Biopharmconsortium Blog

Expert commentary from Haberman Associates biotechnology and pharmaceutical consulting.

Posts filed under: Chemistry

Forma Therapeutics’ expanded R&D collaboration with Celgene


Ubiquitin pathway. Source: Rogerdodd, English language Wikipedia

Ubiquitin pathway. Source: Rogerdodd, English language Wikipedia

On April 1, 2014, Forma Therapeutics (Watertown MA) announced that it had entered into an expanded strategic collaboration with Celgene (Summit, NJ).

Under the new agreement, Forma has received an upfront cash payment of $225 million. The initial collaboration between the two companies under the new agreement will be for 3 1⁄2 years. Celgene will also have the option to enter into up to two additional collaborations with terms of two years each for additional payments totaling approximately $375 million. Depending on the success of the collaborations and if Celgene elects to enter all three collaborations, the combined duration of the three collaborations may be at least 7 1⁄2 years.

Under the terms of the new agreement, Forma will control projects from the research stage through Phase 1 clinical trials. For programs selected for licensing, Celgene will take over clinical development from Phase 2 to commercialization. Forma will retain U.S. rights to these products, and Celgene will have the rights to the products outside of the U.S. For products not licensed to Celgene, FORMA will maintain worldwide rights.

During the term of the third collaboration, Celgene will have the exclusive option to acquire Forma, including the U.S. rights to all licensed programs, and worldwide rights to other wholly owned programs within Forma at that time.

The April 2013 agreement between Forma and Celgene

The new collaboration between Forma and Celgene builds on an earlier agreement between the two companies. On April 29, 2013, the two companies entered into a collaboration aimed at discovery, development, and commercialization of drug candidates to modulate targets involved in protein homeostasis.

Protein homeostasis, also known as proteostasis, involves a tightly regulated network of pathways controlling the biogenesis, folding, transport and degradation of proteins. The ubiquitin pathway (illustrated in the figure above) is one of these pathways. We recently discussed how the ubiquitin pathway is involved in the mechanism of action of thalidomide and lenalidomide (Celgene’s Thalomid and Revlimid).

Targeting protein homeostasis has application to discovery and development of drugs for oncology, neurodegenerative disease, and other disorders. However, the April 2013 Forma/Celgene agreement focused on cancer. Under that agreement, Forma received an undisclosed upfront payment. Upon licensing of preclinical drug candidates by Celgene, Forma was to be eligible to receive up to $200 million in research and early development payments. FORMA was also to be eligible to receive $315 million in potential payments based upon development, regulatory and sales objectives for the first ex-U.S. license, as well as  up to a maximum of $430 million per program for further licensed products, in addition to post-sales royalties.

On October 8, 2013, Forma announced that it had successfully met the undisclosed first objective under its April 2013 strategic collaboration agreement with Celgene. This triggered an undisclosed payment to Forma. Progress in the April 2013 collaboration was an important basis for Celgene’s decision to enter into a new, broader collaboration with Forma a year later.

The scope of the new April 2014 Forma/Celgene collaboration

Unlike the April 2013 agreement, the April 2014 agreement between Forma and Celgene is not limited to protein homeostasis, or to oncology. The goal of the new collaboration is to “comprehensively evaluate emerging target families for which Forma’s platform has exceptional strength” over “broad areas of chemistry and biology”.  The expanded collaboration will thus involve discovery and development of compounds to address a broad range of target families and of therapeutic areas.

According to Celgene’s Thomas Daniel, M.D. (President, Global Research and Early Development), Celgene’s motivation for signing the new agreement is based not only on the early success of the existing Forma/Celgene collaboration, but also on “emerging evidence of the power of Forma’s platform to generate unique chemical matter across important emerging target families”.

According to Forma’s President and CEO, Steven Tregay, Ph.D., the new collaboration with Cegene enables Forma to maintain its autonomy in defining its research strategy and conducting discovery through early clinical development. It also aligns Forma with Celgene’s key strengths in hematology and in inflammatory diseases.

Forma Therapeutics in Haberman Associates publications

We have been following Forma on the the Biopharmconsortium Blog since July 2011. At that time, I was a speaker at Hanson Wade’s World Drug Targets Summit (Cambridge, MA). At that meeting, Mark Tebbe, Ph.D. (then Vice President, Medicinal and Computational Chemistry at Forma) was also a speaker. At the conference, Dr. Tebbe discussed FORMA’s technology platforms, which are designed to be enabling technologies for discovery of small-molecule drugs to address challenging targets such as protein-protein interactions (PPIs).

In particular, Dr. Tebbe discussed Forma’s Computational Solvent Mapping (CS-Mapping) platform, which enables company researchers to interrogate PPIs in intracellular environments, to define hot spots on the protein surfaces that might constitute targets for small-molecule drugs. FORMA has been combining CS-Mapping technology with its chemistry technologies (e.g., structure guided drug discovery, diversity orientated synthesis) for use in drug discovery.

We also discussed Forma’s earlier fundraising successes as of January 2012, and cited Forma as a “built to last” research-stage platform company in an interview for Chemical & Engineering News (C&EN).

Finally, we discussed Forma and its technology platform in our book-length report, Advances in the Discovery of Protein-Protein Interaction Modulators, published by Informa’s Scrip Insights in 2012. (See also our April 25, 2012 blog article.)

In our report, we discussed Forma as a company that employs “second-generation technologies” for the discovery of small-molecule PPI modulators. This refers to a suite of technologies designed to overcome the hurdles that stand in the way of the accelerated and systematic discovery and development of PPI modulators. Such technologies are necessary to make targeting of PPIs a viable field.

Forma’s website now has a brief explanation of its drug discovery engine, as it is applied to targeting PPIs. This includes links to web pages describing:

Our 2012 book-length report discusses technologies of these types, as applied to discovery of PPI modulators, in greater detail than the Forma website.

According to Dr. Daniel: “Progress in our existing [protein homeostasis] collaboration, coupled with emerging evidence of the power of FORMA’s platform to generate unique chemical matter across important emerging target families” led Celgene to enter into its new, expanded collaboration with Forma in April 2014. This suggests that Celgene is especially impressed by Forma’s chemistry and chemical biology platforms. it also suggests that chemistry technology platforms developed to address PPIs may be applicable to areas of drug discovery beyond PPIs as well.

Concluding remarks

Despite the enthusiasm for Forma and its drug discovery engine shown by Celgene, Forma’s other partners, and various industry experts, it must be remembered that Forma is still a research-stage company. The company has not one lone drug candidate in the clinic, let alone achieving proof-of-concept in humans. It is clinical proof-of-concept, followed by Phase 3 success and approval and marketing of the resulting drugs, that is the “proof of the pudding” of a company’s drug discovery and development efforts.

We await the achievement of such clinical milestones by Forma Therapeutics.

From a business strategy point of view, we have discussed Forma’s efforts to build a stand-alone, independent company for the long term in this blog and elsewhere. Now Forma has entered into an agreement with Celgene that might—in around 7-10 years—result in Forma’s acquisition. This would seem to contradict Forma’s “built to last” strategy.

However, in the business environment that has prevailed over the past several years, several established independent biotech companies, notably Genentech and Genzyme, have been acquired by larger companies. Even several Big Pharmas (e.g., Schering-Plough and Wyeth) have been acquired.

Nevertheless, we do not know what the business environment in the biotech/pharma industry will be like in 7-10 years, despite the efforts of strategists to predict it. And Celgene might forgo its option to acquire Forma, for any number of reasons. So the outlook for Forma’s status as an independent or an acquired company (which also depends on its success in developing drugs) is uncertain.


As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

Chemokine receptors and the HIV-1 entry inhibitor maraviroc



In April 2012, Informa’s Scrip Insights published our book-length report, “Advances in the Discovery of Protein-Protein Interaction Modulators.” We also published a brief introduction to this report, highlighting the strategic importance of protein-protein interaction (PPI) modulators for the pharmaceutical industry, on the Biopharmconsortium Blog.

The report included a discussion on discovery and development of inhibitors of chemokine receptors. Chemokine receptors are members of the G-protein coupled receptor (GPCR) superfamily. GPCRs are seven-transmembrane (7TM) domain receptors (i.e. integral membrane proteins that have seven membrane-spanning domains). Compounds that target GPCRs represent the largest class of drugs produced by the pharmaceutical industry. However, in the vast majority of cases, these compounds target GPCRs that bind to natural small-molecule ligands.

