Drug discovery beyond the ‘rule-of-five’

Ming-Qiang Zhang and Barrie Wilkinson

Although a very useful guideline for orally bioavailable small- nitrogen and oxygen atoms in a molecular is greater than 10
molecule drug design, the ‘rule-of-five’ (also known as ‘Lipinski’s [1 ]. Natural products and drugs that are substrates of
rule of drug-likeness’) has to some extent been overemphasized. biological transporters are exceptions to the rule. It was an
Firstly, only 51% of all FDA-approved small-molecule drugs are attempt to rationalize compound design (in particular
both used orally and comply with the ‘rule-of-five’. This does not compound library design) so as not to make too polar,
even include the increasing number of biologicals of which floppy, and large molecules which have a lower chance of
several have reached ‘blockbuster’ status. Secondly, it does not exhibiting desirable pharmaceutical properties and oral
cover natural product and semisynthetic natural product drugs, bioavailability. It has raised the awareness of the import-
which constitute over one-third of all marketed small-molecule ance of ADME (absorption, distribution, metabolism, and
drugs. A more balanced and programmatic approach to drug elimination) and physico-chemical properties for the suc-
discovery should be more productive than to rely on an cess of drug discovery among medicinal chemists, and
overemphasis of ‘rule-of-five’ compliance. Rather it should helped to front-load ADME (and later ADME/toxicity)
consider proactively the development of parenteral drugs in screening in the industrial drug discovery process. It is fair
parallel to oral drugs and to consider the development of to say that the ‘rule-of-five’ has had a major impact on the
therapeutic antibodies in parallel to small-molecule drugs. These daily practice of medicinal chemistry across the pharma-
are particularly relevant for efforts against ‘first-in-class’ and/or ceutical industry and served as a very useful guideline for
particularly challenging targets such as proteases and those orally bioavailable small-molecule drug discovery.
involving protein–protein interactions. In addition, more effort
should be invested in natural product research. Emerging novel
However, during the past 10 years of incorporating the
technologies such as synthetic biology (genetic engineering of
‘rule-of-five’ into routine drug discovery practice, there has
living organisms to produce small-molecule therapeutics) may
been a tendency to overemphasize this simple guide-line, in
address several challenging issues of natural product-based drug
particular its association with ‘drug-likeness’ [2]. The
discovery including synthetic feasibility and ligand efficiency.
worrying signs for misuse and overemphasis of the ‘rule-
of-five’ for determining drug-likeness can be seen in many
Addresses companies and drug discovery conferences. Some tales the
Biotica Technology Ltd., Chesterford Research Park, Little authors have heard include those of senior managers in
Chesterford nr Saffron Walden, Essex CB10 1XL, United Kingdom some companies not accepting an otherwise highly
Corresponding author: Zhang, Ming-Qiang ([email protected])
promising development candidate because it did not fulfill
all ‘rule-of-five’ criteria. Others take pride in the fact that
the average molecular size of the compounds their
Current Opinion in Biotechnology 2007, 18:478–488 company has synthesized is smaller (or has better
compliance with the ‘rule-of-five’) than those of their
This review comes from a themed issue on
Chemical biotechnology competitors. This type of overemphasis has the danger-ous
Edited by Gregory L. Challis and David A. Hopwood potential to overfilter compounds that may otherwise be
good drug candidates, and inevitably results in reduced
Available online 26th November 2007
productivity for discovering new drugs.
0958-1669/$ – see front matter
# 2007 Elsevier Ltd. All rights reserved. The major limitations of dogmatic adherence to the ‘rule-
of-five’ are twofold: it overemphasizes oral bioavailability
DOI 10.1016/j.copbio.2007.10.005
and excludes natural products.

