The Development of New Anti-infectivesfor Drug Resistant Infections

Omonike Arike Olaleye, Ph.D. MPH.Professor of Pharmacology

Program Co-Director MCH STARSRCMI Collaborations and Partnership Core, Leader

Texas Southern University, Houston, TX

Objectives:

1.To define, describe and compare the different approaches to the discovery of new anti-infective agents for drug resistant infections.

2.To describe the identification and characterization of a novel antibacterial target: Methionine Aminopeptidase

3.To describe and evaluate the process of discovery and pre-clinical development of novel anti-infectives targeting drug-resistant infections.

4.To develop and propose a plan for prevention of drug-resistant infections using population health.

The Development of New Anti-infectivesfor Drug Resistant Infections

Antimycobacterial Drug Discovery Methodologies

I. Structural Development of Existing Agents.

II. Combination of Existing Drugs with Lead Compounds

III. Whole Cell and Phenotypic Screens: Lead to Mechanism.

IV. Target-based : Methionine Aminopeptidase.

V. Chemical Genetics: Combination of Target and Whole Cell.

VI. Re-purposing of FDA Approved Drugs or New Compounds.

VII. Inhibitors of Tuberculosis and Co-Infections: HIV & VL/CL

Spectrum of MetAP Inhibitors Targeting Clinically-relevant Pathogens

AntibacterialsTB

HAIs

AntiviralsHIV

AntiparasiticsLmPf

Inhibitors of Co-infection

Growth of Infectious Diseases Research at TSU

1. Discovery of Novel Therapeutics & Targets

2. Development of Lead Compounds (PD, PK and Formulation)

3. Advancing Undergraduate Research in Drug Discovery

4. Maternal and Child Health Research

5.Pharmacoepidemiology

Bridge Global Health & Drug Discovery - Development

Infectious Diseases Projects

III. Pre-Clinical Drug Development and Chemical Biology.

7.Target Validation and Animal Models of Infection (UTEP). 8. Targeting of NME of E. faecalis in C. elegans model of infection (UTHealth).9. Cytotoxicity Studies for MetAP Inhibitors (UST).

IV. Funded Student Training Programs

10. Maternal and Child Health Student Training Fellowship Program (US, HRSA-BCM)11. Advancing Undergraduate Research through a Novel Drug Discovery Scholars Mentoring Program (NIH-UTEP)

V. Global Health Program12. Effect of Prophylactic Antimalarial on Birth Outcomes among Pregnant Women in Botswana (HSPH).

I. Target Based: Methionine Aminopeptidase.

1. Mycobacterium tuberculosis (JHU,UTMB, TMHRI).2. Enterococcus faecalis (JHU). 3. Leishmania major (UT El Paso, and RCMI-RTRN).4. Trypanosoma brucei (UT El Paso, and RCMI-RTRN).

II. Whole Cell, Re-purposing of Approved Drugs and New Comp.

5.Inhibitors of Co-Infections (UTMB, BCM-UT Health CFAR). 6. Development of an In Vitro HTS Assay for TB-HIV (UTMB).

Molecular & BiochemicalCharacterizationXray Structure

Selective Toxicity

HTSBacteria MICIn Vitro Screen

VirologyChemical Genetics

SARIn Vivo

Pharmacology

Lead

Potential Drug Candidates

Kaufmann, S.H.E. (2005). Robert Koch, the Nobel Prize, and the Ongoing Threat of Tuberculosis. N ENGL J MED 353, 2423-2426.

HISTORY OF TUBERCULOSIS

Bedaquiline, for MDR-TB

WHO GLOBAL TB REPORT 2017

•10.4 million people fell ill with TB and >1.0 million (10%) were HIV-positive.(The African Region accounted for 74% of these cases)

•600 000 cases of DRTB (490,000 MDR-TB)

•TB killed 1.3 million people (HIV-negative) and 0.4 million (HIV-positive).

•Globally, only 54% of DR-TB patients were successfully treated.

Estimated HIV prevalence in new and relapse TB cases, 2016

Countries In The Three High-burden Country Lists For TB, TB/HIV And MDR-TB

WHO GLOBAL TB REPORT 2017

TB Drug Therapy•Drugs used in the treatment of tuberculosis can be divided into two major categories: First line and second line

• First-line agents are isoniazid, rifampin, ethambutol, and pyrazinamide. In the first 2 months: isoniazid, rifampin, ethambutol, and pyrazinamide are given, followed by isoniazid and rifampin for the remaining 4 months.