Chemokine receptors, however, bind to small proteins, the chemokines. These proteins constitute a class of small cytokines that guide the migration of immune cells via chemotaxis. Chemokine receptors are thus a class of GPCRs that function by forming PPIs. Direct targeting of interactions between chemokines and their receptors (unlike targeting the interactions between small-molecule GPCR ligands and their receptors) thus involves all the difficulties of targeting other types of PPIs.

However, GPCRs–including chemokine receptors–appear to be especially susceptible to targeting via allosteric modulators. Allosteric sites lie outside the binding site for the protein’s natural ligand. However, modulators that bind to allosteric sites change the conformation of the protein in such a way that it affects the activity of the ligand binding site. (Direct GPCR modulators that bind to the same site as the GPCR’s natural ligands are known as orthosteric modulators.) In the case of chemokine receptors, researchers can in some cases discover small-molecule allosteric modulators that activate or inhibit binding of the receptor to its natural ligands. Discovery of such allosteric activators is much easier than discovery of direct PPI modulators.

Chemokines bind to sites that are located in the extracellular domains of their receptors. Allosteric sites on chemokine receptors, however, are typically located in transmembrane domains that are distinct from the chemokine binding sites. Small-molecule allosteric modulators that bind to these sites were discovered via fairly standard medicinal chemistry and high-throughput screening, sometimes augmented with structure-based drug design. This is in contrast to attempts to discover small molecule agents that directly inhibit binding of a chemokine to its receptor, which has so far been extremely challenging.

Our report describes several allosteric chemokine receptor modulators that are in clinical development, as well as the two agents that have reached the market. One of the marketed agents, plerixafor (AMD3100) (Genzyme’s Mozobil), is an inhibitor of the chemokine receptor CXCR4. It is used in combination with granulocyte colony-stimulating factor (G-CSF) to mobilize hematopoietic stem cells to the peripheral blood for autologous transplantation in patients with non-Hodgkin lymphoma and multiple myeloma. The other agent, which is the focus of this blog post, is maraviroc (Pfizer’s Selzentry/Celsentri).

Maraviroc is a human immunodeficiency virus-1 (HIV-1) entry inhibitor. This compound is an antagonist of the CCR5 chemokine receptor. CCR5 is specific for the chemokines RANTES (Regulated on Activation, Normal T Expressed and Secreted) and macrophage inflammatory protein (MIP) 1α and 1β.  In addition to being bound and activated by these chemokines, CCR5 is a coreceptor (together with CD4) for entry of the most common strain of HIV-1 into T cells. Thus maraviroc acts as an HIV entry inhibitor; this is the drug’s approved indication in the U.S. and in Europe. Maraviroc was discovered via a combination of high-throughput screening and optimization via standard medicinal chemistry.

New structural biology studies of the CCR5-maraviroc complex

Now comes a report in the 20 September 2013 issue of Science on the structure of the CCR5-maraviroc complex. This report was authored by a mainly Chinese group led by Beili Wu, Ph.D. (Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai); researchers at the University of California at San Diego and the Scripps Research Institute, San Diego were also included in this collaboration. A companion Perspective in the same issue of Science was authored by P. J. Klasse, M.D., Ph.D. (Weill Cornell Medical College, Cornell University, New York, NY).

As described in the Perspective, the outer surface of the HIV-1 virus displays numerous envelope protein (Env) trimers, each including the outer gp120 subunit anchored in the viral membrane by gp41. When gp120 binds to the cell-surface receptor CD4, this enables interaction with a specific chemokine receptor, either CCR5 or CXCR4. Interaction with both CD4 and the chemokine receptor triggers complex sets of changes in the Env complex, eventually resulting in the fusion of the viral membrane and the cell membrane, and the entry of the virus particle into the host cell.

HIV-1 gp120 makes contact with CCR5 at several points. The interactions between CCR5 and the variable region of gp120 called V3 are especially important for the tropism of an HIV-1 strain, i.e., whether the virus is specific for CCR5 (the “R5 phenotype”) or CXCR4 (the “X4 phenotype”). In the case of R5-tropic viruses, the tip of the V3 region interacts with the second extracellular loop (ECL2) of CCR5, while the base of V3 interacts with the amino-terminal segment of CCR5. Modeling of the interactions between the V3 domain of gp120 of either R5 or X4-tropic viruses with CCR5 or CXCR4 explains coreceptor use, in terms of forming strong bonds or–conversely–weak bonds and steric hindrance.

Monogram Biosciences (South San Francisco, CA) has developed and markets the Trofile assay. This is a molecular assay designed to identify the R5, X4, or mixed tropism of a patient’s HIV strain. If a patient’s strain is R5-tropic, then treatment with maraviroc is appropriate. However, a patient’s HIV-1 strain may undergo a tropism switch, or may mutate in other ways to become resistant to maraviroc.

Dr Wu and her colleagues determined the high-resolution crystal structure of the complex between maraviroc and a solubilized engineered form of CCR5. This included determining the CCR5 binding pocket for maraviroc, which was determined both by Wu et al’s X-ray crystallography, and by site-directed mutagenesis (i.e., to determine amino acid residues that are critical for maraviroc binding) that had been published earlier by other researchers.

The structural studies of Dr. Wu and her colleagues show that the maraviroc-binding site is different from the recognition sites for gp120 and for chemokines, as expected for an allosteric inhibitor. The X-ray structure shows that maraviroc binding prevents the helix movements that are necessary for binding of g120 to induce the complex sequence of changes that result in fusion between the viral and cellular membranes. (These helix movements are also necessary for induction of signal transduction by binding of chemokines to CCR5.)

Structural studies of CXCR4 and its inhibitor binding sites

In addition to their structural studies of the CCR5-maraviroc complex, Dr. Wu and her colleagues also published structural studies of CXCR4 complexed with small-molecule and cyclic peptide inhibitors in Science in 2010. These inhibitors are IT1t, a drug-like orally-available isothiourea developed by Novartis, and CVX15, a 16-residue cyclic peptide that had been previously characterized as an HIV-inhibiting agent. IT1t and CVX15 bind to overlapping sites in CXCR4. Other researchers have found evidence that the binding site for plerixafor also overlaps with the IT1t binding site.

As discussed in Wu et al’s 2013 paper, CCR5 and CXCR4 have similar, but non-identical structures. The binding site for IT1t in CXCR4 is closer to the extracellular surface than is the maraviroc binding site in CCR5, which is deep within the CCR5 molecule. The entrance to the CXCR4 ligand-binding pocket is partially covered by CXC4’s N terminus and ECL2, but the CCR5 ligand-binding pocket is more open.

Mechanisms of CXCR4 and CCR5 inhibition, and implications for discovery of improved HIV entry inhibitors

The chemokine that specifically interacts with the CXCR4 receptor is known as CXCL12 or stromal cell-derived factor 1 (SDF-1). Researchers have proposed a hypothesis for how CXCL12 interacts with CXCR4; this hypothesis appears to be applicable to the interaction between other chemokines and their receptors as well. This hypothesis is know as the “two-step model” or the “two-site model” of chemokine-receptor activation. Under the two-site model, the core domain of a chemokine binds to a site on its receptor (known as the “chemokine recognition site 1” or “site 1”) defined by the receptor’s N-terminus and its ECLs. In the second step, the flexible N-terminus of the chemokine interacts with a second site (known as “chemokine recognition site 2” or “site 2” or the “activation domain”) deeper within the receptor, in transmembrane domains. This result in activation of the chemokine receptor and intracellular signaling.

Under the two-site model, CXCR4 inhibitors (e.g., IT1t, CVX15, and  plerixafor), which bind to sites within the ECLs of CXCR4, are competitive inhibitors of binding of the core domain of CXCL12 to CXCR4 (i.e.., step 1 of chemokine/receptor interaction). They are thus orthosteric inhibitors of CXCR4. (This is contrary to the earlier assignment of plerixafor as an allosteric inhibitor of CXCR4.)  The CCR5 ligand maraviroc, however, binds within a site within the transmembrane domains of CCR5, which overlaps with the activation domain of CCR5. Dr. Wu and her colleagues propose two alternative hypotheses: 1. Maraviroc may inhibit CCR5 activation by chemokines by blocking the second step of chemokine/chemokine receptor interaction, i.e., receptor activation. 2. Maraviroc may stabilize CCR5 in an inactive conformation. It is also possible that maraviroc inhibition of CCR5 may work via both mechanisms.

