As described above, conventional drug discovery strategies targeting proteases have proved useful, at least for the limited number of successful protease inhibitors currently on the market. We have discussed reasons for the limited success, based primarily on conventional approaches that frequently limit the achievable specificity required for successful therapy. A simple formula to decide whether a drug lead is worth pursuing would be desirable; for example, a selectivity ratio in which a paralogue is inhibited at less than 1% compared with the main target. However, in reality this may be difficult to achieve, with the result that decisions are made empirically (BOX 6
). Related to this consideration is the realization that protease orthologues in animal models do not always have the same specificity as the human enzyme, thus questioning the validity of such animal models. The need to develop approaches for understanding human proteases in their natural context is a priority that will probably dominate protease inhibitor research, if not most drug research, in the future.
Box 6. Aminopeptidases: targets for antimalarials
An interesting challenge is posed by drug development for proteases with overlapping substrate specificity, originating from two different organisms, with the aim of inhibiting only one. Such scenarios are encountered in parasite or pathogen infections in humans. An example for this is provided by the aminopeptidases (APNs). The malaria parasite Plasmodium falciparum
produces eight different APNs — four methionine APNs, two neutral and one prolyl and one aspartyl — some of which are orthologues of human leucine APN (LAP, class M17) and alanine APN (CD13, class M1)115
. These parasite APNs generate a pool of free amino acids through digestion of the peptides formed by other proteases from human haemoglobin in the digestive vacuole of the malaria parasite.
Some substrate specificity studies using fluorogenic substrates or substrate-based inhibitors (phosphonates) suggest substantial specificity overlap of both human and malarial APNs116,117
. Phosphinate inhibitors of mammalian porcine kidney LAP have a similar inhibitory potency towards the P. falciparum
orthologue (inhibition constant (Ki
) = 13.2 nM and Ki
= 66.0 nM towards malaria LAP (Pf
LAP) and mammalian LAP (pk
LAP) respectively). But in a Plasmodium chabaudi
murine malaria model, these inhibitors reduced the parasite burden by 92% without any serious toxicity118
. These data show that in such circumstances, the degree of specificity and selectivity is not as crucial. The key factor seems to be the delivery mechanism responsible for drug distribution in the target cells118
. The chemical structures represent the most potent phosphinate dipeptide analogue inhibitors directed against the P. falciparum
M17 leucine APN as reported in REF. 118
With regard to the broad range of technologies that are now available for protease inhibitor design, the question arises as to which delivers the best hits. Targeting the active site using a combination of specificity-based knowledge and structure-based design is a tried and proven strategy. However, the burst in technologies using combinatorial libraries, HTS or fragment-based screening began only in the early 1990s. Although only a minor fraction of approved drugs have been developed with these methods, this is not surprising considering the time necessary for the development of the technology and the subsequent application, and so the question of which strategy might be best remains open.
In the quest to optimize the specificity of protease inhibitors, alternative targets to the active site should also be considered. So far, no allosteric protease inhibitors or inhibitors that target the exosite have reached advanced development. Highly successful protease inhibitors all target the active site, so why risk pursuing allosteric regulators? One important point to consider is that active-site directed inhibitors are inherently competitive with endogenous substrates. Most proteases in vivo
are thought to operate in an environment in which they are substrate saturated, and this will slow down and/or weaken competitive inhibitors by the factor of 1 + S/ΣKm
, in which ΣKm
is the sum of all values for all the substrates in a cell. In addition to the perennial problems of tissue penetration and pharmacokinetic considerations, this concept — backed up by a recent publication on HIV protease inhibitors that operate allosterically92
— could explain why the inhibitory concentration of a protease inhibitor drug in vivo
is always several orders larger than in a purified system. By contrast, allosteric or exosite inhibitors need not be affected by substrate concentration.