•Second-line agents includes moxifloxacin or gatifloxacin, ethionamide, aminosalicylic acid, cycloserine, amikacin, kanamycin, capreomycin, and linezolid

•Many health authorities are recommending Daily Observed Therapy: DOTs for short.

•Streptomycin was the first drug used successfully to treat tuberculosis

•The goals of therapy are to shorten the clinical course, prevent complications, prevent the development of latency and/or subsequent recurrences, and decrease transmission.

•For patients with latent infection, the goal of therapy is to prevent progression of disease.

“Current” TB Drugs Developed >50yrs ago

Zhang, Y. Annu. Rev. Pharmacol. Toxicol. 45, 529-564 (2005).

Name, Year Structure M.I.C. (μg/mL)

Isoniazid (INH), 1952 0.01-0.2

Inhibition of cell wall mycolic acid synthesis & other effects on DNA, lipids, carbohydrates, & NAD Metabolism. Multiple targets including acyl carrier protein reductase (InhA)

Rifampin (RMP), 1966 0.05-0.5Inhibition of RNA synthesis, Targets RNA Polymerase β subunit

Ethambutol (EMB), 1961 1-5 Inhibition of cell wall arabinogalactan synthesis, Targets Arabinosyl transferase

Pyrazinamide (PZA), 1952 20-100 Membrane energy metabolism

Mechanism of Action and Target

•Chemotherapy: First 2 months: INH, RIF, ETH & PZA ; INH & RIF for the remaining 4 months.

Antibacterial Agents Currently In Phases 1–3 of Clinical Development

PPL, Priority Pathogens List. 42 new therapeutics, 9 biologicals, 7 –TB

Criteria for Innovation:1. No Cross-resistance To Existing Antibiotics2. New Chemical Class3. New Target 4. New Mechanism Of Action

Antibacterial agents in clinical development: an analysis of the antibacterial clinical development pipeline, including tuberculosis. Geneva: World Health Organization; 2017 (WHO/EMP/IAU/2017.12)

Adapted from Antibacterial agents in clinical development: an analysis of the antibacterial clinical development pipeline, including tuberculosis. Geneva: World Health Organization; 2017 (WHO/EMP/IAU/2017.12)

TB is the leading cause from a single infectious agent worldwide

1. TB-HIV: TB is the leading cause of death among people living with HIV.

2. MDR-, XDR-, and TDR-TB: Threatens the survival of HIV patients.

3. THERAPEUTIC CHALLENGES IN THE MANAGEMENT OF HIV-TB CO-INFECTION

4. TB, MINORITY HEALTH AND HEALTH DISPARITIES: “TB adversely affects minority populations and about 87% of the TB cases reported in the United States in 2015 were in racial and ethnic minorities-CDC.”

“We can’t fight AIDS unless we do much more to fight TB”-Nelson Mandela, Bangkok, 14 July, 2004.

HIV-Associated TB, Facts 2013. TB Alliance.

TB-HIV & Drug Resistant TB Infections

Therapeutic Challenges in the Management of HIV-TB Co-infection

Highly active antiretroviral therapy (HAART)

PI & NNRTI are metabolized by CYP3A4 isoenzyme.

RIFAMYCINS are potent inducers of CYP3A4

REDUCTION IN EFFICACY OF HAART REGIME

John SF, Aniemeke E, Ha NP, Chong CR, Gu P, Zhou J, Zhang Y, Graviss E, Liu JO, and Olaleye OA. Tuberculosis, 101, S73-77, 2016.Ebeid A, Davis P, Olaleye O, Guinn D, Aniemeke E, Eluwa A, and Finney S.US Pharmacists 63-68, April, 2013.

The lethal synergy of TB-HIV

High pill burden, drug to drug interactions and toxicity.

Emergence of drug resistance

• Dec. 31, 2012 FDA Grants Accelerated Approval for SIRTURO™ (bedaquiline) as Part of Combination Therapy to Treat Adults with Pulmonary MDR-TB.