Dr. Wu and her colleagues further hypothesize that the interaction of  HIV-1 gp120 with CCR5 (or CXCR4) may operate via similar mechanisms to the interaction of chemokines with their receptors. As we discussed earlier in this article, the base (or the stem region) of the gp120 V3 domain interacts with the amino-terminal segment of CCR5. The tip (or crown) of the V3 domain interacts with the ECL2 of CCR5, and–according to Dr. Wu and her colleagues–also with amino acid residues inside the ligand binding pocket; i.e., the activation site of CCR5. The HIV gp120 V3 domain may thus activate CCR5 via a similar mechanism to the two-step  model utilized by chemokines.

Based on their structural biology studies, Dr. Wu and her colleagues have been building models of the CCR5-R5-V3 and CXC4-X4-V3 complexes, and are also planning to determine additional structures needed to fully understand the mechanisms of HIV-1 tropism. The researchers will utilize their studies in the discovery of improved, second-generation HIV entry inhibitors for both R5-tropic and X4-tropic strains of HIV-1.

The bigger picture

The 17 October 2013 issue of Nature contains a Supplement entitled “Chemistry Masterclass”. In that Supplement is an Outlook review entitled “Structure-led design”, by Nature Publishing Group Senior Editor Monica Hoyos Flight, Ph.D. The subject of this article is structure-based drug design of modulators of GPCRs.
This review outlines progress in determining GPCR structures, and in using this information for discovery of orthosteric and allosteric modulators of GPCRs.

According to the article, the number of solved GPCR structures has been increasing since 2008, largely due to the efforts of the Scripps GPCR Network, which was established in that year. Dr. Wu started her research on CXCR4 and CCR5 as a postdoctoral researcher in the laboratory of Raymond C. Stevens, Ph.D. at Scripps in 2007, and continues to be a member of the network. The network is a collaboration that involves over a dozen academic and industrial labs. Its goal has been to characterize at least 15 GPCRs by 2015; it has already solved 13.

Interestingly, among the solved GPCR structures are those for the corticotropin-releasing hormone receptor and the glucagon receptor. Both have peptide ligands, and thus work by forming PPIs.

One company mentioned in the article, Heptares Therapeutics (Welwyn Garden City, UK), specializes in discovering new medicines that targeting previously undruggable or challenging GPCRs. In addition to discovering small-molecule drugs, Heptares, working with monoclonal antibody (MAb) leaders such as MorphoSys and MedImmune, is working to discover MAbs that act as modulators of GPCRs. Among Heptares’ targets are several GPCRs with peptide ligands.

Meanwhile, Kyowa Hakko Kirin Co., Ltd. has developed the MAb drug mogamulizumab (trade name Poteligeo), which is approved in Japan for treatment of relapsed or refractory adult T-cell leukemia/lymphoma. Mogamulizumab targets CC chemokine receptor 4 (CCR4).

Thus, aided in part by structural biology, the discovery of novel drugs that target GPCRs–including those with protein or peptide targets such as chemokine receptors–continues to make progress.


As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company,  please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

Developing resistance-free antibiotics by targeting quorum sensing


Quorum sensing synthetic biology project

Way back in May 2000, Decision Resources published my short report entitled “New approaches to small-molecule antibacterial drug discovery” as part of its Spectrum Life Sciences series. As might be expected, the report is now out of print.

The report was a brief review of then-novel approaches to antibacterial drug discovery, in the face of the increasing level of antibiotic resistance in pathogenic bacteria. These approaches included genomics and such technologies as high-throughput screening against bacterial-specific targets.

However, the most interesting part of the report was a section on using the study of bacterial physiology to identify targets that are important for the ability of bacteria to cause disease, but are not essential for bacterial proliferation or survival. The hypothesis behind these studies was that it might be possible to develop compounds that prevent these bacteria from causing disease, without selecting for resistant strains of the bacteria.

Antibiotics typically kill or prevent proliferation of bacteria by targeting biomolecules involved in such essential processes as cell wall synthesis, DNA proliferation, or protein synthesis. Treating large populations of bacteria with such agents inevitably selects for a few resistant mutant cells. These proliferate, mutate further, and give rise to antibiotic resistant populations. However, if a therapeutic targets a nonessential pathway that is involved in pathogenesis, resistant populations might not be selected for. That was the hypothesis.

This field of bacterial physiology for drug discovery focused on two related areas–virulence factors and quorum sensing. Virulence factors are not expressed by a strain of pathogenic bacteria in vitro, but are expressed only when the bacteria infect a host. Once expressed, they enable the bacteria to colonize the host and cause disease. Examples of such virulence factors include secretion systems that deliver bacterial effector proteins into host cells. These effector proteins may, for example, kill host cells, inhibit cytokine production or phagocytosis, or may mediate bacterial entry into the host cells.

Quorum sensing is a system by which certain bacteria can monitor their own population density. They accomplish this by secreting specific autoinducer molecules. When the concentration of an autoinducer reaches a critical threshold value (as the result of an increase in bacterial population density), it triggers specific response systems, causing the induction of sets of genes that are only expressed at high population density.

For example, many gram-negative bacteria (e.g., Pseudomonas aeruginosa, Vibrio cholerae, and Escherichia coli) use specific acyl homoserine lactones (AHSLs) as their autoinducers. P. aeruginosa has two quorum sensing systems that use the AHSL autoinducers butyrylhomoserinelactone and 3-oxododecanoylhomoserinelactone, respectively. These systems (operating via specific receptors for the auotoinducers and interacting with each other) control the induction of several genes, some of which are virulence factors. Some of these genes enable the bacteria, when they are at sufficient density, to form biofilms (slimy mats of bacteria and polysaccharide matrix).

P. aeruginosa is an opportunistic pathogen, causing infection in the lungs of people with cystic fibrosis, burn patients, and other hospitalized patients. These infections cause death in over 80% of cystic fibrosis patients. The ability to form biofilms renders the bacteria resistant to antibiotics and to the patient’s own immune system.

Other gram-negative bacteria that form biofilms have been implicated in dental caries, peridontitis, osteomyelitis, and numerous nosocomial infections. Bacterial biofilms can also form on the surface of implanted medical devices, such as catheters and mechanical heart valves, and cause device-related infections.

The gram-positive human pathogen Staphylococcus aureus also has a quorum sensing system. However, it does not use an AHSL as an autoinducer. The S. aureus autoinducers are peptides that contain an unusual thiolactone structure (i.e., a thol ester-linked cyclic structure). The S. aureus quorum sensing system controls the synthesis of virulence factors responsible for the pathogenicity of this organism in vivo. Although specific peptides induce virulence factors in a given strain of S. aureus, there are other specific peptides that inhibit the induction of virulence in strains of the organism other than the one secreting the inhibitory peptides. That finding suggested that researchers should be able to develop specific agents to shut down S. aureus pathogenesis by targeting the quorum sensing system.

Interestingly, quorum sensing-based systems have been used in projects for the International Genetically Engineered Machine (iGEM) competition, an annual undergraduate synthetic biology competition. See the figure above, which was taken from the 2009 Chiba University (Japan) iGEM project.  []

Quorum Sciences and Vertex Pharmaceuticals’ research on quorum sensing

At the time of the writing and publication of our antibacterial drug discovery report, there was a company, Quorum Sciences (Iowa City, IA) that had been established to commercialize the findings of leading researchers on bacterial quorum sensing. As the result of two successive acquisitions in 2000 and 2001, Quorum Sciences passed into the hands of Vertex Pharmaceuticals (Cambridge, MA). In 2006, Vertex researchers and their academic collaborators published a report on the discovery of novel specific inhibitors of the P. aeruginosa quorum sensing system. The last author of this report was quorum sensing pioneer E. Peter Greenberg, formerly of the University of Iowa and chief scientific officer at Quorum Sciences, and from 2005 to the present at the University of Washington School of Medicine. The compounds identified in the 2006 report, discovered via high-throughput screening of a diverse 200,000-compound chemical library, resembled the natural AHSL that binds to the P. aeruginosa quorum sensing receptor LasR. (LasR is a transcription factor that when bound to its specific AHSL, mediates the expression of a set of downstream genes, including those that encode virulence factors.) The researchers concluded that the novel quorum sensing inhibitors might be useful chemical tools, but not drug leads.

In 2010, other academic researchers published a report on the discovery of novel antagonists and agonists of the P. aeruginosa quorum sensing receptor LasR, which were of lower molecular weight and otherwise structurally distinct from the natural P. aeruginosa AHSL. However, these compounds were still deemed to be scaffolds for chemical tools, not drug leads. Nevertheless, the researchers speculated that the compounds “could, with further development, provide a pathway for the design of novel antivirulence agents”. Other researchers are continuing studies aimed at discovery of quorum sensing receptor antagonists, whether synthetic organic molecules or natural products. These involve studies with quorum sensing systems of both gram-positive and gram-negative bacteria.