Oral bioavailability and drug discovery

Introduction productivity
The ‘rule-of-five’ (also known as ‘Lipinski’s rule’) for Although oral bioavailability is a very important criterion
‘drug-likeness’ [1 ] was originally proposed in response to for a successful drug, many of the therapeutics used in
the large number of randomly made compound libraries, today’s medical practice are given parenterally, that is, not
mostly because of synthetic feasibility and sometimes the by oral administration. A recent analysis of 1204 US FDA-
single-minded chase for potency, by com-binatorial approved small-molecule drugs [3] showed that 803 drugs
chemistry in the early 1990s. It states that poor absorption can be dosed orally, 421 can be administered parenterally,
or permeability of a compound is more likely when there and 275 can be used as topical agents. Of all 1204 drugs
are >5 hydrogen-bond donors, the molecular mass is >500, only 73% (885 drugs) passed the ‘rule-of-five’ test of
calculated log P is >5, and the sum of which 70% (619 drugs) are actually used orally, that is,

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Drug discovery beyond ‘Rule-of-Five’ Zhang and Wilkinson 479
just over half of all FDA-approved small-molecule drugs It is therefore more productive when the industry takes a
are both orally administered and satisfy the ‘rule-of-five’. programmatic approach to drug discovery. That is, when
On the other hand, 20% of all oral drugs fail at least one of the discovery of orally bioavailable drugs proves difficult,
the ‘rule-of-five’ parameters. for example because of difficult targets such as proteases or
those involving protein–protein interactions, the decision to
Therefore, overemphasis on orally bioavailable drugs progress initial discovery efforts toward par-enteral drugs
following the ‘rule-of-five’ can be counter-productive. One may be more productive. This not only provides medically
can argue that penicillin G might not be marketed if it were needed drugs more quickly but also helps to validate a
to be discovered today because of the emphasis on oral potential ‘first-in-class’ target more quickly before
bioavailability. The first orally bioavailable peni-cillin intensive effort on oral bioavailability is put in place. For
(phenoxymethylpenicillin, penicillin V) was dis-covered example, during an attempt to develop anti-HIV vaccines,
>15 years after the discovery of penicillin G. Although it Jiang et al. discovered that a peptide derived from the HIV-
might not take 15 years to find an orally bioavailable 1 surface glycoprotein gp41 C-terminal heptad repeat
analog of penicillin G with modern tech-nologies, the region showed anti-HIV activity when incubated with
delayed availability of such an important drug would human T cells [6]. This discovery was initially counter-
inevitably result in unnecessary patient suffering. intuitive — why evolution should have produced a viral
surface peptide that is inhibitory to its own replication.
Further studies of the fusion process between HIV-1 and
The discovery of orally bioavailable drugs is not always CD4 cells showed that the peptide and its congeners acted
easy and can be extremely challenging. An illustrative as decoys of their natural analogs and inhibited the entry of
example can be seen through the efforts undertaken to HIV-1 into T cells. One of these congeners enfuvirtide [7],
discover orally bioavailable thrombin inhibitors [4]. a 36-amino acid pep-tide corresponding to positions 643–
Heparin, an indirect inhibitor of thrombin, was originally 678 in gp41, was later developed by Trimeris and Roche as
discovered in 1916 and has since been one of the most the ‘first-in-class’ HIV fusion inhibitor and was approved
frequently used anticoagulants for the treatment of venous by the US FDA in 2003 — only 10 years after the first
thrombosis. Heparin is not a single substance but a family report on the anti-HIV activity of a gp41 peptide fragment
of sulfated glycosaminoglycans. The most frequently used and despite of the challenging 106-step total synthesis
‘low-molecular-weight heparins’ (LMWHs) in clinical required for its man-ufacture. Enfuvirtide is administered
treatment have molecular weights ranging from 4000 to 15 by subcutaneous injection. Very soon after the report of
000 and are not orally bioavail-able. They are administered anti-HIV activity of gp41 peptides, efforts were initiated to
intravenously or subcu-taneously, which is difficult for discover orally bioavailable gp41 inhibitors [8], but till
long-term treatment. Therefore, there is a need for orally date, there has been little reported success. This is not
bioavailable thrombin inhibitors. More than 80 years after surprising given the general difficulties in inhibiting
the discovery of heparin, during which period almost all protein–protein inter-actions with small molecules and the
major pharma-ceutical companies had had a thrombin lack of well-defined binding pockets on the surface of
inhibitor project, the first orally bioavailable thrombin gp41.
inhibitor ximelaga-tran from AstraZeneca was approved in
2003, but sadly had to be withdrawn from the market in Similar experience has been encountered in the discovery
2006 because of liver toxicities [5]. Of course the of other modulators of protein–protein interactions. For
pharmaceutical industry will continue its efforts to discover example, using fragment-based drug design (or SAR by
orally bioavailable thrombin inhibitors despite this major NMR) [9 ] Abbott scientists were able to discover a potent
setback. low nanomolar inhibitor ABT-737 of Bcl-2 and Bcl-xL,
antiapoptotic proteins that help cancer cells to evade
In the meantime, the discovery of parenteral thrombin programmed cell death and are important targets for
inhibitors has been much more successful. Several drugs, anticancer drug discovery. ABT-737 is not orally
including the direct thrombin inhibitors lepirudin (launched bioavailable and does not comply with the ‘rule-of-five’
1998), argatroban (launched 2000), and bivalir-udin (MW 803, 13 heteroatoms N, O, and S) [10]. Its clinical
(launched 2001), and the indirect thrombin inhibitor trials are conducted by parenteral administration. A further
fondaparinux (launched 2002), have been introduced into Bcl-2 inhibitor, obatoclax (GX15-070, Gemin X), a
clinical practice. Their availability has significantly derivative of the naturally occurring prodiginines is also
improved the treatment of patients undergoing percuta- under clinical development by parenteral admin-istration
neous coronary intervention and also patients who are [11].
contraindicated with heparin. Had the industry only relied
on the discovery of orally bioavailable thrombin inhibitors, This difficulty in finding orally bioavailable inhibitors of
no or little improvement would have been made in the past protein–protein interactions may be because of the large
80 years for the treatment of these patients. interaction surface areas demanded of the small-molecule
inhibitors to interrupt such interactions. Recently, Abbott