“However, 5 times as many patients given the experimental drug died—10 of 79, vs 2 of 81 in the control group. Five of the 10 deaths in patients given the new drug were caused by tuberculosis, indicating treatment failure, as were the 2 deaths in the control group. No single cause explained the remaining excess deaths in the bedaquiline group, but several could have been related to hepatotoxicity. Bedaquiline was also found to cause QT prolongation, which can result in fatal ventricular fibrillation; now that the drug is approved, that fact and the excess mortality will be noted in a black box warning on its label.” Jerry Avorn, MD

JAMA, April 3, 2013—Vol 309, No. 13 1349

Approval of a Tuberculosis Drug Based on a Paradoxical Surrogate Measure

Bedaquiline

Methionine Aminopeptidases

• N-terminal processing of proteins is important for localization, stability, and post-translational modifications

• Genetic studies revealed the lethality of the deletion of MetAP(s) and MetAP belongs to the minimal eubacteria genome requirement

Chang, S. Y., McGary, E.C., & Chang, S. J. Bacteriol. 171, 4071-4072 (1989).Giglione, C., Vallon, O., & Meinnel, T. The EMBO Journal, 22, 13-23 (2003).Miesel, L., Greene, J. & Black, T. Nature Review Genetics, 4, 442-456 (2003)

MetAP as a Promising Drug Target

Characterization of clioquinol and analogues as novel inhibitors of methionine aminopeptidases from Mycobacterium tuberculosisOmonike Olaleye a,b,*, Tirumalai R. Raghunand d, f, Shridhar Bhat a, Curtis Chong a,g, Peihua Gu e,Jiangbing Zhou e, Ying Zhang e, William R. Bishai d, Jun O. Liu a,c,**

Classes of Methionine Aminopeptidase

MetAP is conserved in all life forms from bacteria to humans.

Eukaryotes posses both classes.

Prokaryotes possess homologues of either MetAP1(eubacteria) or MetAP2(archeabacteria).

Addlagatta, A., Quillin, M., Omotoso, O. Liu, J. O., & Matthews, B. W. Biochemistry, 44, 7166-7174 (2005). Lowther, W. T., and Matthews, B. W. (2000). Biochimica et Biophysica Acta. 1477, 157-167. Types of MetAP ( Courtesy of Isichei A, & John S)

Two MetAPs from M. tuberculosis MetAP1a --------------MRPLARLRGRRVVPQRSAG------------ELDAMAAAGAVVAAA 34 MetAP1c MPSRTALSPGVLSPTRPVPNWIARPEYVGKPAAQEGSEPWVQTPEVIEKMRVAGRIAAGA 60 **:.. .* :.*. :: * .** :.*.* MetAP1a LRAIRAAAAPGTSSLSLDEIAESVIRESGATPSFLGYHGYPASICASINDRVVHGIPSTA 94 MetAP1c LAEAGKAVAPGVTTDELDRIAHEYLVDNGAYPSTLGYKGFPKSCCTSLNEVICHGIP-DS 119 * *.***.:: .**.**.. : :.** ** ***:*:* * *:*:*: : **** : MetAP1a EVLAPGDLVSIDCGAVLDGWHGDAAITFGVGALSDADEALSEATRESLQAGIAAMVVGNR 154 MetAP1c TVITDGDIVNIDVTAYIGGVHGDTNATFPAGDVADEHRLLVDRTREATMRAINTVKPGRA 179 *:: **:*.** * :.* ***: ** .* ::* .. * : ***: .* :: *. MetAP1a LTDVAHAIETGTRAAELRYGRSFGIVAGYGGHGIGRQMHMDPFLPNEGAPGRGPLLAAGS 214 MetAP1c LSVIGRVIES----YANRFG--YNVVRDFTGHGIGTTFHNGLVVLHYDQPAVETIMQPGM 233 *: :.:.**: *:* :.:* .: ***** :* . .: : . *. .:: .* MetAP1a VLAIEPMLTLGTTKTVVLDDKWTVTTADGSRAAHWEHTVAVTDDGPRILTLG 266 MetAP1c TFTIEPMINLGALDYEIWDDGWTVVTKDRKWTAQFEHTLLVTDTGVEILTCL 285 .::****:.**: . : ** ***.* * . :*::***: *** * .***

• MtMetAP1a and MtMetAP1c share about 33% identity

• Both mycobacterium isoforms have less than 48% and 30% similarity to hMetAP1 and hMetAP2 respectively

MetAPs as Targets for TB• M. tuberculosis uses MtMetAP1s for N-terminal excision of essential proteins.• Therefore, we propose to inhibit Mycobacterium tuberculosis using MetAP inhibitors.