The 2006 report appears to be the last Vertex publication on quorum sensing. However, Vertex continues to conduct research on antibacterial agents. And the company has a facility in the University of Iowa BioVentures Center (Coralville, IA),  which is a continuation of the old Quorum Sciences Iowa facility. As of 2009, Vertex’s Iowa-based team consisted of seven full-time scientists, working on development of antibacterials, and agents to treat hepatitis C and cystic fibrosis, among other areas. The Iowa group participated in the development of Vertex’ now-marketed anti-hepatitis C virus (HCV) agent Incivek (telaprevir).

The May 2012 article “Freezing Time” in The Scientist, and discovery of novel quorum sensing inhibitors

The May 2012 issue of The Scientist contains an article entitled “Freezing Time”, by Vern L Schramm, Ph.D. (Albert Einstein College of Medicine (Bronx, NY). The article focused on design of “transition state analogues”, i.e., compounds with a chemical structure that resembles the transition state of a substrate in an enzyme-catalyzed reaction. Transition state analogs usually act as enzyme inhibitors by blocking the enzyme’s active site. They are exquisitely potent and specific inhibitors, which act at extremely small doses. This makes these compounds potentially attractive as drugs.

A transition state analogue inhibitor that was designed by Dr. Schramm and his colleagues in the early 2000s as an early proof-of-concept molecule is immucillin-H, or forodesine. This is a transition-state analog inhibitor of purine nucleoside phosphorylase.  Forodesine is being developed by BioCryst Pharmaceuticals for treatment of relapsed B-cell chronic lymphocytic leukemia, and the results of a Phase 2 trial were published in 2010.

As described in Dr. Schramm’s May 2012 article, his laboratory has been applying their transition-state analogue technology to the field of quorum sensing in bacteria. Instead of targeting the recognition of AHSLs by quorum sensing receptors such as LasR, the researchers targeted the key enzyme in the AHSL biosynthesis pathway in gram-negative bacteria, known as 5′-methylthioadenosine nucleosidase (MTAN). The biosynthetic pathway for the production of AHSLs, including the key role of MTAN, had been elucidated by Dr. Greenberg and his colleagues in the late 1990s.

Dr. Schramm and his colleagues published the results of studies of three transition state analogues that potently inhibited MTANs of gram-negative bacteria. For example, they inhibited the Vibrio cholerae MTAN with dissociation constants of 73, 70, and 208 pM, respectively. They inhibited MTAN in cell of a virulent strain of V. cholerae with IC50 values of 27, 31, and 6 nM respectively, disrupting autoinducer production in a dose-dependent manner without affecting bacterial growth. The compounds were also potent inhibitors of autoinducer production in an enterohemorrhagic strain of Escherichia coli. The transition-state analogues did not inhibit growth in either V. cholerae or E. coli, but one such compound reduced biofilm production by 18% in E. coli and 71% in V. cholerae.

Moreover, the MTAN inhibitors did not appear to select for bacterial resistance in vitro. When V. cholerae bacteria were grown for 26 generations in the presence of a large excess of MTAN inhibitors, subsequent generations of these bacteria were equally sensitive to inhibition by these compounds as bacteria that had not been previously exposed to the inhibitors. These results are consistent with the hypothesis that agents that inhibit targets that are important in the ability of bacteria to cause disease, but are not essential for bacterial proliferation or survival might not select for drug resistance.

As Dr. Schramm said in the May 2012 article in The Scientist, it remains to be seen whether the MTAN-targeting transition-state analogs developed in his laboratory can translate into novel antibiotics that do not select for resistant pathogens. As of March 2009, Dr. Schramm’s team had developed over 20 potent MTAN inhibitors, which will be specific for bacteria and should have no effect on human metabolism. These compounds have been licensed to Pico Pharmaceuticals (Melbourne, Australia), which plans to develop and initiate clinical trials. Dr. Schramm is a Pico Pharmaceuticals co-founder and chairman of its scientific advisory board. Pico claims that one of its quorum sensing inhibitors, designated as PC0208, has demonstrated proof-of-concept in preclinical studies, and now has “pre-IND” status.

Lessons from these studies

Dr. Schramm’s discovery of novel quorum sensing inhibitors was made possible by a strategy that involved a combination of biology-driven drug discovery and sophisticated chemistry technology. The biology-driven drug discovery strategy involved a combination of 1. Building on the quorum sensing studies of Dr. Greenberg and others, and adopting the strategy, as reviewed in our 2000 Spectrum report, of targeting the quorum sensing system in order to discover agents that would have the possibility of not triggering resistance, and 2. Targeting a critical, bacterial-specific pathway enzyme that is upstream of the recognition of AHSLs by quorum sensing receptors (the usual target of most researchers in this area). This enzyme, MTAN, has a key role in the biosynthesis of AHSLs.

The sophisticated chemical technology employed by Dr. Schramm and his colleagues was of course the transition state analogue technology developed in his own laboratory. Combined with the biology-driven strategy described in the last paragraph, Dr. Schramm’s approach has succeeded in the discovery of compounds that are potential drug candidates, while approaches based on high-throughput screening for AHSL antagonists have so far failed to produce any such compounds. Dr. Scharamm’s laboratory has also obtained evidence that treatment with their compounds should not result in the selection of resistant strains of pathogenic bacteria.

It is possible that other chemistry approaches might be successfully employed to discover quorum sensing inhibitors, both for gram-negative bacteria and gram-positive organisms such as S. aureus.

As we have discussed in numerous articles on this blog, biology-driven drug discovery strategies, often coupled with innovative approaches to chemistry (in the case of small-molecule drug discovery) are applicable to very many different targets involved in a whole range of human diseases. (Biology-driven drug discovery has also been central to discovery and development of many successful large-molecule drugs.) The quorum sensing case study in this article is a simple, understandable, and elegant example of such a strategy.

In addition to the scientific, clinical, and medical aspects of antibacterial drug discovery, the other major issue is the business of antibacterial discovery and development. The economics of drug discovery and development have shifted pharmaceutical industry investment away from the development of drugs targeting short course therapies for acute diseases (such as antibacterials) and towards long-term treatment of chronic conditions.  At the same time, discovery of novel antibacterials has gotten more difficult. As a result, during the 2000-2010 period, such companies as Wyeth, Aventis, Eli Lilly, GlaxoSmithKline, Bristol-Myers Squibb, Abbott Laboratories, Proctor & Gamble, and Merck have either deprioritized anti-bacterial R&D or left the field altogether. Meanwhile, antibiotic resistance, which was a problem in 2000, has become an even greater problem in 2012, in some cases reaching crisis proportions [e.g, methicillin resistant S. aureus (MRSA) that is also resistant to the drug of last resort, vancomycin].

As a result of these economic, scientific, and medical challenges, a €223.7 consortium of five pharmaceutical companies and leading academics, called NewDrugs4BagBugs (ND4BB) was launched in Europe in May 2012. The program is envisioned to involve a three-stage approach – to improve the understanding of antimicrobial resistance, to design and implement efficient clinical trials, and finally, to take novel drug candidates through clinical development.

And at least one venture capitalist has observed that biotechs that specialize in antibacterial drug development (as well as those that specialize in other areas that have been deemphasized by Big Pharmas) have provided “contrarian opportunities” in biotech venture. According to a June 2 2012 article by Bruce Booth of Atlas Venture published in Forbes, what has been deprioritized by some (or several) Big Pharmas, are likely be re-prioritized by others several years later. Such antibacterial drug developers as Calixa, Cerexa, Novexel, Neutec, Paratek, Pennisula, Protez, and Vicuron have produced some of the best returns in biotech venture capital from merger/acquisition exits. These biotechs included companies that were built around compounds outlicensed from Big Pharma, and others that conducted new research on novel targets, especially for MRSA and other resistant bacteria.  By taking advantage of a strategic depriorization in Pharma, these biotechs and their venture backers were able to create considerable value in the past decade out of antibacterial drug development.

Meanwhile, antibiotic specialist Cubist Pharmaceuticals (Lexington, MA) remains an independent, and profitable, biotech company that is continuing to conduct R&D, including on discovery and development of agents to treat pathogens that are resistant to current antibiotics. It has expanded into development and marketing of peripheral mu-opioid receptor antagonists (including via acquisition of Adolor in 2011), and has recently expanded its R&D facilities.

Can Pico Pharmaceuticals (which has oncology programs in addition to antibacterials) experience similar success?