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480 Chemical biotechnology
has announced that the company is progressing an orally their direct semisynthetic derivatives [15 ]. The impact of
bioavailable Bcl-2 inhibitor ABT-263 toward clinical trials, natural products is even more profound among drugs that
which again does not comply with the ‘rule-of-five’ are used to treat severe and/or life-threatening diseases such
[10,12]. as cancer and infectious diseases. Among all approved
small-molecule anticancer drugs (total 155), 47% are either
A major technology advancement made in the past 10 years natural products or direct semisynthetic derivatives of
is the emergence of biologicals which promise to transform them. Similar dominance of natural products is observed
the pharmaceutical industry. Although the concept of among the anti-infectives, and amazingly >75% of
therapeutic monoclonal antibodies (MAbs) has been around approved antibacterials are natural products or their semi-
for a much longer period, it is only within the past 10 years synthetic derivatives (74 out of 98). It is impossible to
that we have witnessed their major success as therapeutics. imagine where human health would be without these
For example, 10 years ago there were only two MAb drugs natural products! Furthermore, during the period 2000–
on the world market, whereas now there are 19 FDA- 2006, when high-throughput synthesis should have had
approved MAb thera-peutics — including 6 blockbuster sufficient time in operation to produce approved drugs,
drugs. The market in 2005 was worth >$13 billion approximately 50% of the approved small-molecule drugs
worldwide, representing an astounding growth of 37% from were still those related to natural products [15 ].
mid-1990s. Biotech pipe-lines are overflowing with >160
MAb-based drugs [13]. The high productivity of natural product-based drug
discovery may be related to the fact that their chemical
It is not only productive to develop MAbs as therapeutics structures have been biologically prevalidated by evol-
per se but they could also be used to lead the development utionary selection which defines structural prerequisites for
of small-molecule drugs, for example to generate clinical binding to proteins. For example, an analysis of over 154
proof-of-concept for a potentially ‘first-in-class’ target 000 natural products showed that the majority of them have
because of their high specificity against biological targets, molecular volumes ranging from 100 to
˚3

