class B are the most prevalent enzymes in this case. These clavulanic-acid resistant enzymes have a large spectrum of hydrolysis including penicillins, cephalosporins (third and fourth generations), carbapenems but not monobactams. They are responsible for acquired resistance in several Gram negative species of clinical relevance in human medicine. IMP-1 was the first reported as acquired in Japan, mostly from Serratia marcescens and Pseudomonas aeruginosa isolates, and has been detected in Europe recently. Several variants of IMP-1 (IMP-2 to -9) have been characterized, possessing 85 to 99% amino acid identity, mostly from P. aeruginosa isolates. In addition, VIM-1 to -3 ß-lactamases have also been described, first in Europe (Italy, France, and Greece) and now in Korea. The VIM series shares 30% amino acid identity with the IMP-series. Most of these class B enzymes have genes that are integron- and plasmid-located. Finally, a few Ambler class A (SME-1, NMC-A, IMI-1, KPC-1) and class D (OXA-23 to -27) ß-lactamases involved in carbapenem hydrolysis have been reported also from rare isolates of Gramnegative rods. This review underlines the worldwide spread of carbapenem-hydrolyzing βlactamases as representing an important threat for efficacy of antibiotics in the near future.

Repair of tooth-supporting structures destroyed by the chronic inflammatory disease periodontitis is a major goal of oral therapy. The field of tissue engineering combines materials science and biology to repair tissues and organs. Periodontal tissue engineering has been achieved with limited success by the utilization of guiding tissue (cell occlusive) membranes and bone grafting techniques. Over the past decade investigators have begun to utilize signaling molecules such as growth factors to restore lost tooth support due to periodontitis, the most common bone disease affecting humans. This review will provide information on the status of growth factor therapies being applied in periodontology to treat advanced alveolar bone loss.

The human genome sequence has given us the view of the internal genetic scaffold around which human life is molded. We have inherited this heritage from our ancestors and through it we are connected to all life on earth. The sequencing of the human genome, amongst others, has led to the newer areas of healthcare and medicine. The human population is heterogeneous and consists of populations of immense ethnic diversity. There are considerable allelic differences between human populations as well as individuals within each ethnic group as a result of molecular heterogeneity of the genome. This, in turn, is responsible for differential allelic expression of genes endowing them with polymorphic characters. The molecular diversity within genes is responsible amongst others, of disease resistance or susceptibility or for that matter drug response.The objective of this article is to understand the nuances of the genetic repertoire and correlate it with disease gene identification, genes that have been or can be used as drug targets, identify candidate genes for drug development and recent trends in drug discovery.As regular clinical trials for drugs does not take into account the ethnic variations, it sometimes results in the differential response with respect to the efficacy and / or adverse reaction of the drug. Therefore the diverse ethnic populations of the world pose a challenge to the pharma industry. The concept of the personal medicine seems to be the answer to this problem. But it is a Herculean task requiring immense innovation in technology, is time consuming and is not a financially viable proposition at this point of time. An alternate approach would be to divide the populations in genetic cohorts and design drugs according to their genetic profile and haplotype. In addition, the ethical and legal bindings have also to be taken into consideration.

Cancer is a multigenic disorder involving mutations of both tumor suppressor genes and oncogenes. A large body of preclinical data, however, has suggested that cancer growth can be arrested or reversed by treatment with gene transfer vectors that carry a single growth inhibitory or pro-apoptotic gene or a gene that can recruit immune responses against the tumor. Many of these gene transfer vectors are modified viruses that retain the capability of the virus for efficient gene delivery but are safer than the native virus due to modifications that eliminate or alter one or more essential viral functions. The field of viral-based gene transfer vectors for the treatment of cancer has now entered the final stage of clinical testing prior to possible product approvals. Three viral vectors are currently undergoing this Phase III or Phase II / III clinical testing for cancer treatment. All three of these vectors are based on adenovirus, a common human virus that in its native state can cause cold or flu-like symptoms. In two of these vectors, genes essential for viral replication have been replaced with the wild-type p53 tumor suppressor gene, a gene that is deleted or mutated in over 50% of human cancers and which, when transferred into tumor cells, can induce tumor cell death. The third vector retains more of the natural adenoviral functions and relies on replication in tumor cells to induce cell killing. These three vectors represent two of the approaches now being taken to develop viral-based gene transfer vectors for cancer treatment. Additional approaches include the transfer of genes capable of converting non-toxic prodrugs into toxic forms, using anti-angiogenic gene transfer to block the formation of tumor blood vessels, inhibiting the activity of oncogenes through blocks to transcription or translation, stimulating the body's own immune system with immunomodulatory genes, and “cancer vaccination” with genes for tumor antigens.The data derived to date from clinical trials with viral-based vector systems are promising. The vectors have been generally well-tolerated without the severe toxicities common to standard cancer treatments. Many of these vectors have been demonstrated to have anti-tumor activity in a clinical setting and to lead to tumor regressions or to reductions in the rate of tumor growth. Furthermore, the safety profile of these vector systems has allowed for their clinical testing in combination with conventional cancer treatments to determine their benefit in multi-modality therapy. As part of their clinical development, all three of the vectors in Phase III and Phase II / III clinical trials are being tested for their benefit in combination with chemotherapy in randomized trials.The field is also looking to future product opportunities with improvements that will further increase potency, safety, and / or ease of administration. These developments may expand the number of cells that are susceptible to infection or may target the vector to particular tumor types following an intravenous administration. Some of these approaches are already in clinical testing. Additional opportunities may utilize multigene strategies to target multiple pathways in cancer cells or expand the use of cytotoxic or cytostatic gene transfer in combination with immunotherapy.

Microdialysis has been used extensively in animal studies for decades and in human pharmacokinetic studies for about 10 years.Microdialysis samples from the interstitial space which is a defined, anatomical compartment there is no net loss of body fluid the sample is “purified” and no enzymatic degradation takes place because proteins do not pass through the probe membrane into the dialysate microdialysis data relate to the intact molecule time resolution is high compared to biopsy and skin blister techniques radioabelling or induction of a magnetic response is not needed microdialysis is also an alternative method to determine protein binding of a compound in vivo microdialysis can readily be set up in clinical research units without expensive infrastructure.Microdialysis has been used to measure tissue concentrations of endogenous compounds and to investigate the tissue penetration of drugs in a variety of tissues in humans in vivo in both healthy volunteers and patients. Microdialysis data have also been used in PK-PD modelling and to obtain concentration-response relationships locally in tissues in vivo. There are also studies combining microdialysis with imaging techniques, e.g. PET.Microdialysis data may be used in early studies to select the appropriate compound, to optimise dosing regimens and to investigate the kinetic and dynamic consequences in the tissues of drug-drug and drug-disease interactions. Microdialysis can also be used in late phase studies to provide tissue concentration data in support of therapeutic efficacy trials or to create a niche for an already marketed drug.FDA and CPMP documents emphasise the value and importance of human tissue drug concentration data and support the use of microdialysis in humans to obtain such information. Microdialysis can satisfy regulatory requirements by providing data on drug concentrations in a well-defined anatomical tissue compartment at or close to the effect target site.Microdialysis is a versatile technique because of its multifaceted utility, low cost, ease of use, adaptability to different types of compounds and its feasibility for a number of organs and tissues. Equipment and probes for use in various organs have been commercially available for years.