As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

Advances in the Discovery of Protein-Protein Interaction Modulators published by Informa’s Scrip Insights



On April 13, 2012, Informa’s Scrip Insights announced the publication of a new book-length report, Advances in the Discovery of Protein-Protein Interaction Modulators, by Allan B. Haberman, Ph.D.

Protein-protein interactions (PPIs) are of central importance in biochemical pathways, including pathways involved in disease processes. However, PPIs have been considered the prototypical “undruggable” or “challenging” targets. The discovery of small-molecule drugs that can serve as antagonists or agonists of PPIs, and which are capable of being successfully taken into human clinical trials, has been extremely difficult. Among the theoretical reasons for this is that contact surfaces involved in PPIs are usually large and flat, and lack the types of cavities present in the surfaces of proteins that bind to small-molecule ligands.

Nevertheless, over the last twenty years, researchers have developed a set of technologies and strategies that have enabled them, in a several cases, to discover developable small-molecule PPI modulators. One direct PPI agonist, the thrombopoietin mimetic eltrombopag (Ligand/GlaxoSmithKline’s Promacta/Revolade), has reached the market. The chemical structure of this compound is illustrated in the figure above. Several other small-molecule PPI modulators are in clinical trials. Despite this progress, the discovery and development of small-molecule PPI modulators has been one-at-a-time, slow and laborious.

The new strategic importance of protein-protein interactions as drug targets

Meanwhile, PPIs as potential drug targets have acquired a key strategic importance for the success of the pharmaceutical industry. Over at least the last decade, pharmaceutical R&D has failed to develop enough high-valued new drugs to make up for or exceed revenues from blockbusters that are losing patent protection. As we have discussed in previous publications and in articles on this blog, this low productivity is mainly due to pipeline attrition. There are several factors (ranging from target selection through drug design, preclinical studies, identification and use of biomarkers, and design of clinical trials) that can influence pipeline attrition.

However, increasing numbers of industry leaders and analysts identify target selection as the key factor that is limiting the productivity of pharmaceutical R&D. For example, I served as a workshop leader at Hanson Wade’s “World Drug Targets Summit”  last summer, which took that point of view. There are at least several such conferences throughout the year, which are organized at the request of industry leaders.

Industry experts who identify poor target selection as a major cause of pharma R&D’s productivity woes conclude that the main issue is that companies are running out of “druggable” targets that have not already been addressed by marketed drugs. As of 2011, only 2% of human proteins have been targeted with drugs. Most of the remaining disease-relevant proteins, including transcription factors and many other types of signaling proteins, work via interacting with other proteins in PPIs. Therefore, in order to reverse its R&D slump, the pharmaceutical industry needs to develop technologies and strategies to address PPIs and other hitherto “undruggable” targets.

Contents of the report

Our report discusses technologies and strategies that enable the discovery of drugs targeting PPIs, including both small-molecule and synthetic peptidic modulators. It includes case studies on the discovery of compounds that address specific target classes, with emphasis on agents that have reached human clinical studies. This includes addressing the issue of the need to produce PPI modulatory agents that have pharmacological properties that will enable them to be good clinical candidates.

The report also includes discussions of second-generation technologies for the discovery of small-molecule and peptidic PPI modulators, which have been developed by such companies as Forma, Ensemble, and Aileron, and by academic laboratories. The field of PPI modulator discovery has represented a “premature technology”, i.e., a field of biomedical science in which consistent practicable therapeutic applications are in the indefinite future, due to difficult technological hurdles. We have discussed premature technologies on earlier articles on this blog. The second-generation technologies are designed to overcome the hurdles and to thus enable a more accelerated and systematic approach to PPI drug discovery and development.

In part as the result of the development of these technologies, and of the increasing strategic importance of PPI modulator development, companies have been moving into the field. Examples include Bristol-Myers Squibb, Pfizer, Novartis, and Roche. A key issue is to what extent the new technologies for PPI modulator R&D will enable this area to be commercially successful, and to meet the strategic needs of the industry for expanding the universe of targets for which drugs can be developed.

For more information about Advances in the Discovery of Protein-Protein Interaction Modulators, or to order the report, see the Scrip Insights website.


As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

Forma Therapeutics enters into two Big Pharma alliances in succession for the New Year!


RNase/RNase inhibitor protein-protein interaction. Dcrjsr

We mentioned Forma Therapeutics in two previous articles on this blog. In one article, we focused on Forma’s R&D efforts in discovering small-molecule inhibitors of protein-protein interactions (PPIs).  The other article included a discussion on Forma’s efforts in cancer metabolism.

This month–January 2012–when the new year had barely started–Forma signed two new Big Pharma alliances, covering both of these areas.

On January 5, Forma announced that it had entered into an R&D collaboration with Boehringer Ingelheim, focusing on discovery and development of small molecule drugs to address oncology-relevant PPIs. Under the terms of the agreement, Forma will receive a total of $65 million in up-front payments and research funding, and could be eligible for up to $750 million in pre-commercial milestone payments for development programs resulting from the collaboration.

As with the Genentech deal in cancer metabolism that we discussed in an earlier article, the new Boehringer Ingelheim agreement provides Forma and its shareholders several opportunities to realize early return through assets developed under the collaboration. However, details of how this might occur were not disclosed. According to a January 6, 2012 article in BioWorld Today, flexibility and liquidity (without the need for a IPO or an acquisition) are importance goal of Forma’s business development activity in general. Nevertheless, Forma CEO Steven Tregay does not rule out a future acquisition, and says that large pharmaceutical companies are interested in such a deal.

On January 10, 2012, Forma announced an exclusive alliance with Janssen Biotech (a Johnson & Johnson company), in which the companies will collaborate on the discovery, development and commercialization of novel small molecule drug candidates that target mechanisms of tumor metabolism.

Under the terms of the agreement, Forma will discover and develop drugs against a panel of tumor metabolism targets. Forma may receive up to $700 million in project and milestone funding. In addition, FORMA may receive royalties on revenues from products commercialized as a result of the collaboration. Moreover, if certain milestones are achieved during the initial phase of the collaboration, FORMA will have the opportunity to co-develop and maintain North American commercial rights to one program selected by Janssen. The two companies may also expand the collaboration to include other targets, including those in areas beyond tumor metabolism.

Once again, Dr. Tregay sees the opportunity to maintain North American rights to a product resulting from the collaboration as in line with the company’s strategy to create long-term shareholder value within Forma.

In December 2011, Forma moved its operations from Cambridge MA to Watertown MA, in the process gaining double the amount of space it had before. This will allow for the company’s growth in new internal and partnered R&D projects, and for the growth in staff that this will entail.

As we discussed in earlier articles on this blog, PPIs have been considered “undruggable” targets. However, given that researchers have been able to discover and in at least one case develop small-molecule agents to address this class of targets, it is best to think of this area as a premature technology. As discussed in our July 27, 2011 article, Forma believes that it has developed a set of enabling technologies to move the PPI field up the technology curve, similar to what happened to the monoclonal antibody field in the 1990s. Apparently, several partner organizations–not only Boehringer Ingelheim, but also Novartis and the Leukemia & Lymphoma Society–agreed with Forma enough to invest in partnerships in this area.

Forma is not the only Boston-area biotech to have a major program in discovery of drugs that modulate PPIs. Ensemble Therapeutics (Cambridge, MA), has internal programs and partnerships in discovery of small-molecule compounds that target PPIs, and Aileron Therapeutics (Cambridge, MA), which we discussed in our November 27th 2009 and our August 24th 2010 blog articles, is developing peptide compounds designed to target PPIs in internal and partnered programs.

As for cancer metabolism, Forma is once again not the only Boston-area biotech to have major programs in drug discovery in this area. We have discussed Agios Pharmaceuticals, which specializes in that area, in our December 31, 2009, April 23, 2010, and November 30, 2011  Biopharmconsortium Blog articles.

In our December 22, 2010 blog article, we discussed the field of intermediary metabolism, asking “Will intermediary metabolism be a hot field of biology again?” In the 1920s through the 1950s, intermediary metabolism was a hot field of biology, but the field was eclipsed by molecular biology starting with the Watson and Crick paper in 1953. However, largely as the result of research that combines intermediary metabolism and molecular biology, metabolism is coming to the forefront of biomedicine again. In the area of cancer metabolism, researchers such as signal-transduction pioneer (and Agios scientific founder) Lewis Cantley have been combining the two fields in order to understand cancer disease pathways, with implications for drug discovery and development.