coupled with the relatively straightforward nature of their 500 A [16 ]. In the meantime, the volumes found in over 18
manufacturing. This is probably best illustrated by the 000 binding cavities of protein targets range from
˚3

development of tyrosine kinase inhibitors for the treat-ment 300 to 800 A . Therefore, the average volumes of natural
of cancer [14]. Earlier encouraging data generated with products correlate with the average dimensions of protein
MAbs such as cetuximab and trastuzumab has been cavities (note that protein ligands often do not fill the entire
beneficial to the development of small-molecule inhibi-tors volume of a given protein cavity). Although many natural
such as gefitinib, erlotinib, and lapatinib. products from plants and microorganisms are not meant to
bind to human proteins, many human proteins consist of the
It therefore appears sensible to adopt a more program-matic same building blocks and contain similar structural
approach in discovering new drugs instead of wholly domains to the targets with which natural products have
concentrating on oral bioavailability. That is: coevolved.

to progress initially a parenteral candidate before too Natural products therefore represent privileged chemical
much effort and resources have to be invested for an oral starting points for drug discovery, and yet they have quite
candidate; and distinct structural characteristics from synthetic molecules.
to progress initially a therapeutic antibody wherever For example, when analyzed by either a size-independent
possible (most probably by parenteral administration) ‘chemistry space filter’ or ‘support-vector-machine’
before too much effort and resources have to be invested approach, natural products exhibit better scores of ‘drug-
for a small-molecule candidate. likeness’ than synthetic compounds [17]. Further, natural
products contain on average twice as many oxygen atoms
These may be particularly relevant for ‘first-in-class’ drug and three times fewer nitrogen atoms than synthetic drug
discovery efforts against difficult targets such as proteases molecules [17]. They also contain a slightly higher number
and those involving protein–protein interactions. of hydrogen-bond donors than do synthetic drugs. Natural
products contain approximately four times more chiral
Natural products and drug discovery centers and far fewer aromatic rings, a fact which may
productivity engender upon natural products better selectivity when
A major class of drug molecules that are excluded from the binding to stereo-defined sites.
original analysis which generated the ‘rule-of-five’ are natural
products. This is a serious defect as we know that a large Better exploration of the biologically prevalidated struc-
percentage of marketed drugs are natural products and their tural scaffolds of natural products could increase our
semisynthetic derivatives. A recent analysis of all drugs success rate of finding more active leads. For example, the
approved worldwide between 1981 and 2006 showed that 34% research group of Waldmann, in collaboration with
of all small-molecule drugs are natural products or Novartis, have devised a hierarchical structural

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Drug discovery beyond ‘Rule-of-Five’ Zhang and Wilkinson 481
classification of natural products (SCONP) based on binding cores of different proteins [18], researchers would
structural similarities and the number of rings in their be able to select a structurally less complex natural product
scaffolds [16 ]. Each node of this hierarchical system scaffold that could potentially bind to the same protein
contained a core structure found in a number of natural target of the complex lead. This structurally less complex
products and was annotated with biological sources and and synthetically more feasible scaffold could then be used
activities wherever they were known. The utility of such a as a novel chemotype or chemical starting point to build
system is that one can navigate and link a structurally targeted compound libraries. The attrac-tiveness and
complex natural product leading to a set of synthetically difference of this approach to fragment-based approach is
more feasible scaffolds, for example those most frequently that the less complex scaffolds are themselves found in
found in natural products containing two to four rings. In natural products (not necessarily linked to the complex lead
combination with protein structure sim-ilarity clustering by evolution or biosynthesis), and are therefore also
(PSSC), which identifies similar ligand- biologically prevalidated.