Olaleye, OA., Bishai, WR., and Liu, JO. Targeting the Role of N-terminal Methionine Processing Enzymes in Mycobacterium tuberculosis (2009). Tuberculosis (Edinb). 89, Suppl 1:S55-9.

Mtb PDF

Formyl-Met-Polypeptide Met-Polypeptide Polypeptide or Protein

MtMetAP1aor

MtMetAP1c

Some Essential and Non-Essential Mtb Functional Proteins

Mtb Survival and Dormancy during Host Infection

•Post-translational modifications•Stability•Localization•Targeted protein degradation

Inhibitors

Inhibitors

Specific Aims and Research Strategy

2. I. Structure Activity/Toxicity RelationshipII. Formulation and Physiochemical Studies

3. I. TB Drug sensitive Mouse ModelII. TB Drug resistant Mouse Model Target Validation

Pre-Clinical Drug Development

Clone, Over-express & Purify MetAP

Biochemical Characterization

MetAP High-throughput Screening (175,000cmpds)Drug sensitive Mycobacterial Culture Screen

TB-HIV Assay Development

1. I. Drug Resistant mycobacterial Culture ScreenII. Isolation of Resistant Mutants

Phas

e 1

Phas

e 2

MtMetAPs as Antimycobacterial TargetsMW 1 2 3MW 1 2 3

177.3110.779.861.047.835.9

24.5

18.7

13.9

MtMetAP1a (~28 kDa) MtMetAP1c (~32 kDa)

177.3110.7

79.861.0

47.835.9

24.518.7

Olaleye O, Raghunand TR, Bhat S, He J, Tyagi S, Lamichhane G, Gu P, Zhou J, Zhang Y, Grosset J, Bishai WR, Liu JO. Cell Press, Chem Biol.17:86-97,2010.Olaleye O, Raghunand, TR, Bhat, S., Chong CR, Gu, P, Zhou, J, Zhang, Y, Bishai, WR, and Liu, JO. Tuberculosis 91 Suppl 1:S61-5, 2011.Bhat S, Olaleye O, Meyer KJ, Shi W, Zhang, Y, and Liu, JO. Bioorg. Med. Chem. 20: 4507-4513, 2012.

0 250 500 750 10000.0

2.5

5.0

7.5

MtMetAP1a

MtMetAP1c

Met-Pro-pNA Concentration (µM)

V (µ

M/m

in)

OTB6

IC50 (μM)

MtMetAP1c: 5.3MtMetAP1a: 4.9HsMetAP1: 315.5HsMetAP2: 145.6

MIC (μg/mL)

Mtb: 5.0 – 10.0DMtb:1.82

Development of Novel Models and Inhibitors Targeting TB and HIV Co-infection

JHU-NAQMtI-2

IC50 (µM)MtMetAP1c : 8.7MtMetAP1a : 4.0HsMetAP1¶ : > 30HsMetAP2§ : > 100 Mn2+-HsMetAP2§ : > 10

M.I.C. (µg/mL)Mtb : 25.0DMtb : 23.8

JHU-NAQMtI-4

IC50 (µM)MtMetAP1c : 6.6MtMetAP1a : 3.3HsMetAP1¶ : 1.1HsMetAP2§ : > 100 Mn2+-HsMetAP2§ : 0.91

M.I.C. (µg/mL)Mtb : 10.0DMtb : 5.7-11.4

JHU-NAQMtI-2

IC50 (µM)MtMetAP1c : 8.7MtMetAP1a : 4.0HsMetAP1¶ : > 30HsMetAP2§ : > 100 Mn2+-HsMetAP2§ : > 10

M.I.C. (µg/mL)Mtb : 25.0DMtb : 23.8

JHU-NAQMtI-4

IC50 (µM)MtMetAP1c : 6.6MtMetAP1a : 3.3HsMetAP1¶ : 1.1HsMetAP2§ : > 100 Mn2+-HsMetAP2§ : 0.91

M.I.C. (µg/mL)Mtb : 10.0DMtb : 5.7-11.4

JHU-NAQMtI-2

IC50 (µM)MtMetAP1c : 8.7MtMetAP1a : 4.0HsMetAP1¶ : > 30HsMetAP2§ : > 100 Mn2+-HsMetAP2§ : > 10