All of the companies mentioned in this article are research-stage companies, with no drug candidates yet beyond the preclinical stage. The strategies of these companies, and the compounds that have resulted from them, thus must be validated in clinical studies. Nevertheless, we are encouraged by these companies’ success so far, and the interest show in them and their science and technology platforms by large pharmaceutical companies. The success of these companies also provides an object lesson–premature technologies and neglected fields may at least in some cases provide opportunities for drug developers.


As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

World Drug Targets Summit, Cambridge MA, July 19-21


Hanson Wade’s World Drug Targets Summit took place on July 20-21, 2011, with pre-conference workshops on July 19. The conference was held in the Sheraton Commander Hotel in Harvard Square in Cambridge, MA.

I led the first workshop on the 19th, on “Developing Improved Animal Models in Oncology and CNS Diseases to Increase Drug Discovery and Development Capabilities”. The workshop was well-attended, with good questions and discussion from those in attendance. For a description of the workshop, see our July 5, 2011 blog post. The second workshop, on “Exploiting Kinase Signaling Pathways: Opportunities for Drug Development”, was led by Kamal D Puri and Heather Webb, both of Gilead Sciences (Foster City, CA).

The main conference included speakers from both Big Pharmas (Novartis, UCB Pharma, Merck, Pfizer, AstraZeneca, Boehringer Ingelheim, Bayer Schering Pharma) and such biotech companies as Gilead, Infinity Pharmaceuticals, Merrimack Pharmaceuticals, NeurAxon, and FORMA Therapeutics, as well as a couple of researchers from Harvard Medical School and its teaching hospitals. Attendees who were not speakers included people from these same companies and from other Big Pharmas, as well as from such up-and-coming biotechs as Aileron Therapeutics and Proteostasis Therapeutics (both in Cambridge, MA and both mentioned on our blog), and other companies in the U.S. and in Europe.

In addition to case studies and strategies for identifying and validating drug targets that would be likely to yield safe, efficacious, and commercializable drugs, there was a section on strategies for fostering outsourcing and collaboration in target identification and validation. These included Bayer’s Grants 4 Targets program and Tempero Pharmaceuticals’ collaborative programs. (Tempero is a wholly owned subsidiary of GlaxoSmithKline located in Cambridge, MA.)

One highlight of the Summit was a section on “undruggable” targets (and hard targets known as “high-hanging fruit”); this section occurred at the end of the conference. John Andrews of NeurAxon (Mississauga, Ontario Canada) gave an overview of companies working on “undruggables”, which included not only protein-protein interactions (PPIs), but also what we have called areas of “premature technology” such as RNAi therapeutics and, up until the mid-1990s, monoclonal antibody drugs. (See our blog articles located here, here, and here.) He then presented NeurAxon’s own work on developing a first-in-class neuronal nitric oxide synthase (nNOS) inhibitor for treatment of migraine. nNOS inhibitors represent “high-hanging fruit” because of the difficulty of designing drug-like compounds that are selective for nNOS as opposed to endothelial NOS (eNOS).

At the end of Dr. Andrews’ presentation, I briefly outlined the concept of “premature technologies”, and the development of enabling technologies to overcome technological prematurity. MAb drugs constitute a classic case. I then asked if researchers were developing enabling technologies to make possible the efficient discovery of small-molecule drugs to address PPIs, as opposed to the case-by-case development of such drugs as occurs now. (See this article on our blog for an example.)

The chairman for the day, David Winkler of Infinity Pharmaceuticals, instead of having Dr. Andrews answer the question, moved on to the final speaker of the day, Mark Tebbe of FORMA Therapeutics (Cambridge, MA). Dr. Tebbe discussed FORMA’s technology platforms, which are designed to be enabling technologies for discovery of small-molecule drugs to address PPIs, thus answering my question.

In particular, Dr. Tebbe cited FORMA’s CS-Mapping platform, which enables company researchers to interrogate PPIs in intracellular environments, to define hot spots on the protein surfaces that might constitute targets for small-molecule drugs. (For an example of hot spots that are critical for binding in a PPI in the Wnt signaling pathway, see this research report, which we cited in our PPI blog article.) FORMA combines CS-Mapping technology with its chemistry technologies (e.g., structure guided drug discovery, diversity orientated synthesis) to discover drugs.

As an example of hot spot determination, Dr. Tebbe cited the GTP/GDP biding site of the RAS protein. RAS is a notoriously “undruggable” target that is important in a large percentage of human cancers.

FORMA also has a collaboration with the Leukemia & Lymphoma Society to discover and develop small-molecule compounds that target the interaction between the transcriptional repressor Bcl-6 and the SMRT co-repressor. This interaction is key to signaling pathways that are involved in diffuse large B cell lymphoma, a type of aggressive non-Hodgkin’s lymphoma.

FORMA has several executives and board members with Novartis backgrounds, and Novartis is an investor in FORMA and collaborates with FORMA in the area of small-molecule drugs for PPIs in oncology. As discussed in the blog article mentioned earlier on development of small-molecule drugs to target PPIs, Novartis has also been collaborating with researchers at Harvard teaching hospitals in that area. These collaborations show the interest of Novartis in the PPI area, which many pharmaceutical companies shun because of its difficulty and high risk.

The World Drug Targets Summit was a relatively small conference, but had a high concentration of pharmaceutical and biotechnology company R&D leaders, especially in target identification and validation. This provided excellent opportunities to ask questions of the speakers, and to interact with speakers and other attendees during breaks, and in the “speed networking” session and at the conference’s networking dinner. All and all, it was a good conference.

Haberman Associates Multitargeted Therapies report published by CHI Insight Pharma Reports


On June 1, 2011, Cambridge Healthtech Institute’s (CHI’s) Insight Pharma Reports announced the publication of our new book-length report, Multitargeted Therapies: Promiscuous Drugs and Combination Therapies.

In the past 20 years or so, pharmaceutical and biotechnology industry R&D has been increasingly aimed at developing drugs to treat complex diseases such as cancer, cardiovascular disease, type 2 diabetes, and Alzheimer’s disease. However, the one drug-one target-one disease paradigm that has become dominant in the post-genomic era has proven to be inadequate to address complex diseases, which have multiple “causes”, and each of which may be more than one disease. This has been a major cause of clinical failure and the low productivity of the pharmaceutical industry.

Moreover, researchers have found that most of the successful, FDA-approved small-molecule drugs that were developed prior to the year 2000 are promiscuous, i.e., they are single drugs that address multiple targets. In addition, the great majority of kinase inhibitors, one of the most successful drug classes of the early 21st century, are also promiscuous.

The study of small-molecule drug promiscuity has spawned the emerging field of network pharmacology, which can be applied both to study drug promiscuity and to rationally design small-molecule multitargeted drugs. (Researchers can discover or design multitargeted kinase inhibitors without the use of network pharmacology, however.)

Meanwhile, the development of targeted drugs such as kinase inhibitors and monoclonal antibodies has resulted in the need to develop multitargeted combination therapies. This has been especially true in cancer, where disease causation may involve multiple signaling pathways. In particular, the development of resistance to targeted antitumor drugs has spawned the need to develop second-generation treatments, many of which are multitargeted combination therapies.

Our report covers both discovery and design of small-molecule promiscuous/multitargeted drugs, and of multitargeted combination therapies.

The design of multitargeted combination therapies is one of the hottest areas of cancer R&D today, especially with respect to developing means to overcome resistance to targeted therapies. This area was the focus of many key presentations at the 2011 American Society of Clinical Oncology (ASCO) Annual Meeting, which was held in Chicago on June 3-7. For example, treatment with vemurafenib (PLX4032) of metastatic melanoma patients whose tumors carry the B-Raf(V600E) mutation has produced spectacular overall response rates and increased survival. However, in nearly all cases, the tumors relapse. The latest results with vemurafenib were discussed at ASCO 2011, as well as strategies to overcome resistance to therapy. Our new report also discusses strategies for overcoming vemurafenib resistance, all of which involve design of multitargeted combination therapies.

Another topic discussed at ASCO 2011 was antitumor strategies based on synthetic lethality. We discussed this strategy in an earlier article on this blog, especially with respect to poly(ADP) ribose polymerase (PARP) inhibitors such as KuDOS/AstraZenaca’s olaparib. At a session at the ASCO meeting entitled “PARP Inhibitors, DNA Repair, and Beyond: Theory Meets Reality in the Clinic”, speakers reviewed current progress in developing PARP inhibitors, of which six are now in clinical development.