Table 1
US FDA-approved polyketide drugs (up to December 2006)

Compound Indication Compound class Producing organism Semi-synthetic

Oxytetracycline Anti-bacterial Tetracyclines Actinomycete N
Methacycline Anti-bacterial Tetracyclines Actinomycete Y
Democycline Anti-bacterial Tetracyclines Actinomycete Y
Clomocycline Anti-bacterial Tetracyclines Actinomycete Y
Minocycline Anti-bacterial Tetracyclines Actinomycete Y
Lymecycline Anti-bacterial, Tetracyclines Actinomycete Y
anti-malarial,
anti-protozoal
Tetracycline Anti-bacterial Tetracyclines Actinomycete N
Doxycycline Anti-bacterial, Tetracyclines Actinomycete Y
anti-malarial
Tigecycline Anti-bacterial Tetracyclines Actinomycete Y
Rifabutin Anti-bacterial Ansa-macrolide Actinomycete Y
Rifampin Anti-bacterial Ansa-macrolide Actinomycete Y
Rifaximin Anti-bacterial Ansa-macrolide Actinomycete Y
Clarithromycin Anti-bacterial Macrolide (14 membered) Actinomycete Y
Azithromycin Anti-bacterial Macrolide (14 membered) Actinomycete Y
Telithromycin Anti-bacterial Macrolide (14 membered) Actinomycete Y
Dithromycin Anti-bacterial Macrolide (14 membered) Actinomycete Y
Erythromycin Anti-bacterial Macrolide (14 membered) Actinomycete N
Roxithromycin Anti-bacterial Macrolide (14 membered) Actinomycete Y
Mupirocin Anti-bacterial Mupirocin Pseudomonad N
Amphotericin B Anti-fungal Polyene Actinomycete N
Candicidin Anti-fungal Polyene Actinomycete N
Natamycin Anti-fungal Polyene Actinomycete N
Nystatin Anti-fungal Polyene Actinomycete N
Idarubicin Anti-neoplastic Anthracycline Actinomycete Y
Doxorubicin Anti-neoplastic Anthracycline Actinomycete N
Epirubicin Anti-neoplastic Anthracycline Actinomycete Y
Daunorubicin Anti-neoplastic Anthracycline Actinomycete N
Valrubicin Anti-neoplastic Anthracycline Actinomycete Y
Rapamycin Immunosuppresive, FKBP12-binding Actinomycete N
anti-proliferative
FK506 Immunosuppresive, FKBP12-binding Actinomycete N
anti-proliferative
Pimecrolimus Anti-inflammatory FKBP12-binding Actinomycete Y
Simvastatin Anti-cholesterimic Statin Fungi Y
Pravastatin Anti-cholesterimic Statin Fungi Y
Lovastatin Anti-cholesterimic Statin Fungi N
Ivermectin Anthelmintic, Avermectin derivative Streptomyces Y
anti-nematode,
anti-protazoal
Bleomycin Anti-neoplastic Bleomycin Streptomyces N
Hesperetin Anti-cholesterimic Hesperetin Plant N
Etoposide Anti-neoplastic Etoposide Plant Y

Data was complied through analysis of DrugBank (http://redpoll.pharmacy.ualberta.ca/drugbank/ [38]).

www.sciencedirect.com Current Opinion in Biotechnology 2007, 18:478–488
482 Chemical biotechnology
Synthetic biology for natural product drug their components to address various problems that cannot
discovery be solved using naturally occurring entities [20]. In relation
Indeed one of the greatest challenges for natural product- to drug discovery, the relevance of synthetic biology can be
based drug discovery has been their structural complexity considered largely synonymous with ‘metabolic or
and low synthetic feasibility. However, recent progress in biosynthetic engineering’ which engineers the genetics of
organic chemistry [19] and the emergence of novel tech- living systems (e.g. microorganisms) using recombinant
nologies such as synthetic biology (biosynthetic engin- DNA technology toward desired end pro-ducts [21].
eering) may help to overcome this issue.