M.I.C. (µg/mL)Mtb : 25.0DMtb : 23.8

JHU-NAQMtI-4

IC50 (µM)MtMetAP1c : 6.6MtMetAP1a : 3.3HsMetAP1¶ : 1.1HsMetAP2§ : > 100 Mn2+-HsMetAP2§ : 0.91

M.I.C. (µg/mL)Mtb : 10.0DMtb : 5.7-11.4

SB1602A

IC50 (µM)MtMetAP1c : 0.40MtMetAP1a : 34.68HsMetAP1¶ : > 30HsMetAP2§ : > 30

RH01190SC

IC50 (µM)MtMetAP1c : 0.36MtMetAP1a : < 50HsMetAP1¶ : > 30HsMetAP2§ : > 30

N

SNH N

OHN

SNH N

OH

OCH3

SB1602A

IC50 (µM)MtMetAP1c : 0.40MtMetAP1a : 34.68HsMetAP1¶ : > 30HsMetAP2§ : > 30

RH01190SC

IC50 (µM)MtMetAP1c : 0.36MtMetAP1a : < 50HsMetAP1¶ : > 30HsMetAP2§ : > 30

N

SNH N

OHN

SNH N

OH

OCH3

Pharmacology of MtMetAP1 Inhibitors

Antibacterial agents in clinical development: an analysis of the antibacterial clinical development pipeline, including tuberculosis. Geneva: World Health Organization; 2017 (WHO/EMP/IAU/2017.12)

II. Lead Identification•High-throughput Screening & Validation

•IC50 determination•SAR: Optimization of Potency & Selectivity

III. Mycobacterial culture Screen• M. tuberculosis

•Spectrum of activity•Synthesis of Novel derivatives

IV. In vivo Target Validation• Over-expression of target in Mtb•Knockdown (Test for essentiality)

•Drug Resistant Mtb•In Vivo Pharmacology

•Pre-clinical Studies

I. Target Identification•Cloning, Over-expression & Purification

•Biochemical & Structural Characterization

MtMetAPs as Novel Anti-TB Targets We have successfully screened, characterized and validated novel classes of inhibitors of MetAPs.

The insights gained from these studies will reveal the physiological role of MetAP in pathogenesis of infectious diseases and facilitate the future design of novel antibacterial agents.

We have developed a novel assay for mycobacteria-HIV drug HTS screening.

A Multiplex Approach to Infections

TSU RCMI Core Capabilities

Acknowledgement

Funding:•Baylor–UT Health NIAID Center for AIDS Research•Texas Southern University Seed Research Grant•John S. Dunn Gulf Coast Consortium for Chemical Genomics•NIH Research Centers in Minority Institutions, RCMI •NIH RTRN Small Grants Program•U.S. Department of Health and Human Services, HRSA•NIH UTEP BUILDing Scholars Program

•Prof. Jun Liu•Dr. William Bishai•Dr. Shridar Bhat•Dr. Jacques Grosset•Dr. Ying Zhang•Dr. Raghunand Tirumalai•Dr. Gyanu Lamichhane•Sandeep Tyagi

Ph.D.•Manvir Kaur•Collins Onyenaka•Marie Toukam•Maria Rincon-Nigro•Fadia Boughaba•Sarah Finney•Ada IsicheiPharm. D. Grads•Emmanuel Aniemeke•Veronica Ajewole•Fomanyi Fossoh•Hien Huynh•Nishat Farooqui•Robyn Butler•Vy Phillip•Uloma Okonkwo•Olubusola Sokale

•Dr. Rosa Maldonado•Elizabeth Calzada•Carylinda Serna• Miguel A. Vasquez

• Dr. Hamisu Salihu•Dr. Kiara Spooner•Dr. Jason Salemi•Dr. Christina Nance•Dr. Edward Siwak•Melinda D’Souza

Pharmaceutical Sciences•Dr. Dong Liang•Dr. Huan Xie•Dr. Angabin Matin•Dr. Jing Ma•Dr. Yuan Chen

•Dr. Janice Endsley•Sudhamathi Vijayakumar•Matthew Huante

•Dr. Rosemarie Rosell•Emily Erwin•Angela Addison•UST Team – Rosell Lab

•Dr. Edward Gravis•Ngan Ha

Pharmacy Continuing Education

Credit code: 6GDS Pharmacists: to receivecditre for participation in

this live educational session, you must claim your credit via the TSHP Education Portal (http://tshp.wcea.education/) no later than Monday, March 9, 2019.