This session also included a presentation by Michael B. Kastan, MD, PhD (St. Jude Children’s Research Hospital, Memphis TN) on other ways of using the synthetic lethally strategy, for example by targeting kinases involved in DNA repair pathways such as ATM (Ataxia-Telangiectasia Mutated) or Chk1 checkpoint kinase, or even utilizing features of the tumor microenviroment such as hypoxia. Such strategies might be used to design multitargeted combination therapies that specifically target cancer cells with defects in DNA repair and/or in hypoxic solid tumors, and/or to sensitize cancer cells to radiation.

Our new report includes a chapter on using the synthetic lethality strategy to design combination therapies of a cytotoxic drug with a chemosensitizing agent, and to develop therapies for p53-negative cancers. (The key tumor suppressor p53 is deleted, mutated, or inactivated in the majority of human cancers).

Although design of multitargeted combination therapies, as well as discovery and design of kinase inhibitors, are of key importance for current oncology R&D and are also being applied to other diseases, design of single small-molecule multitargeted drugs via network pharmacology is an early-stage, and perhaps a premature, technology. Nevertheless, given the current pharmaceutical company R&D business model that emphasizes outsourcing early-stage R&D, academic research groups and biotechnology companies that are active in this area may be able to forge partnerships with pharmaceutical companies.

For more information on Multitargeted Therapies: Promiscuous Drugs and Combination Therapies, or to order it, see the Insight Pharma Reports website.

Aileron Therapeutics partners with Roche


On November 27, 2009, we posted an article on this blog about the use of stapled peptides in targeting intracellular pathways. This technology was originally developed by Dr. Gregory Verdine (Department of Chemical Biology, Harvard University, Cambridge MA, and the Dana-Farber Cancer Institute, Boston MA) and his colleagues. A biotechnology company, Aileron Therapeutics (Cambridge, MA) was founded (with Dr. Verdine among its founders) in 2005 to develop and commercialize stapled peptide drugs. Aileron’s most advanced compounds, which are being developed for the treatment of solid and hematological tumors, are only in the preclinical stage.

On August 24, 2010, Aileron and Roche announced that they had entered into a collaboration to discover, develop, and commercialize stapled peptide drugs designed to address up to five undisclosed targets. These targets are selected from Roche’s key therapeutic areas of interest–oncology, viral diseases, inflammation, metabolic diseases, and central nervous system diseases.

Under the agreement, Roche will provide Aileron guaranteed funding of at least $25 million in R&D support and technology access fees. Aileron will also be eligible to receive up to $1.1 billion in discovery, development, regulatory, and commercialization milestone payments, if drug candidates are developed against all five targets. Aileron will also receive royalties on any future sales of marketed products that result from the collaboration.

In our November 2009 article, we discussed the design of stapled peptides, in which hydrocarbon moieties are used to constrain, or “staple” peptide sequences into an α-helical conformation. These sequences are designed to mimic key binding domains of proteins that are involved in intracellular signaling pathways. We gave two examples of pathways that were addressed by specific stapled peptides: the Notch pathway and a Bcl-2-related apoptotic pathway. In both cases, the stapled peptides modulated protein-protein interactions that are considered “undruggable” by conventional small-molecule drugs.

According to Roche, It is “as yet intractable” intracellular protein-protein interactions that are of special interest to the company in collaborating with Aileron.

According to Aileron, the new alliance with Roche validates the broad potential of their stapled peptide technology platform across multiple therapeutic areas and classes of targets. The alliance also provides Aileron with capital to advance its internal R&D.

The Roche agreement represents Aileron’s first Big Pharma strategic alliance. However, a venture capital consortium that included GlaxoSmithKline, Novartis, Roche, and Lilly invested $40 million in Aileron in June 2009.

As we said in our November 2009 article, stapled peptides represent an exciting and innovative technology with the potential to address “undruggable” protein-protein interactions, even though the therapeutic value of stapled peptides has not yet been confirmed in the clinic. (We have discussed several other means of addressing protein-protein interactions in various articles in this blog–these targets represent an area of opportunity for companies that are innovative enough to pursue it.) And as we discussed in a more recent article, Roche is one of the Big Pharma companies that continues to be focused on innovative drug discovery and development, in an era of Big Pharma R&D retrenchment. The Aileron-Roche partnership therefore appears to be an ideal match.

Plexxikon’s discovery of PLX4032, a selective targeted therapeutic for metastatic melanoma

In our March 2, 2010 blog post, we focused on a Phase I clinical trial of Plexxikon/Roche’s PLX4032, in which clinical researchers led by Keith T. Flaherty achieved a dramatic breakthrough in treatment of metastatic melanoma. Now we shall discuss the discovery of the drug itself, PLX4032.

In 2002, a research consortium based at the Wellcome Trust Sanger Institute in the U.K. found B-Raf somatic missense mutations in 66% of malignant melanomas (as well as in a subset of other cancers). V600E (valine substituted by glutamic acid at position 600) accounted for 80% of these mutant forms of B-Raf. The V600E mutation causes destabilization of the inactive conformation of B-Raf kinase, shifting the equilibrium toward the catalytically active conformation.

B-Raf is a serine/threonine protein kinase that is a component of an intracellular pathway that mediates signals from growth factors. B-Raf is regulated by binding to Ras. In turn, B-Raf activates MEK (mitogen-activated protein kinase kinase), which activates ERK (extracellular signal-regulated kinase). Activated ERK goes on to upregulate transcriptional pathways that promote cellular proliferation and survival.

Growth factors → →Ras→ B-Raf→ MEK→ ERK→ →upregulation of cell proliferation and survival

Growth factor signaling via Ras also controls other signaling pathways that upregulate cell proliferation, notably the PI3K-Akt (phosphatidylinositol-3-OH kinase-Akt) pathway.

The Sanger researchers found evidence that cells carrying B-Raf(V600E) no longer require Ras function for proliferation. This would mean that melanoma cells carrying this mutation could proliferate independently of growth factor signaling, resulting in the runaway proliferation characteristic of the malignant phenotype.

These results suggested that B-Raf(V600E) would be a good target for novel kinase inhibitors to treat malignant melanoma. The first such kinase inhibitors to be developed, although they had inhibitory activities at low nanomolar concentrations against B-Raf (both wild-type and mutant), were not successful in the clinic, due to their inhibition of multiple nonspecific targets and/or their poor bioavailability. Plexxikon researchers therefore set out to discover inhibitors that are highly selective for B-Raf(V600E). The result was the discovery of PLX4032.

The discovery of PLX4720 (a tool compound or chemical probe related to PLX4032) by Plexxikon researchers and their academic colleagues, and its preclinical validation, is described in a 2008 publication, Tsai et al. Plexxikon used its proprietary “scaffold-based drug design” technology platform to discover PLX4720. Scaffold-based drug design involves synthesizing sets of low-molecular weight “scaffold-like’” compounds. These compounds interact (typically at low affinity) with many members of a protein family by targeting their conserved regions.

In the B-Raf study, the researchers identified protein kinase scaffolds by screening a select library of 20,000 150-350-dalton compounds for inhibition of a set of three structurally characterized protein kinases at a concentration of 200 micromolar (μM). Of this library, 238 compounds were selected on the basis of their inhibition of the kinases by at least 30% at the 200 μM concentration. Each of the compounds was cocrystallized with one if the three kinases, Pim-1. Using this method, the researchers found that 7-azaindole bound to the ATP-binding site of Pim-1 kinase. They further modified this compound by adding side chains on the 3 position of 7-azaindole, resulting in a “scaffold candidate” with increased affinity for the ATP binding site of PIm-1 and other kinases. The researchers further modified this scaffold, based on structural data from other kinases. Ultimately, they cocrystallized their modified compounds with wild-type B-Raf and B-Raf(V600E), and optimized the structure of their compounds to give a compound, PLX4720, with selectivity for B-Raf(V600E) and against wild-type B-Raf and other kinases. This process (including the relevant chemical and protein structures) is illustrated in Figure 1 of Tsai et al.

In biochemical assays, the researchers found that PLX4720 inhibited B-Raf(V600E) at low nanomolar concentrations, and was 10-fold more selective for B-Raf(V600E) than for wild-type B-Raf, and was even more selective for B-Raf(V600E) than for other kinases.

Surprisingly, in cellular assays, PLX4720 is over 100-fold (not 10-fold) more selective in inhibiting proliferation of tumor cell lines that bear B-Raf(V600E) as compared to those that bear wild-type B-Raf. Moreover, as first found by researchers at Pfizer and their academic collaborators, a specific inhibitor of MEK (Pfizer’s CI-1040) is also similarly selective for tumor cell lines bearing B-Raf(V600E). This suggests that the B-Raf-MEK-ERK pathway is critical for the proliferation of B-Raf(V600E) cells, but not for cells bearing wild-type B-Raf. [For example, tumor cells that bear wild-type B-Raf might use the PI3K-Akt pathway to upregulate pathways that control cell proliferation independent of ERK signaling, while tumor cells that bear B-Raf(V600E) cannot.]