Synthetic biology is an emerging scientific field which Although the structures of natural products can be highly
seeks to understand and design biological systems and/or complex and diverse, their biosynthesis can be accom-

Figure 1
Polyketide biosynthesis on a modular PKS as exemplified by the biosynthesis of rapamycin. Biosynthesis is initiated by loading of the PKS with 4,5-
dihydroxycyclohexe-1-ene carboxylic acid, with reduction by the loading module ER domain, and subsequent rounds of chain extensions with
malonyl-CoA and methylmalonyl-CoA, in which various levels of reductive processing of the newly formed b-keto groups occur. The modular
organization of the PKS is shown as a series of spherical domains: CoL, CoA-ligase like domain; ER, enoylreductase; ACP, acylcarrier protein; AT,
acyltransferase; DH, dehydratase; KR, b-ketoacylreductase; KS, b-ketoacylsynthase. Biosynthesis is completed by the amidation of the chain with L-
pipecolic acid and macrocylization. The resulting prerapamycin is then converted to rapamycin by the action of several post-PKS acting
monooxygenases and methyltransferase.
Current Opinion in Biotechnology 2007, 18:478–488 www.sciencedirect.com
Drug discovery beyond ‘Rule-of-Five’ Zhang and Wilkinson 483
plished by a remarkably few simple types of reactions. One Island (Rapa Nui). It is a very potent inhibitor of the serine-
example is the biosynthesis of the aliphatic, or reduced, threonine kinase target of rapamycin (TOR) involved in the
polyketides [22], a structurally diverse class of natural phosphatidylinositol 3-kinase (PI3K)/ Akt (protein kinase
products that include drugs such as lovastatin, B) signaling pathway that mediates cell survival and
erythromycin, rifamycin, amphotericin, and rapamycin proliferation [24]. It inhibits TOR by first binding to the
among others (Table 1). The biosynthesis of their poly- immunophilin FK506-binding protein 12 (FKBP-12) and
ketide core structures involves one major type of reac-tion the resulting binary complex then binds to TOR at an
— a decarboxylative Claisen-type condensation in which allosteric site, not competing directly with ATP. It is
various acyl-CoA monomers are selected by a polyketide therefore the most selective kinase inhibitor known till
synthase and added, one by one, to the grow-ing polyketide date.
chain which is tethered covalently to the PKS (Figure 1).
The resulting b-keto esters thus formed can be further The structural complexity of rapamycin has been a major
(optionally) reduced to a hydroxyl group, dehydrated to challenge for analog preparation, and there have been
give an olefin, and finally reduced to a saturated methylene several total syntheses reported over the years [25]. None
unit. These few simple chemical transformations occur with of these, however, is less than 50 steps, nor offers an
specific stereochemical out-comes, and in combination, overall yield >0.5%. It is therefore impractical to use any of
generate tremendous struc-tural diversity. The simplicity of these for lead optimization. As a consequence, the
polyketide biosynthesis is further facilitated by the modular rapamycin analogs that have progressed to market or
organization of the multienzyme PKSs in which highly clinical development are all simple semisynthetic deriva-
similar ‘modules’ — a group of enzymatic domains tives at a single structural point, the secondary hydroxyl
responsible for one ketide extension — act as an assembly functionality at C40 (Figure 2) [24]. The structural sim-
production line, with the structure of the chemical product ilarity of these compounds means they share very similar
‘hardwired’ into the DNA sequence encoding the PKS. biological profiles with little significant improvement, for
Whilst exceptions to the ‘one module for one ketide example, over poor pharmaceutical or pharmacokinetic
extension’ rule do exist [23], in general this simple properties.
template-based process pro-vides a direct and easily
understood relationship between DNA sequence and The biosynthesis of rapamycin is catalyzed by a mixed type
chemical structure, and a paradigm for the rational I PKS/nonribosomal peptide synthetase (NRPS) system
engineering of such systems to generate new natural (Figure 1) [26]. The PKS uses a shikimate-derived 4,5-
product analogs biosynthetically [22]. dihydroxycyclohex-1-enecarboxylic acid starter unit and
carries out a total of 14 successive steps of polyketide chain
As an example, this technology has been successfully used extension. The NRPS then incorporates an L-lysine-derived
to generate novel rapamycin analogs potentially useful as L-pipecolicacid moiety into the chain, followed by cleavage
immunosuppressants, anticancer and anti-inflammatory from the enzyme to produce the key intermediate
drugs. Rapamycin (Figure 2) is a polyketide macrolide prerapamycin [27]. Prerapamycin is further modified by
originally discovered from the culture of Strep-tomyces methyltransferases (RapI, RapM, and RapQ) and cytochrome
hygroscopicus NRRL5491 collected from Easter P450 monooxygenases

Figure 2
Structures of rapamycin and its semisynthetic analogs in clinical use or trials.
www.sciencedirect.com Current Opinion in Biotechnology 2007, 18:478–488
484 Chemical biotechnology

Figure 3

Mutasynthesis of prerapamycin analogs using the genetically engineered strain Streptomyces hygroscopicus MG2-10 and feeding exogenous starter
acid analogs [28].