There is also concern that an allosteric or exosite inhibitor connected to an active-site directed moiety may generate highly specific binding, but will result in an extremely large molecule that will not be viable for commercial inhibitor development. However, as pointed out in , not all exosites or allosteric sites require large binding surfaces, and the proof-of-principle already exists in the form of the thrombin inhibitors based on the exosite binder desirudin. A proof-of-concept application of a small-molecule allosteric inhibitor strategy was reported by Sunesis Pharmaceuticals, who developed low molecular mass inactivators of caspase 3 and caspase 7 using a fragment-based screening technology. Among a selection of molecules were ones that targeted not the active site, but a region at the interface of the caspase dimer, essentially freezing the caspases in their catalytically inactive zymogen conformation72
. For technical reasons based on the low affinity covalent tethering method required for the identification of hits, it is challenging to develop these particular compounds directly into drugs. nevertheless, the message is clear: allosteric regulation by small molecules is now a high priority approach to protease inhibition. The evidence that currently available protease inhibitor drugs are highly specific is quite limited, and specificity is a major issue in active-site directed protease development programmes. Therefore, in our opinion, to achieve high specificity and selectivity one must avoid the active site. However, development has been slow in this area, and given the thermodynamic hot spot that defines protease active sites, unconventional approaches are required to target away from the active site93
Importantly, these considerations also apply to several other potential drug targets that can be regulated by allosteric interactions. Indeed, the hugely successful kinase inhibitor imatinib (Gleevec; novartis) has been shown to operate by a predominantly allosteric mechanism because it does not directly bind to the ATP binding site94
, and allosteric modulators of G protein-coupled receptors are emergent leads for compounds that provide high selectivity for this group of therapeutically tractable proteins95
Given the wealth of mechanistic and structural data on proteases, as well as new fragment screening approaches, we anticipate that strategies to target the allosteric sites of proteases will soon emerge and that research and development in this area is likely to be extensive and profitable. Readers interested in learning more about allosteric inhibitors are directed to an excellent review in REF. 96
. Naturally, an equally important part of protease inhibitor therapy, common to all drugs, is to achieve adequate absorption, distribution, metabolism, excretion and toxicology parameters, and only a skilful combination of these parameters with inhibitor selectivity will provide strong drug candidates.
Finally, although small molecules are still the most popular approach to protease inhibitor therapy, the opportunity of using biologicals is increasingly being realized. Future biotechnology drugs in protease therapy may include allosteric-site interactors, biologicals based on antibodies and highly selective natural protease inhibitors, and even proteases themselves (BOX 7
Box 7. Proteases as therapeutics
The concept of using proteases as therapeutic agents is not new. Proteases are used in wound debridement (collagenase); for defibrinating wounds in hospitals; for dissolving membranes in diphtheria; or for swelling, for fever and for adhesions reduction after surgery (papain); to reduce ruptured spinal discs (chymopapain); in clot dissolving; and recently for the management of acute ischaemic stroke in adults (recombinant tissue plasminogen activator, Activase; Genentech/Boehringer Ingelheim)119
; and in clotting factor replacement (recombinant factor VIIa, NN-1731; Novo Nordisk)120
. However, there are currently highly interesting programmes in academic institutions and in biotechnology companies to engineer proteases for specific therapeutic targets. Pioneered by the concept of making inhibitor-resistant versions of tissue plasminogen activator and tissue factor-independent versions of coagulation factor VIIa, the concept of evolving proteases for specific functions is increasingly popular. Evolved proteases promise at least one major advantage over other biologicals such as antibodies. They are catalytic and can therefore be used in smaller amounts, resulting in potentially high efficacy.
Nascent steps in this direction have been taken in two studies in which the substrate specificity and the catalytic efficiency of proteases was altered using Escherichia coli
surface endopeptidase OmpT as a scaffold121,122
. In these studies, a panel of proteases with new substrate selectivities in the P1 and P1′ positions was generated by screening a library of mutated proteases against specific substrates. An analogous method with similar aims in generating proteases of novel specificity for the treatment of inflammatory and oncogenic diseases is being undertaken by Catalyst Biosciences. Here, the protease platform is oriented towards evolving the protease substrate specificity of a protease scaffold (Alterase) towards the degradation of specific proteins that promote diseases123