The B-Raf-MEK-ERK pathway dependence of B-Raf(V600E) cells may in part be related to feedback inhibition of B-Raf (and other isoforms of Raf). Activated ERK can phosporylate wild-type Raf isoforms at specific inhibitory sites. This results in downregulation of signaling via the Raf-MEK-ERK pathway. However, in cells bearing B-Raf(V600E), this feedback inhibition is disabled, resulting in uncontrolled signaling.

The Plexxikon researchers (Tsai et al.) tested PLX4720 against tumor xenograft models. Oral administration of PLX4720 blocked tumor growth, and in 4 out of 9 cases caused tumor regressions, in mice with tumor xenografts bearing B-Raf(V600E). Treatment with PLX4720 was well tolerated, and showed no adverse effects. Growth of tumor xenografts bearing wild-type B-Raf was not affected by PLX4720. In mice with tumors bearing B-Raf(V600E), PLX4720 blocked B-Raf-MEK-ERK pathway signaling, as demonstrated by immunohistochemical assays.

The exquisite specificity of PLX4720/PLX4032 for B-Raf(V600E) as compared to wild-type B-Raf was made possible by Plexxikon’s structure-guided “scaffold-based drug design” technology. Other structure-guided drug design technologies, such as fragment-based lead design, as is carried out in several companies, might be used to design comparably specific drugs.

The discovery of PLX4720/PLX4032 is an example of the use of new-generation chemistry technologies (or the revival of the old, and now disused natural products chemistry approach), coupled with biology-driven drug discovery strategies, to discover promising new drugs. We have discussed this strategy in several articles on this blog. (For example, see here and here).

Despite the promising results seen in Phase I clinical trials of PLX4032, it must be emphasized that the establishment of the efficacy and safety of this compound awaits the completion of the ongoing Phase III trials. Moreover, despite the dramatic regressions and increased survival seen in the Phase I trials, all the patients apparently eventually suffered relapses. Dr. Flaherty, as discussed in our earlier blog post, sees the need for combination therapies to effectively combat metastatic melanoma. In early 2009, Dr. Flaherty and his colleague Keiran S Smalley published a mini-review on potential strategies for developing such combination therapies.

Small-molecule drugs for targeting an intracellular signaling pathway: research and development of new oncology drugs

In our November 27th blog post, we discussed an innovative new technology, stapled peptides, for use in targeting intracellular protein-protein interactions. In the example we gave, the target was a transcription factor complex in the Notch pathway. As we stated, protein-protein interactions are deemed to be “undruggable”, since they cannot be readily addressed with small molecule drugs.

Nevertheless, in some cases, small molecules have been discovered that do address key protein-protein interactions, and which may become clinical candidates.

Back in February 2006, Decision Resources published our report, “Protein-Protein Interactions: Are They Now Druggable Targets?” Among the case studies we discussed in that report was one in which researchers were attempting to discover small-molecule agents that targeted the Wnt pathway. The researchers discovered small-molecule agents that, as with the stapled-peptide example we discussed in our previous blog post, targeted a transcription factor complex. As of late 2009, two of these compounds are in preclinical development for treatment of various cancers.

Mutations that mediate deregulation of the Wnt pathway are causative factors in several types of cancer, most notably colorectal cancer, as well as multiple myeloma (MM), hepatocellular carcinoma (HCC), and B-cell chronic lymphocytic leukemia B-CLL). In the canonical Wnt pathway, soluble extracellular factors that are members of the Wnt family activate the pathway. A complex that includes the protein adenomatous polyplosis coli (APC) is central to the Wnt pathway. When Wnt receptors are not engaged by their ligands, kinases in the APC complex phosphorylate β-catenin, a multifunctional protein that is involved both in signal transduction and in adhesion between cells. Phosphorylation targets β-catenin for degradation.

When Wnt proteins bind to their receptors, the kinase activity of the APC complex is inactivated. This results in the accumulation of β-catenin, which moves into the nucleus. There it binds to proteins of the T cell factor (Tcf) family. β-catenin binding changes Tcf from a transcriptional repressor into a transcriptional activator. Downstream genes controlled by the β-catenin/Tcf complex include the oncogene Myc and other genes that mediate cell proliferation.

In precancerous colonic adenomas or the colorectal cancers that they may evolve into, APC is usually mutated. This results in constitutive stabilization of β-catenin and constitutive activation of Tcf and its downstream genes. In other types of cancer that involve constitutive Wnt pathway activation, β-catenin also becomes stabilized, via other means. This makes the Tcf/β-catenin a tempting target for drug discovery. However, it is a protein-protein interaction, and is thus deemed “undruggable”.

In 2004, A group led by Ramesh Shivdasani (Harvard Medical School, Dana-Farber Cancer Institute, and Brigham and Women’s Hospital, Boston MA), including researchers from the Novartis Institutes for BioMedical Research (Cambridge, MA), discovered several small-molecule inhibitors of the interaction between human Tcf4 and human β-catenin.

Dr. Shivdasani’s group, among others, had previously determined crystal structures of Tcf-β-catenin complexes. The interaction between the two proteins occurs over a large surface area. It is the large, and usually hydrophobic, interface between proteins in protein-protein interactions that forms the theoretical basis for the difficulty of addressing these interactions with small molecules. However, there is a small hydrophobic pocket that is critical for binding (as also confirmed by site-specific mutation studies), which might accommodate a small molecule inhibitor.

Therefore, the researchers screened approximately 7,000 purified natural products from public and proprietary libraries using an enzyme-linked immunosorbent (ELISA) assay involving release of a labeled Tcf4 binding fragment from its complex with a β-catenin fragment absorbed to an ELISA plate. Eight compounds were found that gave reproducible, concentration-dependent release of the Tcf4 fragment at less than 10 micromolar concentration. The structures and purity of these compounds (most of which are complex, multi-ringed planar compounds with multiple hydroxy groups) were then determined. The sources of these compounds include fungi, actinomycetes, and a marine sponge.

The researchers performed several additional biochemical assays to confirm the compounds’ specific disruption of the Tcf/β-catenin complex, and also performed cellular assays and an in vivo assay in the Xenopus (frog) embryo to study the activities of these compounds against β-catenin-mediated cellular effects. Each of the eight compounds shows different levels of potency in the different assays used in this study, and the compounds differ from each other in their activities in the different assays.

Two fungal-derived compounds, PKF115-854 and CGP04909, gave the best results in all the assays. It is those compounds that have been tested in preclinical studies as potential oncology drug candidates. In a study published in PNAS in 2007, researchers at the Dana-Farber and at Brigham and Women’s Hospital tested PKF115-584 in human MM cells in vitro and in xenograft models. The compound blocked expression of Wnt target genes, induced cytotoxicity in MM cells in vitro, and inhibited tumor growth and prolonged survival in the xenograft model. In a study in HCC at the Asian Liver Center at Stanford University School of Medicine, PKF115-584, CGP049090, and another of the Shivdasani group’s compounds, PKF118-310, also induced cytotoxicity in human HCC cell lines in vitro, and suppressed tumor growth and induced apoptosis in tumor cells in a human HCC xenograft model. Finally, in an abstract presented at the American Society of Hematology (ASH) meeting in December 2009, researchers at the Novartis Institute for Biomedical Research in Basel and their academic collaborators presented data that showed that CGP04090 and PKF115-584 potently inhibited the survival of primary human B-CLL cells in vitro and in vivo. In all three cases, the compounds showed no significant cytotoxicty against normal cells.

In the conclusion of the ASH meeting abstract, the authors stated that further investigations are warranted to determine the feasibility of testing these compounds in human clinical trials.

Many medicinal chemists remain skeptical about the ability of researchers to develop small-molecule drugs that target protein-protein interactions, which have satisfactory pharmacokinetics and can advance through clinical trials and reach the market. However, at least one nonpeptide small-molecule compound that targets a protein-protein interaction, the thrombopoietin receptor agonist eltrombopag (Ligand/GSK’s Promacta), has reached the market. (The FDA approved it in November 2008.) Several other small-molecule drugs that target protein-protein interactions are in clinical development. And Cambridge Healthtech Institute will be sponsoring a conference on this subject, which is scheduled for April 2010. This conference is in its third year. Thus, as also shown by the development of stapled peptides, there is renewed interest in discovering and developing drugs that address these “hard targets”.