(RapJ and RapN) to yield the fully processed rapamycin tory potency against TOR kinase activity and exhibits
molecule [28]. potent anticancer activities in various in vitro and in vivo
assays. It is not a substrate of the P-gp efflux pump and has
Interestingly, when a region of DNA including the genes good oral bioavailability. Of particular interest is the
thought to encode those post-PKS acting methyltransfer- effective penetration of the blood–brain barrier by BC210.
ases and monooxygenases (rap KIJMNOQL) were excised, When given intravenously (i.v.) 3 mg/kg in mice, it
the resultant mutant S. hygroscopicus MG2-10 was unable ‘accumulates’ in the brain resulting in the brain to blood
to produce prerapamycin unless fed with exogenous area under curve (AUC) ratio of 1.6, a property which is
pseudo-starter acid 3,4-dihydroxycyclohexane-carboxylic unique among published rapamycin analogs (cf. the ratio
acid or complemented with the gene rapK (Figure 3) [29 ]. for rapamycin is 0.25). This pharmacokinetic property of
This indicated that the product of rapK is involved in starter BC210 makes the compound a highly promising candi-date
acid biosynthesis and/or its regulation. as a potential therapeutic for the treatment of brain tumors
such as glioblastoma multiformes, or neurodegenerative
The production of prerapamycin by feeding exogenous diseases, either alone or in combi-nation with other
starter acid to the MG2-10 mutant, combined with the therapeutic agents [31].
restoration of post-PKS structural changes by expressing all
or selected post-PKS genes (rapI,J,M,N,O,Q), opened up The utility of such genetically engineered biosynthesis
the possibility of the combinatorial biosynthesis of a wide approaches for drug discovery has additional benefit
range of rapamycin analogs [29 ]. For example, it was compared to the more widely used semisynthetic
demonstrated that feeding other carboxylic acids in place of approaches. For example, semisynthetic derivatization
the starter unit acid led to the production of various
often produces analogs with larger molecule sizes than the
rapamycin analogs. One of these analogs, BC210 (Figure
4), which has the metabolically labile C39-meth-oxy group natural product lead, leading to reduced ligand effi-ciency
of rapamycin removed from its structure has shown [32]. Biosynthetic modifications need not suffer from this
drawback. The 39-desmethoxy rapamycin BC210 (Figure
significantly improved metabolic stability (t 1/2 59 min
4) has similar FKBP-12/TOR binding affinity to rapamycin
when incubated with human liver microsomes) and Caco-2
but its molecular weight is slightly less than rapamycin. On
permeability (Papp 29 nm/s) compared to rapamycin (t 1/2 40 the contrary, all semisynthetic derivatives of rapamycin
min with human liver microsomes and P app 2 nm/s) [30]. currently marketed or under clinical development (Figure
BC210 has low nanomolar inhibi- 2) have larger molecular

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 Drug discovery beyond ‘Rule-of-Five’ Zhang and Wilkinson 485

Figure 4

Design and biosynthetic preparation of BC210: removal of the methoxy moiety at C39 of rapamycin using mutasynthesis provides a compound
with enhanced metabolic stability versus human liver microsomes [29].

weight than rapamycin and equal or less affinity to the 1.2 mM, MW 561) (Figure 5) [34]. A further advantage of
targets FKBP-12/TOR. the nonquinone derivative produced by biosynthetic
engineering over the semisynthetic tanespimycin is that it
This advantage of being able to maintain or increase ligand does not undergo redox cycling and should have signifi-
efficiency by biosynthesis-based lead optimization has also cantly reduced off-target toxicity and consequently an
been observed in several other cases. For example, increased therapeutic window.
optimization of the naturally occurring heat-shock protein-
90 (Hsp90) inhibitor macbecin I by a synthetic biology An increased therapeutic window has also been achieved
approach has generated a nonquinone derivative that has
with a series of borrelidin analogs without increasing their
significantly increased affinity to Hsp90 (K d 0.007 mM) molecular sizes [35]. The biosynthetic derivative BC194
and a reduced molecular weight (MW 519) compared to the exhibited decreased toxicity but increased antiangiogenic
lead molecule (Kd 0.24 mM, MW 559 for macbecin I) activity against human umbi-lical vein endothelial cells
(Figure 5) [33]. On the contrary, semisynthetic (HUVEC) compared to bor-relidin, and its reduced toxicity
optimization of another naturally occurring Hsp90 inhibitor was confirmed in an in vivo study in nude mice. The
geldanamycin has resulted in a 17-ally-lamino derivative, maximum tolerated dose (MTD) of BC194 was >130
tanespimycin, that has similar affinity to Hsp90 (K d 1.3 mg/kg, i.v., whereas the MTD of borrelidin was found to be
mM) but increased molecular weight (MW 586) compared in the range of 5–15 mg/kg, i.v. [35].
to the lead geldanamycin (Kd

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486 Chemical biotechnology

Figure 5

Comparison of semisynthetic and synthetic biology-based lead optimization approaches for natural products, exemplified by optimization of
ansamycin-based Hsp90 inhibitors. Binding efficiency indices (BEI) = pK d/MW (kDa). For a small-molecule inhibitor of MW 500 and Kd of 1.0
nM, its BEI = 18 [31].
Conclusions parenteral drug candidate in parallel to an oral drug
Currently, the majority of industrial efforts in drug dis- candidate, in some cases before the availability of an oral
covery, especially in large pharmaceutical companies, are drug candidate and second, to consider the development of
being invested in discovering small molecule, orally bioa- a therapeutic antibody wherever possible in parallel to a
vailable drugs that comply with the ‘rule-of-five’. How- small-molecule drug candidate. These are particularly
ever, only half of all FDA-approved small-molecule drugs relevant for effort against ‘first-in-class’ drug targets and/
are both used orally and compliant with the ‘rule-of-five’ or particularly challenging targets such as proteases and
[3]. In other words, nearly half of all small-molecule drugs those involving protein–protein interactions, where, to
are either not used for oral administration or do not comply discover an orally bioavailable drug candidate can be more
with the ‘rule-of-five’. This incompatibility between huge problematic and slow.
industrial effort and the lower percentage of relevant drugs
in clinical use has almost certainly something to do with In addition, more effort should be invested in natural
economic considerations — oral drugs are more likely to be product research. Novel technologies such as synthetic
‘blockbusters’. Hopefully this is changing with the emer- biology may address many of the challenging issues of
gence of blockbuster biologicals and target-based antic- natural product-based drug discovery, for example struc-
ancer drugs [36], and the drive to develop drugs for niche tural complexity and synthetic feasibility. Synthetic biology
indications [37]. approaches can in practice be simple and straight-forward
in analog preparation. Genetic alterations can be made in
It seems to us that in order to increase drug discovery such a way as to biosynthesize analogs with predesigned
productivity and to address seriously un-met medical needs structural modifications, in very much the same way as
a programmatic approach to bioavailability could be achieved by conventional medicinal chem-istry. Unlike the
adopted. That is first, to consider the development of a total synthesis of complex natural pro-

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Drug discovery beyond ‘Rule-of-Five’ Zhang and Wilkinson 487
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Acknowledgement This latest review together with the previous two by the same authors
We would like to thank our colleagues at Biotica for their contribution to the (cited in the paper) presents a comprehensive analysis on the impact of
work cited in this review. natural products to drug discovery. This includes not only the naturally
occurring compounds and their direct semisynthetic derivatives but also
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